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Morishita H, Kawai K, Egami Y, Honda K, Araki N. Live-cell imaging and CLEM reveal the existence of ACTN4-dependent ruffle-edge lamellipodia acting as a novel mode of cell migration. Exp Cell Res 2024; 442:114232. [PMID: 39222868 DOI: 10.1016/j.yexcr.2024.114232] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2024] [Revised: 08/26/2024] [Accepted: 08/29/2024] [Indexed: 09/04/2024]
Abstract
α-Actinin-4 (ACTN4) expression levels are correlated with the invasive and metastatic potential of cancer cells; however, the underlying mechanism remains unclear. Here, we identified ACTN4-localized ruffle-edge lamellipodia using live-cell imaging and correlative light and electron microscopy (CLEM). BSC-1 cells expressing EGFP-ACTN4 showed that ACTN4 was most abundant in the leading edges of lamellipodia, although it was also present in stress fibers and focal adhesions. ACTN4 localization in lamellipodia was markedly diminished by phosphoinositide 3-kinase inhibition, whereas its localization in stress fibers and focal adhesions remained. Furthermore, overexpression of ACTN4, but not ACTN1, promoted lamellipodial formation. Live-cell analysis demonstrated that ACTN4-enriched lamellipodia are highly dynamic and associated with cell migration. CLEM revealed that ACTN4-enriched lamellipodia exhibit a characteristic morphology of multilayered ruffle-edges that differs from canonical flat lamellipodia. Similar ruffle-edge lamellipodia were observed in A549 and MDA-MB-231 invasive cancer cells. ACTN4 knockdown suppressed the formation of ruffle-edge lamellipodia and cell migration during wound healing in A549 monolayer cultures. Additionally, membrane-type 1 matrix metalloproteinase was observed in the membrane ruffles, suggesting that ruffle-edge lamellipodia have the ability to degrade the extracellular matrix and may contribute to active cell migration/invasion in certain cancer cell types.
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Affiliation(s)
- Haruka Morishita
- Department of Histology and Cell Biology, School of Medicine, Kagawa University, Kagawa, 761-0793, Miki, Japan
| | - Katsuhisa Kawai
- Department of Histology and Cell Biology, School of Medicine, Kagawa University, Kagawa, 761-0793, Miki, Japan
| | - Youhei Egami
- Department of Histology and Cell Biology, School of Medicine, Kagawa University, Kagawa, 761-0793, Miki, Japan
| | - Kazufumi Honda
- Department of Bioregulation, Graduate of Medicine, Nippon Medical School, Sendagi, Bunkyo-ku, 113-8602, Tokyo, Japan
| | - Nobukazu Araki
- Department of Histology and Cell Biology, School of Medicine, Kagawa University, Kagawa, 761-0793, Miki, Japan.
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2
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Kumari R, Ven K, Chastney M, Kokate SB, Peränen J, Aaron J, Kogan K, Almeida-Souza L, Kremneva E, Poincloux R, Chew TL, Gunning PW, Ivaska J, Lappalainen P. Focal adhesions contain three specialized actin nanoscale layers. Nat Commun 2024; 15:2547. [PMID: 38514695 PMCID: PMC10957975 DOI: 10.1038/s41467-024-46868-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2024] [Accepted: 03/13/2024] [Indexed: 03/23/2024] Open
Abstract
Focal adhesions (FAs) connect inner workings of cell to the extracellular matrix to control cell adhesion, migration and mechanosensing. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton. By using super-resolution iPALM microscopy, we identify two additional nanoscale layers within FAs, specified by actin filaments bound to tropomyosin isoforms Tpm1.6 and Tpm3.2. The Tpm1.6-actin filaments, beneath the previously identified α-actinin cross-linked actin filaments, appear critical for adhesion maturation and controlled cell motility, whereas the adjacent Tpm3.2-actin filament layer beneath seems to facilitate adhesion disassembly. Mechanistically, Tpm3.2 stabilizes ACF-7/MACF1 and KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Tpm3.2 depletion leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during migration. Thus, FAs are composed of distinct actin filament layers, and each may have specific roles in coupling adhesions to the cytoskeleton, or in controlling adhesion dynamics.
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Affiliation(s)
- Reena Kumari
- HiLIFE Institute of Biotechnology, University of Helsinki, FI-00014, Helsinki, Finland
| | - Katharina Ven
- HiLIFE Institute of Biotechnology, University of Helsinki, FI-00014, Helsinki, Finland
| | - Megan Chastney
- Turku Bioscience Centre, University of Turku and Åbo Akademi University, FI-20520, Turku, Finland
| | - Shrikant B Kokate
- HiLIFE Institute of Biotechnology, University of Helsinki, FI-00014, Helsinki, Finland
| | - Johan Peränen
- HiLIFE Institute of Biotechnology, University of Helsinki, FI-00014, Helsinki, Finland
| | - Jesse Aaron
- Advanced Imaging Center, HHMI Janelia Research Campus, Ashburn, VA, 20147, USA
| | - Konstantin Kogan
- HiLIFE Institute of Biotechnology, University of Helsinki, FI-00014, Helsinki, Finland
| | - Leonardo Almeida-Souza
- HiLIFE Institute of Biotechnology, University of Helsinki, FI-00014, Helsinki, Finland
- Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
| | - Elena Kremneva
- HiLIFE Institute of Biotechnology, University of Helsinki, FI-00014, Helsinki, Finland
| | - Renaud Poincloux
- Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, Toulouse, France
| | - Teng-Leong Chew
- Advanced Imaging Center, HHMI Janelia Research Campus, Ashburn, VA, 20147, USA
| | - Peter W Gunning
- School of Biomedical Sciences, UNSW Sydney, Wallace Wurth Building, Sydney, NSW 2052, Australia
| | - Johanna Ivaska
- Turku Bioscience Centre, University of Turku and Åbo Akademi University, FI-20520, Turku, Finland
- Department of Life Technologies, University of Turku, FI-20520, Turku, Finland
- InFLAMES Research Flagship Center, University of Turku, Turku, Finland
- Foundation for the Finnish Cancer Institute, Tukholmankatu 8, FI-00014, Helsinki, Finland
| | - Pekka Lappalainen
- HiLIFE Institute of Biotechnology, University of Helsinki, FI-00014, Helsinki, Finland.
- Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland.
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3
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Chinnathambi S. α-Linolenic Acid Vesicles-Mediated Tau Internalization in Microglia. Methods Mol Biol 2024; 2816:117-128. [PMID: 38977593 DOI: 10.1007/978-1-0716-3902-3_11] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/10/2024]
Abstract
In Alzheimer's disease, the synaptic loss is prominent due to the accumulation of Amyloid βeta (Aβ) protein in synapses, which affect neurotransmission, and thus ultimately causes neuronal loss. Tau, a microtubule-associated protein, is a vital protein of intracellular neurofibrillary tangles (NFTs) in AD. Along with the accumulation of aberrant proteins, glial cells, mainly astrocytes and microglia, play a major role in impairing neuronal network. Microglia have the ability to phagocytose Tau and rerelease in exosomes, which causes further spreading of Tau. Reduction in exosome synthesis can reduce spreading of Tau. Modulating microglia to clear the extracellular Tau seeds by its imported degradation would resolve the disease condition in Alzheimer's disease. In this study, we have shown the ability of α-linolenic acid (ALA) to inhibit the Tau aggregation and modulate their internalization property in microglial cells.
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Affiliation(s)
- Subashchandrabose Chinnathambi
- Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences (NIMHANS), Institute of National Importance, Bangalore, Karnataka, India.
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4
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Gélin M, Schaeffer A, Gaillard J, Guérin C, Vianay B, Orhant-Prioux M, Braun M, Leterrier C, Blanchoin L, Théry M. Microtubules under mechanical pressure can breach dense actin networks. J Cell Sci 2023; 136:jcs261667. [PMID: 37870087 DOI: 10.1242/jcs.261667] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2023] [Accepted: 09/25/2023] [Indexed: 10/24/2023] Open
Abstract
The crosstalk between the actin network and microtubules is essential for cell polarity. It orchestrates microtubule organization within the cell, driven by the asymmetry of actin architecture along the cell periphery. The physical intertwining of these networks regulates spatial organization and force distribution in the microtubule network. Although their biochemical interactions are becoming clearer, the mechanical aspects remain less understood. To explore this mechanical interplay, we developed an in vitro reconstitution assay to investigate how dynamic microtubules interact with various actin filament structures. Our findings revealed that microtubules can align and move along linear actin filament bundles through polymerization force. However, they are unable to pass through when encountering dense branched actin meshworks, similar to those present in the lamellipodium along the periphery of the cell. Interestingly, immobilizing microtubules through crosslinking with actin or other means allow the buildup of pressure, enabling them to breach these dense actin barriers. This mechanism offers insights into microtubule progression towards the cell periphery, with them overcoming obstacles within the denser parts of the actin network and ultimately contributing to cell polarity establishment.
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Affiliation(s)
- Matthieu Gélin
- Université Paris cité, CEA, INSERM, Institut de Recherche Saint Louis, UMR976 HIPI, CytoMorpho Lab, Avenue Claude Vellefaux, 75010 Paris, France
| | - Alexandre Schaeffer
- Université Paris cité, CEA, INSERM, Institut de Recherche Saint Louis, UMR976 HIPI, CytoMorpho Lab, Avenue Claude Vellefaux, 75010 Paris, France
| | - Jérémie Gaillard
- Université Grenoble-Alpes, CEA, CNRS, INRA, Interdisciplinary Research Institute of Grenoble, UMR5168-LPCV, CytoMorpho Lab, Avenue des Martyrs, 38054 Grenoble, France
| | - Christophe Guérin
- Université Grenoble-Alpes, CEA, CNRS, INRA, Interdisciplinary Research Institute of Grenoble, UMR5168-LPCV, CytoMorpho Lab, Avenue des Martyrs, 38054 Grenoble, France
| | - Benoit Vianay
- Université Paris cité, CEA, INSERM, Institut de Recherche Saint Louis, UMR976 HIPI, CytoMorpho Lab, Avenue Claude Vellefaux, 75010 Paris, France
| | - Magali Orhant-Prioux
- Université Grenoble-Alpes, CEA, CNRS, INRA, Interdisciplinary Research Institute of Grenoble, UMR5168-LPCV, CytoMorpho Lab, Avenue des Martyrs, 38054 Grenoble, France
| | - Marcus Braun
- Institute of Biotechnology, Czech Academy of Sciences, BIOCEV, 25250 Vestec, Prague West, Czech Republic
| | - Christophe Leterrier
- Aix Marseille Université, CNRS, INP UMR7051, NeuroCyto, 13385, Marseille, France
| | - Laurent Blanchoin
- Université Paris cité, CEA, INSERM, Institut de Recherche Saint Louis, UMR976 HIPI, CytoMorpho Lab, Avenue Claude Vellefaux, 75010 Paris, France
- Université Grenoble-Alpes, CEA, CNRS, INRA, Interdisciplinary Research Institute of Grenoble, UMR5168-LPCV, CytoMorpho Lab, Avenue des Martyrs, 38054 Grenoble, France
| | - Manuel Théry
- Université Paris cité, CEA, INSERM, Institut de Recherche Saint Louis, UMR976 HIPI, CytoMorpho Lab, Avenue Claude Vellefaux, 75010 Paris, France
- Université Grenoble-Alpes, CEA, CNRS, INRA, Interdisciplinary Research Institute of Grenoble, UMR5168-LPCV, CytoMorpho Lab, Avenue des Martyrs, 38054 Grenoble, France
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5
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Sarantelli E, Mourkakis A, Zacharia LC, Stylianou A, Gkretsi V. Fascin-1 in Cancer Cell Metastasis: Old Target-New Insights. Int J Mol Sci 2023; 24:11253. [PMID: 37511011 PMCID: PMC10379093 DOI: 10.3390/ijms241411253] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2023] [Revised: 06/29/2023] [Accepted: 07/07/2023] [Indexed: 07/30/2023] Open
Abstract
As metastasis is responsible for most cancer-related deaths, understanding the cellular and molecular events that lead to cancer cell migration and invasion will certainly provide insights into novel anti-metastatic therapeutic targets. Fascin-1 is an actin-bundling protein fundamental to all physiological or pathological processes that require cell migration. It is responsible for cross-linking actin microfilaments during the formation of actin-rich cellular structures at the leading edge of migrating cells such as filopodia, lamellipodia and invadopodia. While most epithelial tissues express low levels of Fascin-1, it is dramatically elevated in the majority of cancers and its expression has been associated with more aggressive disease and decreased overall survival. Hence, it has been proposed as a potential anti-cancer target. In the present review, we studied recent literature with regard to Fascin-1 expression in different cancers, its role in altering the mechanical properties of cancer cells, promoting cancer cell migration, invasion and metastasis and the effect of its inhibition, via various pharmacological inhibitors, in eliminating metastasis in vitro and/or in vivo. Recent studies corroborate the notion that Fascin-1 is critically involved in metastasis and prove that it is a valuable anti-metastatic target that is worth investigating further.
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Affiliation(s)
- Eleonora Sarantelli
- Biological Sciences Program, Department of Life Sciences, School of Sciences, European University Cyprus, Nicosia 2404, Cyprus
| | - Apostolis Mourkakis
- Cancer Metastasis and Adhesion Laboratory, Basic and Translational Cancer Research Center (BTCRC), European University Cyprus, Nicosia 2404, Cyprus
| | - Lefteris C Zacharia
- Department of Health Sciences, School of Life and Health Sciences, University of Nicosia, Nicosia 2417, Cyprus
| | - Andreas Stylianou
- Cancer Mechanobiology and Applied Biophysics Laboratory, Basic and Translational Cancer Research Center (BTCRC), European University Cyprus, Nicosia 2404, Cyprus
| | - Vasiliki Gkretsi
- Cancer Metastasis and Adhesion Laboratory, Basic and Translational Cancer Research Center (BTCRC), European University Cyprus, Nicosia 2404, Cyprus
- Biomedical Sciences Program , Department of Life Sciences, School of Sciences, European University Cyprus, Nicosia 2404, Cyprus
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6
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Yeon M, Bertolini I, Agarwal E, Ghosh JC, Tang HY, Speicher DW, Keeney F, Sossey-Alaoui K, Pluskota E, Bialkowska K, Plow EF, Languino LR, Skordalakes E, Caino MC, Altieri DC. Parkin ubiquitination of Kindlin-2 enables mitochondria-associated metastasis suppression. J Biol Chem 2023; 299:104774. [PMID: 37142218 PMCID: PMC10236456 DOI: 10.1016/j.jbc.2023.104774] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2023] [Revised: 04/17/2023] [Accepted: 04/24/2023] [Indexed: 05/06/2023] Open
Abstract
Mitochondria are signaling organelles implicated in cancer, but the mechanisms are elusive. Here, we show that Parkin, an E3 ubiquitination (Ub) ligase altered in Parkinson's disease, forms a complex with the regulator of cell motility, Kindlin-2 (K2), at mitochondria of tumor cells. In turn, Parkin ubiquitinates Lys581 and Lys582 using Lys48 linkages, resulting in proteasomal degradation of K2 and shortened half-life from ∼5 h to ∼1.5 h. Loss of K2 inhibits focal adhesion turnover and β1 integrin activation, impairs membrane lamellipodia size and frequency, and inhibits mitochondrial dynamics, altogether suppressing tumor cell-extracellular matrix interactions, migration, and invasion. Conversely, Parkin does not affect tumor cell proliferation, cell cycle transitions, or apoptosis. Expression of a Parkin Ub-resistant K2 Lys581Ala/Lys582Ala double mutant is sufficient to restore membrane lamellipodia dynamics, correct mitochondrial fusion/fission, and preserve single-cell migration and invasion. In a 3D model of mammary gland developmental morphogenesis, impaired K2 Ub drives multiple oncogenic traits of EMT, increased cell proliferation, reduced apoptosis, and disrupted basal-apical polarity. Therefore, deregulated K2 is a potent oncogene, and its Ub by Parkin enables mitochondria-associated metastasis suppression.
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Affiliation(s)
- Minjeong Yeon
- Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA
| | - Irene Bertolini
- Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA
| | - Ekta Agarwal
- Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA
| | - Jagadish C Ghosh
- Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA
| | - Hsin-Yao Tang
- Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA; Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA
| | - David W Speicher
- Proteomics and Metabolomics Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA; Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA
| | - Frederick Keeney
- Imaging Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania, USA
| | - Khalid Sossey-Alaoui
- Department of Medicine, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio, USA
| | - Elzbieta Pluskota
- Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA
| | - Katarzyna Bialkowska
- Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA
| | - Edward F Plow
- Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA
| | - Lucia R Languino
- Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
| | - Emmanuel Skordalakes
- Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, Pennsylvania, USA
| | - M Cecilia Caino
- Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado, USA
| | - Dario C Altieri
- Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.
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7
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Zhang Z, Lv Y, Harati J, Song J, Du P, Ou P, Liang J, Wang H, Wang PY. Submicron-Grooved Films Modulate the Directional Alignment and Biological Function of Schwann Cells. J Funct Biomater 2023; 14:jfb14050238. [PMID: 37233348 DOI: 10.3390/jfb14050238] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2023] [Revised: 04/12/2023] [Accepted: 04/21/2023] [Indexed: 05/27/2023] Open
Abstract
Topographical cues on material surfaces are crucial for guiding the behavior of nerve cells and facilitating the repair of peripheral nerve defects. Previously, micron-grooved surfaces have shown great potential in controlling nerve cell alignment for studying the behavior and functions of those cells and peripheral nerve regeneration. However, the effects of smaller-sized topographical cues, such as those in the submicron- and nano-scales, on Schwann cell behavior remain poorly understood. In this study, four different submicron-grooved polystyrene films (800/400, 800/100, 400/400, and 400/100) were fabricated to study the behavior, gene expression, and membrane potential of Schwann cells. The results showed that all submicron-grooved films could guide the cell alignment and cytoskeleton in a groove depth-dependent manner. Cell proliferation and cell cycle assays revealed that there was no significant difference between the submicron groove samples and the flat control. However, the submicron grooves can direct the migration of cells and upregulate the expression of critical genes in axon regeneration and myelination (e.g., MBP and Smad6). Finally, the membrane potential of the Schwann cells was significantly altered on the grooved sample. In conclusion, this study sheds light on the role of submicron-grooved patterns in regulating the behavior and function of Schwann cells, which provides unique insights for the development of implants for peripheral nerve regeneration.
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Affiliation(s)
- Zhen Zhang
- Center for Human Tissues and Organs Degeneration, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yuanliang Lv
- Center for Human Tissues and Organs Degeneration, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
| | - Javad Harati
- Center for Human Tissues and Organs Degeneration, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jianan Song
- Oujiang Laboratory, Key Laboratory of Alzheimer's Disease of Zhejiang Province, Institute of Aging, Wenzhou Medical University, Wenzhou 325000, China
| | - Ping Du
- Center for Human Tissues and Organs Degeneration, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
| | - Peiyan Ou
- Center for Human Tissues and Organs Degeneration, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jiaqi Liang
- Center for Human Tissues and Organs Degeneration, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
| | - Huaiyu Wang
- Center for Human Tissues and Organs Degeneration, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Peng-Yuan Wang
- Oujiang Laboratory, Key Laboratory of Alzheimer's Disease of Zhejiang Province, Institute of Aging, Wenzhou Medical University, Wenzhou 325000, China
- Shenzhen Key Laboratory of Biomimetic Materials and Cellular Immunomodulation, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
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8
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S100A8/S100A9 Integrates F-Actin and Microtubule Dynamics to Prevent Uncontrolled Extravasation of Leukocytes. Biomedicines 2023; 11:biomedicines11030835. [PMID: 36979814 PMCID: PMC10045313 DOI: 10.3390/biomedicines11030835] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2023] [Revised: 03/03/2023] [Accepted: 03/07/2023] [Indexed: 03/12/2023] Open
Abstract
Immune reactions are characterized by the rapid immigration of phagocytes into sites of inflammation. Meticulous regulation of these migratory processes is crucial for preventing uncontrolled and harmful phagocyte extravasation. S100A8/S100A9 is the major calcium-binding protein complex expressed in phagocytes. After release, this complex acts as a proinflammatory alarmin in the extracellular space, but the intracellular functions of these highly abundant proteins are less clear. Results of this study reveal an important role of S100A8/S100A9 in coordinated cytoskeleton rearrangement during migration. We found that S100A8/S100A9 was able to cross-link F-actin and microtubules in a calcium- and phosphorylation-dependent manner. Cells deficient in S100A8/S100A9 showed abnormalities in cell adhesion and motility. Missing cytoskeletal interactions of S100A8/S100A9 caused differences in the surface expression and activation of β1-integrins as well as in the regulation of Src/Syk kinase family members. Loss of S100A8/S100A9 led to dysregulated integrin-mediated adhesion and migration, resulting in an overall higher dynamic activity of non-activated S100A8/S100A9-deficient phagocytes. Our data suggest that intracellular S100A8/S100A9 is part of a novel regulatory mechanism that ensures the precise control necessary to facilitate the change between the quiescent and activated state of phagocytes.
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9
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Rodgers NC, Lawrence EJ, Sawant AV, Efimova N, Gonzalez-Vasquez G, Hickman TT, Kaverina I, Zanic M. CLASP2 facilitates dynamic actin filament organization along the microtubule lattice. Mol Biol Cell 2023; 34:br3. [PMID: 36598814 PMCID: PMC10011731 DOI: 10.1091/mbc.e22-05-0149] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2022] [Revised: 12/14/2022] [Accepted: 12/21/2022] [Indexed: 01/05/2023] Open
Abstract
Coordination between the microtubule and actin networks is essential for cell motility, neuronal growth cone guidance, and wound healing. Members of the CLASP (cytoplasmic linker-associated protein) family of proteins have been implicated in the cytoskeletal cross-talk between microtubules and actin networks; however, the molecular mechanisms underlying the role of CLASP in cytoskeletal coordination are unclear. Here, we investigate CLASP2α's cross-linking function with microtubules and F-actin. Our results demonstrate that CLASP2α cross-links F-actin to the microtubule lattice in vitro. We find that the cross-linking ability is retained by L-TOG2-S, a minimal construct containing the TOG2 domain and serine-arginine-rich region of CLASP2α. Furthermore, CLASP2α promotes the accumulation of multiple actin filaments along the microtubule, supporting up to 11 F-actin landing events on a single microtubule lattice region. CLASP2α also facilitates the dynamic organization of polymerizing actin filaments templated by the microtubule network, with F-actin forming bridges between individual microtubules. Finally, we find that depletion of CLASPs in vascular smooth muscle cells results in disorganized actin fibers and reduced coalignment of actin fibers with microtubules, suggesting that CLASP and microtubules contribute to higher-order actin structures. Taken together, our results indicate that CLASP2α can directly cross-link F-actin to microtubules and that this microtubule-CLASP-actin interaction may influence overall cytoskeletal organization in cells.
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Affiliation(s)
- N. C. Rodgers
- Chemical and Physical Biology Graduate Program, Vanderbilt University, Nashville, TN 37232
| | - E. J. Lawrence
- Department of Cell and Development Biology, Vanderbilt University, Nashville, TN 37232
| | - A. V. Sawant
- Department of Cell and Development Biology, Vanderbilt University, Nashville, TN 37232
| | - N. Efimova
- Department of Cell and Development Biology, Vanderbilt University, Nashville, TN 37232
| | - G. Gonzalez-Vasquez
- Interdisciplinary Graduate Program, Vanderbilt University, Nashville, TN 37232
| | - T. T. Hickman
- Quantitative and Chemical Biology Graduate Program, Vanderbilt University, Nashville, TN 37232
| | - I. Kaverina
- Department of Cell and Development Biology, Vanderbilt University, Nashville, TN 37232
| | - M. Zanic
- Department of Cell and Development Biology, Vanderbilt University, Nashville, TN 37232
- Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN 37232
- Department of Biochemistry, Vanderbilt University, Nashville, TN 37232
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10
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Leonov S, Inyang O, Achkasov K, Bogdan E, Kontareva E, Chen Y, Fu Y, Osipov AN, Pustovalova M, Merkher Y. Proteomic Markers for Mechanobiological Properties of Metastatic Cancer Cells. Int J Mol Sci 2023; 24:ijms24054773. [PMID: 36902201 PMCID: PMC10003476 DOI: 10.3390/ijms24054773] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2022] [Revised: 01/26/2023] [Accepted: 02/17/2023] [Indexed: 03/06/2023] Open
Abstract
The major cause (more than 90%) of all cancer-related deaths is metastasis, thus its prediction can critically affect the survival rate. Metastases are currently predicted by lymph-node status, tumor size, histopathology and genetic testing; however, all these are not infallible, and obtaining results may require weeks. The identification of new potential prognostic factors will be an important source of risk information for the practicing oncologist, potentially leading to enhanced patient care through the proactive optimization of treatment strategies. Recently, the new mechanobiology-related techniques, independent of genetics, based on the mechanical invasiveness of cancer cells (microfluidic, gel indentation assays, migration assays etc.), demonstrated a high success rate for the detection of tumor cell metastasis propensity. However, they are still far away from clinical implementation due to complexity. Hence, the exploration of novel markers related to the mechanobiological properties of tumor cells may have a direct impact on the prognosis of metastasis. Our concise review deepens our knowledge of the factors that regulate cancer cell mechanotype and invasion, and incites further studies to develop therapeutics that target multiple mechanisms of invasion for improved clinical benefit. It may open a new clinical dimension that will improve cancer prognosis and increase the effectiveness of tumor therapies.
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Affiliation(s)
- Sergey Leonov
- School of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, Russia
- Institute of Cell Biophysics, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia
| | - Olumide Inyang
- School of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, Russia
| | - Konstantin Achkasov
- School of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, Russia
| | - Elizaveta Bogdan
- School of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, Russia
| | - Elizaveta Kontareva
- School of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, Russia
| | - Yongheng Chen
- Department of Oncology, NHC Key Laboratory of Cancer Proteomics & State Local Joint Engineering Laboratory for Anticancer Drugs, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha 410008, China
| | - Ying Fu
- Department of Oncology, NHC Key Laboratory of Cancer Proteomics & State Local Joint Engineering Laboratory for Anticancer Drugs, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha 410008, China
| | - Andreyan N. Osipov
- State Research Center—Burnasyan Federal Medical Biophysical Center of Federal Medical-Biological Agency, 123098 Moscow, Russia
- N.N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, 119991 Moscow, Russia
- Correspondence:
| | - Margarita Pustovalova
- School of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, Russia
- State Research Center—Burnasyan Federal Medical Biophysical Center of Federal Medical-Biological Agency, 123098 Moscow, Russia
| | - Yulia Merkher
- School of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, Russia
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11
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Zamborlin A, Voliani V. Gold nanoparticles as antiangiogenic and antimetastatic agents. Drug Discov Today 2023; 28:103438. [PMID: 36375738 DOI: 10.1016/j.drudis.2022.103438] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Revised: 10/27/2022] [Accepted: 11/08/2022] [Indexed: 11/13/2022]
Abstract
Angiogenesis and metastasis are two interdependent cancer hallmarks, the latter of which is the key cause of treatment failure. Thus, establishing effective antiangiogenesis/antimetastasis agents is the final frontier in cancer research. Gold nanoparticles (GNPs) may provide disruptive advancements in this regard due to their intrinsic physical and physiological features. Here, we comprehensively discuss recent potential therapeutical strategies to treat angiogenesis and metastasis and present a critical review on the state-of-the-art in vitro and in vivo evaluations of the antiangiogenic/antimetastatic activity of GNPs. Finally, we provide perspectives on the contribution of GNPs to the advancement of cancer management.
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Affiliation(s)
- Agata Zamborlin
- Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro, 12 - 56127 Pisa, Italy; NEST-Scuola Normale Superiore, Piazza San Silvestro, 12 - 56127 Pisa, Italy
| | - Valerio Voliani
- Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro, 12 - 56127 Pisa, Italy; Department of Pharmacy, University of Genoa, Viale Cembrano, 4 - 16148 Genoa, Italy.
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12
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Weier AK, Homrich M, Ebbinghaus S, Juda P, Miková E, Hauschild R, Zhang L, Quast T, Mass E, Schlitzer A, Kolanus W, Burgdorf S, Gruß OJ, Hons M, Wieser S, Kiermaier E. Multiple centrosomes enhance migration and immune cell effector functions of mature dendritic cells. J Cell Biol 2022; 221:e202107134. [PMID: 36214847 PMCID: PMC9555069 DOI: 10.1083/jcb.202107134] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2021] [Revised: 03/01/2022] [Accepted: 09/12/2022] [Indexed: 12/13/2022] Open
Abstract
Centrosomes play a crucial role during immune cell interactions and initiation of the immune response. In proliferating cells, centrosome numbers are tightly controlled and generally limited to one in G1 and two prior to mitosis. Defects in regulating centrosome numbers have been associated with cell transformation and tumorigenesis. Here, we report the emergence of extra centrosomes in leukocytes during immune activation. Upon antigen encounter, dendritic cells pass through incomplete mitosis and arrest in the subsequent G1 phase leading to tetraploid cells with accumulated centrosomes. In addition, cell stimulation increases expression of polo-like kinase 2, resulting in diploid cells with two centrosomes in G1-arrested cells. During cell migration, centrosomes tightly cluster and act as functional microtubule-organizing centers allowing for increased persistent locomotion along gradients of chemotactic cues. Moreover, dendritic cells with extra centrosomes display enhanced secretion of inflammatory cytokines and optimized T cell responses. Together, these results demonstrate a previously unappreciated role of extra centrosomes for regular cell and tissue homeostasis.
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Affiliation(s)
- Ann-Kathrin Weier
- Life and Medical Sciences Institute, Immune and Tumor Biology, University of Bonn, Bonn, Germany
| | - Mirka Homrich
- Life and Medical Sciences Institute, Immune and Tumor Biology, University of Bonn, Bonn, Germany
| | - Stephanie Ebbinghaus
- Life and Medical Sciences Institute, Immune and Tumor Biology, University of Bonn, Bonn, Germany
| | - Pavel Juda
- BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic
| | - Eliška Miková
- BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic
| | - Robert Hauschild
- Institute of Science and Technology Austria, Klosterneuburg, Austria
| | - Lili Zhang
- Life and Medical Sciences Institute, Quantitative Systems Biology, University of Bonn, Bonn, Germany
| | - Thomas Quast
- Life and Medical Sciences Institute, Molecular Immunology and Cell Biology, University of Bonn, Bonn, Germany
| | - Elvira Mass
- Life and Medical Sciences Institute, Developmental Biology of the Immune System, University of Bonn, Bonn, Germany
| | - Andreas Schlitzer
- Life and Medical Sciences Institute, Quantitative Systems Biology, University of Bonn, Bonn, Germany
| | - Waldemar Kolanus
- Life and Medical Sciences Institute, Molecular Immunology and Cell Biology, University of Bonn, Bonn, Germany
| | - Sven Burgdorf
- Life and Medical Sciences Institute, Cellular Immunology, University of Bonn, Bonn, Germany
| | - Oliver J. Gruß
- Institute of Genetics, University of Bonn, Bonn, Germany
| | - Miroslav Hons
- BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic
| | - Stefan Wieser
- Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, Castelldefels, Spain
| | - Eva Kiermaier
- Life and Medical Sciences Institute, Immune and Tumor Biology, University of Bonn, Bonn, Germany
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13
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Mossakowska BJ, Fabisiewicz A, Tudek B, Siedlecki JA. Possible Mechanisms of Resistance Development to Photodynamic Therapy (PDT) In Vulvar Cancer Cells. Int J Mol Sci 2022; 23:ijms232314689. [PMID: 36499013 PMCID: PMC9741432 DOI: 10.3390/ijms232314689] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2022] [Revised: 11/14/2022] [Accepted: 11/16/2022] [Indexed: 11/27/2022] Open
Abstract
Photodynamic therapy (PDT) is a low-invasive treatment method that can be used to treat VIN patients. A photosensitizer (PS) applied to a patient is activated with use of the appropriate wavelength of light, which in an oxygen environment leads to the formation of a reactive oxygen species (ROS) that destroys the tumor. However, cells can protect themselves against these cytotoxic products by increasing their antioxidant mechanisms and repair capacity. Changes in the cytoskeleton may also influence resistance to PDT. Our results revealed that PDT-resistant cells changed the amount of ROS. Cells resistant to PDT A-431 exhibited a decreased ROS level and showed higher viability after oxidizing agent treatment. Resistant Cal-39 cells exhibited a decreased O2- level but increased other ROS. This provides protection from PDT but not from other oxidizing agents. Moreover, PDT leads to alterations in the cytoskeleton that may result in an epithelial-mesenchymal transition (EMT) or increased adhesion. Both EMT and cell adhesion may activate signaling pathways involved in survival. This means that resistance to PDT in vulvar cancer may be at least in part a result of changes in ROS level and alterations in the cytoskeleton.
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Affiliation(s)
- Beata Joanna Mossakowska
- Department of Molecular and Translational Oncology, Maria Skłodowska-Curie National Research Institute of Oncology, 02-781 Warsaw, Poland
- Correspondence:
| | - Anna Fabisiewicz
- Department of Molecular and Translational Oncology, Maria Skłodowska-Curie National Research Institute of Oncology, 02-781 Warsaw, Poland
| | - Barbara Tudek
- Faculty of Biology, Institute of Genetics and Biotechnology, University of Warsaw, 02-106 Warsaw, Poland
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland
| | - Janusz Aleksander Siedlecki
- Department of Molecular and Translational Oncology, Maria Skłodowska-Curie National Research Institute of Oncology, 02-781 Warsaw, Poland
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14
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Mechanical guidance of self-condensation patterns of differentiating progeny. iScience 2022; 25:105109. [DOI: 10.1016/j.isci.2022.105109] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2022] [Revised: 07/25/2022] [Accepted: 09/08/2022] [Indexed: 11/21/2022] Open
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15
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Sala F, Ficorella C, Osellame R, Käs JA, Martínez Vázquez R. Microfluidic Lab-on-a-Chip for Studies of Cell Migration under Spatial Confinement. BIOSENSORS 2022; 12:bios12080604. [PMID: 36004998 PMCID: PMC9405557 DOI: 10.3390/bios12080604] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/20/2022] [Revised: 07/27/2022] [Accepted: 08/01/2022] [Indexed: 11/16/2022]
Abstract
Understanding cell migration is a key step in unraveling many physiological phenomena and predicting several pathologies, such as cancer metastasis. In particular, confinement has been proven to be a key factor in the cellular migration strategy choice. As our insight in the field improves, new tools are needed in order to empower biologists’ analysis capabilities. In this framework, microfluidic devices have been used to engineer the mechanical and spatial stimuli and to investigate cellular migration response in a more controlled way. In this work, we will review the existing technologies employed in the realization of microfluidic cellular migration assays, namely the soft lithography of PDMS and hydrogels and femtosecond laser micromachining. We will give an overview of the state of the art of these devices, focusing on the different geometrical configurations that have been exploited to study specific aspects of cellular migration. Our scope is to highlight the advantages and possibilities given by each approach and to envisage the future developments in in vitro migration studies under spatial confinement in microfluidic devices.
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Affiliation(s)
- Federico Sala
- Institute for Photonics and Nanotechnologies, CNR, Piazza Leonardo da Vinci 32, 20133 Milano, Italy
| | - Carlotta Ficorella
- Peter Debye Institute for Soft Matter Physics, University of Leipzig, 04109 Leipzig, Germany
| | - Roberto Osellame
- Institute for Photonics and Nanotechnologies, CNR, Piazza Leonardo da Vinci 32, 20133 Milano, Italy
| | - Josef A. Käs
- Peter Debye Institute for Soft Matter Physics, University of Leipzig, 04109 Leipzig, Germany
| | - Rebeca Martínez Vázquez
- Institute for Photonics and Nanotechnologies, CNR, Piazza Leonardo da Vinci 32, 20133 Milano, Italy
- Correspondence:
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16
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Mentor S, Makhathini KB, Fisher D. The Role of Cytoskeletal Proteins in the Formation of a Functional In Vitro Blood-Brain Barrier Model. Int J Mol Sci 2022; 23:ijms23020742. [PMID: 35054928 PMCID: PMC8775705 DOI: 10.3390/ijms23020742] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2021] [Revised: 12/27/2021] [Accepted: 01/07/2022] [Indexed: 02/05/2023] Open
Abstract
The brain capillary endothelium is highly regulatory, maintaining the chemical stability of the brain’s microenvironment. The role of cytoskeletal proteins in tethering nanotubules (TENTs) during barrier-genesis was investigated using the established immortalized mouse brain endothelial cell line (bEnd5) as an in vitro blood-brain barrier (BBB) model. The morphology of bEnd5 cells was evaluated using both high-resolution scanning electron microscopy and immunofluorescence to evaluate treatment with depolymerizing agents Cytochalasin D for F-actin filaments and Nocodazole for α-tubulin microtubules. The effects of the depolymerizing agents were investigated on bEnd5 monolayer permeability by measuring the transendothelial electrical resistance (TEER). The data endorsed that during barrier-genesis, F-actin and α-tubulin play a cytoarchitectural role in providing both cell shape dynamics and cytoskeletal structure to TENTs forming across the paracellular space to provide cell-cell engagement. Western blot analysis of the treatments suggested a reduced expression of both proteins, coinciding with a reduction in the rates of cellular proliferation and decreased TEER. The findings endorsed that TENTs provide alignment of the paracellular (PC) spaces and tight junction (TJ) zones to occlude bEnd5 PC spaces. The identification of specific cytoskeletal structures in TENTs endorsed the postulate of their indispensable role in barrier-genesis and the maintenance of regulatory permeability across the BBB.
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Affiliation(s)
- Shireen Mentor
- Neurobiology Research Group, Department of Medical Biosciences, University of the Western Cape, Cape Town 7535, South Africa; (S.M.); (K.B.M.)
| | - Khayelihle Brian Makhathini
- Neurobiology Research Group, Department of Medical Biosciences, University of the Western Cape, Cape Town 7535, South Africa; (S.M.); (K.B.M.)
| | - David Fisher
- Neurobiology Research Group, Department of Medical Biosciences, University of the Western Cape, Cape Town 7535, South Africa; (S.M.); (K.B.M.)
- School of Health Professions, University of Missouri, Columbia, MO 65211, USA
- Correspondence: ; Tel.: +27-21-959-2185
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17
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Liu H, Liu S, Xiao Y, Song W, Li H, Ho LWC, Shen Z, Choi CHJ. A pH-Reversible Fluorescent Probe for in Situ Imaging of Extracellular Vesicles and Their Secretion from Living Cells. NANO LETTERS 2021; 21:9224-9232. [PMID: 34724785 DOI: 10.1021/acs.nanolett.1c03110] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Our knowledge in how extracellular vesicles (EVs) are secreted from cells remains inadequate due to the limited technologies available for visualizing them in situ. We report a pH-reversible boron dipyrromethene (BODIPY) fluorescent probe for confocal imaging of EVs secreted from living cells without inducing severe cytotoxicity. This probe predominantly assumes a non-fluorescent leuco-BODIPY form under basic conditions, but it gradually switches to its fluorescent parent BODIPY form upon acidification; such pH transition empowers the imaging of acidic EVs (such as CD81-enriched exosomes and extracellular multivesicular bodies) in weakly basic culture medium and intracellular acidic precursor EVs in weakly basic cytoplasm, with minimal false positive signals frequently encountered for "always-on" dyes. Joint application of this probe with plasmid transfection reveals the secretion of some EVs from cellular pseudopodia via microtubule trackways. This probe may provide mechanistic insights into the extracellular transport of EVs and support the development of EV-based nanomedicines.
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Affiliation(s)
- Hanzhuang Liu
- Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR
| | - Shaorui Liu
- Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR
| | - Yu Xiao
- Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR
| | - Wenting Song
- State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
| | - Huize Li
- Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR
| | - Lok Wai Cola Ho
- Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR
| | - Zhen Shen
- State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
| | - Chung Hang Jonathan Choi
- Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR
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18
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Lucas ED, Schafer JB, Matsuda J, Kraus M, Burchill MA, Tamburini BAJ. PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity. Cell Rep 2021; 33:108258. [PMID: 33053342 PMCID: PMC7688291 DOI: 10.1016/j.celrep.2020.108258] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2020] [Revised: 08/06/2020] [Accepted: 09/21/2020] [Indexed: 12/26/2022] Open
Abstract
Although the function of the extracellular region of programmed death ligand 1 (PD-L1) through its interactions with PD-1 on T cells is well studied, little is understood regarding the intracellular domain of PD-L1. Here, we outline a major role for PD-L1 intracellular signaling in the control of dendritic cell (DC) migration from the skin to the draining lymph node (dLN). Using a mutant mouse model, we identify a TSS signaling motif within the intracellular domain of PD-L1. The TSS motif proves critical for chemokine-mediated DC migration to the dLN during inflammation. This loss of DC migration, in the PD-L1 TSS mutant, leads to a significant decline in T cell priming when DC trafficking is required for antigen delivery to the dLN. Finally, the TSS motif is required for chemokine receptor signaling downstream of the Gα subunit of the heterotrimeric G protein complex, ERK phosphorylation, and actin polymerization in DCs. Lucas et al. define three residues within the cytoplasmic tail of PD-L1 that are required for proper dendritic cell migration from the skin to the lymph node. These three-amino-acid residues promote chemokine signaling in dendritic cells and productive T cell responses to skin infections.
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Affiliation(s)
- Erin D Lucas
- Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus School of Medicine, Aurora, CO, USA
| | - Johnathon B Schafer
- Department of Medicine, Division of Gastroenterology and Hepatology, University of Colorado Anschutz Medical Campus School of Medicine, Aurora, CO, USA; Molecular Biology Program, University of Colorado Anschutz Medical Campus School of Medicine, Aurora, CO, USA
| | | | - Madison Kraus
- Gates Summer Research Program, University of Colorado Anschutz Medical Campus School of Medicine, Aurora, CO, USA
| | - Matthew A Burchill
- Department of Medicine, Division of Gastroenterology and Hepatology, University of Colorado Anschutz Medical Campus School of Medicine, Aurora, CO, USA
| | - Beth A Jirón Tamburini
- Department of Medicine, Division of Gastroenterology and Hepatology, University of Colorado Anschutz Medical Campus School of Medicine, Aurora, CO, USA; Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus School of Medicine, Aurora, CO, USA; Molecular Biology Program, University of Colorado Anschutz Medical Campus School of Medicine, Aurora, CO, USA.
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19
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Gilpin TE, Walter FR, Herbath M, Sandor M, Fabry Z. Mycobacterium bovis Bacillus Calmette-Guérin-Infected Dendritic Cells Induce TNF-α-Dependent Cell Cluster Formation That Promotes Bacterial Dissemination through an In Vitro Model of the Blood-Brain Barrier. THE JOURNAL OF IMMUNOLOGY 2021; 207:1065-1077. [PMID: 34321229 DOI: 10.4049/jimmunol.2001094] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/24/2020] [Accepted: 06/11/2021] [Indexed: 11/19/2022]
Abstract
CNS tuberculosis (CNSTB) is the most severe manifestation of extrapulmonary tuberculosis infection, but the mechanism of how mycobacteria cross the blood-brain barrier (BBB) is not well understood. In this study, we report a novel murine in vitro BBB model combining primary brain endothelial cells, Mycobacterium bovis bacillus Calmette-Guérin-infected dendritic cells (DCs), PBMCs, and bacterial Ag-specific CD4+ T cells. We show that mycobacterial infection limits DC mobility and also induces cellular cluster formation that has a similar composition to pulmonary mycobacterial granulomas. Within the clusters, infection from DCs disseminates to the recruited monocytes, promoting bacterial expansion. Mycobacterium-induced in vitro granulomas have been described previously, but this report shows that they can form on brain endothelial cell monolayers. Cellular cluster formation leads to cluster-associated damage of the endothelial cell monolayer defined by mitochondrial stress, disorganization of the tight junction proteins ZO-1 and claudin-5, upregulation of the adhesion molecules VCAM-1 and ICAM-1, and increased transmigration of bacteria-infected cells across the BBB. TNF-α inhibition reduces cluster formation on brain endothelial cells and mitigates cluster-associated damage. These data describe a model of bacterial dissemination across the BBB shedding light on a mechanism that might contribute to CNS tuberculosis infection and facilitate treatments.
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Affiliation(s)
- Trey E Gilpin
- Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI; and.,Graduate Training Program of Cellular and Molecular Pathology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI
| | - Fruzsina R Walter
- Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI; and
| | - Melinda Herbath
- Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI; and
| | - Matyas Sandor
- Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI; and
| | - Zsuzsanna Fabry
- Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI; and .,Graduate Training Program of Cellular and Molecular Pathology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI
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20
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Roncato F, Regev O, Yadav SK, Alon R. Microtubule destabilization is a critical checkpoint of chemotaxis and transendothelial migration in melanoma cells but not in T cells. Cell Adh Migr 2021; 15:166-179. [PMID: 34152257 PMCID: PMC8218694 DOI: 10.1080/19336918.2021.1934958] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Microtubules (MTs) control cell shape and intracellular cargo transport. The role of MT turnover in the migration of slow-moving cells through endothelial barriers remains unclear. To irreversibly interfere with MT disassembly, we have used the MT-stabilizing agent zampanolide (ZMP) in Β16F10 melanoma as amodel of slow-moving cells. ZMP-treated B16 cells failed to follow chemotactic gradients across rigid confinements and could not generate stable sub-endothelial pseudopodia under endothelial monolayers. In vivo, ZMP-treated Β16 cells failed to extravasate though lung capillaries. In contrast to melanoma cells, the chemotaxis and transendothelial migration of ZMP-treated Tcells were largely conserved. This is afirst demonstration that MT disassembly is akey checkpoint in the directional migration of cancer cells but not of lymphocytes.
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Affiliation(s)
- Francesco Roncato
- Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
| | - Ofer Regev
- Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
| | | | - Ronen Alon
- Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
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21
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Naffa R, Padányi R, Ignácz A, Hegyi Z, Jezsó B, Tóth S, Varga K, Homolya L, Hegedűs L, Schlett K, Enyedi A. The Plasma Membrane Ca 2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton. Cancers (Basel) 2021; 13:cancers13061354. [PMID: 33802790 PMCID: PMC8002435 DOI: 10.3390/cancers13061354] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 03/08/2021] [Accepted: 03/14/2021] [Indexed: 02/07/2023] Open
Abstract
Simple Summary Earlier we demonstrated that the plasma membrane Ca2+ pump PMCA4b inhibits migration and metastatic activity of BRAF mutant melanoma cells, however, the exact mechanism has not been fully understood. Here we demonstrate that PMCA4b acted through actin cytoskeleton remodeling in generating a low migratory melanoma cell phenotype resulting in increased cell–cell connections, lamellipodia and stress fiber formation. Both proper trafficking and calcium transporting activity of the pump were essential to complete these tasks indicating that controlling Ca2+ concentration levels at specific plasma membrane locations such as the cell front played a role. Our findings suggest that PMCA4b downregulation is likely one of the mechanisms that leads to the perturbed cancer cell cytoskeleton organization resulting in enhanced melanoma cell migration and metastasis. Abstract We demonstrated that the plasma membrane Ca2+ ATPase PMCA4b inhibits migration and metastatic activity of BRAF mutant melanoma cells. Actin dynamics are essential for cells to move, invade and metastasize, therefore, we hypothesized that PMCA4b affected cell migration through remodeling of the actin cytoskeleton. We found that expression of PMCA4b in A375 BRAF mutant melanoma cells induced a profound change in cell shape, cell culture morphology, and displayed a polarized migratory character. Along with these changes the cells became more rounded with increased cell–cell connections, lamellipodia and stress fiber formation. Silencing PMCA4b in MCF-7 breast cancer cells had a similar effect, resulting in a dramatic loss of stress fibers. In addition, the PMCA4b expressing A375 cells maintained front-to-rear Ca2+ concentration gradient with the actin severing protein cofilin localizing to the lamellipodia, and preserved the integrity of the actin cytoskeleton from a destructive Ca2+ overload. We showed that both PMCA4b activity and trafficking were essential for the observed morphology and motility changes. In conclusion, our data suggest that PMCA4b plays a critical role in adopting front-to-rear polarity in a normally spindle-shaped cell type through F-actin rearrangement resulting in a less aggressive melanoma cell phenotype.
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Affiliation(s)
- Randa Naffa
- Department of Transfusiology, Semmelweis University, H-1089 Budapest, Hungary; (R.N.); (S.T.)
| | - Rita Padányi
- Department of Biophysics and Radiation Biology, Semmelweis University, H-1094 Budapest, Hungary;
| | - Attila Ignácz
- Department of Physiology and Neurobiology, Eötvös Loránd University, H-1117 Budapest, Hungary; (A.I.); (K.S.)
| | - Zoltán Hegyi
- Institute of Enzymology, Research Centre for Natural Sciences, Magyar Tudosok krt.2, H-1117 Budapest, Hungary; (Z.H.); (B.J.); (L.H.)
| | - Bálint Jezsó
- Institute of Enzymology, Research Centre for Natural Sciences, Magyar Tudosok krt.2, H-1117 Budapest, Hungary; (Z.H.); (B.J.); (L.H.)
| | - Sarolta Tóth
- Department of Transfusiology, Semmelweis University, H-1089 Budapest, Hungary; (R.N.); (S.T.)
- Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, H-1117 Budapest, Hungary
| | | | - László Homolya
- Institute of Enzymology, Research Centre for Natural Sciences, Magyar Tudosok krt.2, H-1117 Budapest, Hungary; (Z.H.); (B.J.); (L.H.)
| | - Luca Hegedűs
- Department of Thoracic Surgery, Ruhrlandklinik, University Clinic Essen, 45239 Essen, Germany;
| | - Katalin Schlett
- Department of Physiology and Neurobiology, Eötvös Loránd University, H-1117 Budapest, Hungary; (A.I.); (K.S.)
| | - Agnes Enyedi
- Department of Transfusiology, Semmelweis University, H-1089 Budapest, Hungary; (R.N.); (S.T.)
- Correspondence:
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22
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Abstract
In the final stages of apoptosis, apoptotic cells can generate a variety of membrane-bound vesicles known as apoptotic extracellular vesicles (ApoEVs). Apoptotic bodies (ApoBDs), a major subset of ApoEVs, are formed through a process termed apoptotic cell disassembly characterised by a series of tightly regulated morphological steps including plasma membrane blebbing, apoptotic membrane protrusion formation and fragmentation into ApoBDs. To better characterise the properties of ApoBDs and elucidate their function, a number of methods including differential centrifugation, filtration and fluorescence-activated cell sorting were developed to isolate ApoBDs. Furthermore, it has become increasingly clear that ApoBD formation can contribute to various biological processes such as apoptotic cell clearance and intercellular communication. Together, recent literature demonstrates that apoptotic cell disassembly and thus, ApoBD formation, is an important process downstream of apoptotic cell death. In this chapter, we discuss the current understandings of the molecular mechanisms involved in regulating apoptotic cell disassembly, techniques for ApoBD isolation, and the functional roles of ApoBDs in physiological and pathological settings.
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Ferraris S, Spriano S, Scalia AC, Cochis A, Rimondini L, Cruz-Maya I, Guarino V, Varesano A, Vineis C. Topographical and Biomechanical Guidance of Electrospun Fibers for Biomedical Applications. Polymers (Basel) 2020; 12:E2896. [PMID: 33287236 PMCID: PMC7761715 DOI: 10.3390/polym12122896] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2020] [Revised: 11/30/2020] [Accepted: 12/01/2020] [Indexed: 02/07/2023] Open
Abstract
Electrospinning is gaining increasing interest in the biomedical field as an eco-friendly and economic technique for production of random and oriented polymeric fibers. The aim of this review was to give an overview of electrospinning potentialities in the production of fibers for biomedical applications with a focus on the possibility to combine biomechanical and topographical stimuli. In fact, selection of the polymer and the eventual surface modification of the fibers allow selection of the proper chemical/biological signal to be administered to the cells. Moreover, a proper design of fiber orientation, dimension, and topography can give the opportunity to drive cell growth also from a spatial standpoint. At this purpose, the review contains a first introduction on potentialities of electrospinning for the obtainment of random and oriented fibers both with synthetic and natural polymers. The biological phenomena which can be guided and promoted by fibers composition and topography are in depth investigated and discussed in the second section of the paper. Finally, the recent strategies developed in the scientific community for the realization of electrospun fibers and for their surface modification for biomedical application are presented and discussed in the last section.
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Affiliation(s)
- Sara Ferraris
- Department of Applied Science and Technology, Politecnico di Torino, 10129 Torino, Italy;
| | - Silvia Spriano
- Department of Applied Science and Technology, Politecnico di Torino, 10129 Torino, Italy;
| | - Alessandro Calogero Scalia
- Department of Health Sciences, Center for Translational Research on Autoimmune and Allergic Diseases–CAAD, Università del Piemonte Orientale UPO, 28100 Novara, Italy; (A.C.S.); (A.C.); (L.R.)
| | - Andrea Cochis
- Department of Health Sciences, Center for Translational Research on Autoimmune and Allergic Diseases–CAAD, Università del Piemonte Orientale UPO, 28100 Novara, Italy; (A.C.S.); (A.C.); (L.R.)
| | - Lia Rimondini
- Department of Health Sciences, Center for Translational Research on Autoimmune and Allergic Diseases–CAAD, Università del Piemonte Orientale UPO, 28100 Novara, Italy; (A.C.S.); (A.C.); (L.R.)
| | - Iriczalli Cruz-Maya
- Institute for Polymers, Composites and Biomaterials (IPCB), National Research Council of Italy, Mostra d’Oltremare, Pad. 20, V. le J.F. Kennedy 54, 80125 Napoli, Italy; (I.C.-M.); (V.G.)
| | - Vincenzo Guarino
- Institute for Polymers, Composites and Biomaterials (IPCB), National Research Council of Italy, Mostra d’Oltremare, Pad. 20, V. le J.F. Kennedy 54, 80125 Napoli, Italy; (I.C.-M.); (V.G.)
| | - Alessio Varesano
- Institute of Intelligent Industrial Technologies and Systems for Advanced Manufacturing (STIIMA), National Research Council of Italy (CNR), Corso Giuseppe Pella 16, 13900 Biella, Italy; (A.V.); (C.V.)
| | - Claudia Vineis
- Institute of Intelligent Industrial Technologies and Systems for Advanced Manufacturing (STIIMA), National Research Council of Italy (CNR), Corso Giuseppe Pella 16, 13900 Biella, Italy; (A.V.); (C.V.)
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24
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Caruso S, Atkin-Smith GK, Baxter AA, Tixeira R, Jiang L, Ozkocak DC, Santavanond JP, Hulett MD, Lock P, Phan TK, Poon IKH. Defining the role of cytoskeletal components in the formation of apoptopodia and apoptotic bodies during apoptosis. Apoptosis 2020; 24:862-877. [PMID: 31489517 DOI: 10.1007/s10495-019-01565-5] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
During apoptosis, dying cells undergo dynamic morphological changes that ultimately lead to their disassembly into fragments called apoptotic bodies (ApoBDs). Reorganisation of the cytoskeletal structures is key in driving various apoptotic morphologies, including the loss of cell adhesion and membrane bleb formation. However, whether cytoskeletal components are also involved in morphological changes that occur later during apoptosis, such as the recently described generation of thin apoptotic membrane protrusions called apoptopodia and subsequent ApoBD formation, is not well defined. Through monitoring the progression of apoptosis by confocal microscopy, specifically focusing on the apoptopodia formation step, we characterised the presence of F-actin and microtubules in a subset of apoptopodia generated by T cells and monocytes. Interestingly, targeting actin polymerisation and microtubule assembly pharmacologically had no major effect on apoptopodia formation. These data demonstrate apoptopodia as a novel type of membrane protrusion that could be formed in the absence of actin polymerisation and microtubule assembly.
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Affiliation(s)
- Sarah Caruso
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Georgia K Atkin-Smith
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Amy A Baxter
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Rochelle Tixeira
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Lanzhou Jiang
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Dilara C Ozkocak
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Jascinta P Santavanond
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Mark D Hulett
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Peter Lock
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Thanh Kha Phan
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia
| | - Ivan K H Poon
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia.
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25
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Lopez-Guerrero AM, Espinosa-Bermejo N, Sanchez-Lopez I, Macartney T, Pascual-Caro C, Orantos-Aguilera Y, Rodriguez-Ruiz L, Perez-Oliva AB, Mulero V, Pozo-Guisado E, Martin-Romero FJ. RAC1-Dependent ORAI1 Translocation to the Leading Edge Supports Lamellipodia Formation and Directional Persistence. Sci Rep 2020; 10:6580. [PMID: 32313105 PMCID: PMC7171199 DOI: 10.1038/s41598-020-63353-5] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2019] [Accepted: 03/26/2020] [Indexed: 12/18/2022] Open
Abstract
Tumor invasion requires efficient cell migration, which is achieved by the generation of persistent and polarized lamellipodia. The generation of lamellipodia is supported by actin dynamics at the leading edge where a complex of proteins known as the WAVE regulatory complex (WRC) promotes the required assembly of actin filaments to push the front of the cell ahead. By using an U2OS osteosarcoma cell line with high metastatic potential, proven by a xenotransplant in zebrafish larvae, we have studied the role of the plasma membrane Ca2+ channel ORAI1 in this process. We have found that epidermal growth factor (EGF) triggered an enrichment of ORAI1 at the leading edge, where colocalized with cortactin (CTTN) and other members of the WRC, such as CYFIP1 and ARP2/3. ORAI1-CTTN co-precipitation was sensitive to the inhibition of the small GTPase RAC1, an upstream activator of the WRC. RAC1 potentiated ORAI1 translocation to the leading edge, increasing the availability of surface ORAI1 and increasing the plasma membrane ruffling. The role of ORAI1 at the leading edge was studied in genetically engineered U2OS cells lacking ORAI1 expression that helped us to prove the key role of this Ca2+ channel on lamellipodia formation, lamellipodial persistence, and cell directness, which are required for tumor cell invasiveness in vivo.
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Affiliation(s)
- Aida M Lopez-Guerrero
- Department of Biochemistry and Molecular Biology, School of Life Sciences and Institute of Molecular Pathology Biomarkers, University of Extremadura, Badajoz, 06006, Spain
| | - Noelia Espinosa-Bermejo
- Department of Biochemistry and Molecular Biology, School of Life Sciences and Institute of Molecular Pathology Biomarkers, University of Extremadura, Badajoz, 06006, Spain
| | - Irene Sanchez-Lopez
- Department of Biochemistry and Molecular Biology, School of Life Sciences and Institute of Molecular Pathology Biomarkers, University of Extremadura, Badajoz, 06006, Spain
| | - Thomas Macartney
- MRC- Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, United Kingdom
| | - Carlos Pascual-Caro
- Department of Biochemistry and Molecular Biology, School of Life Sciences and Institute of Molecular Pathology Biomarkers, University of Extremadura, Badajoz, 06006, Spain
| | - Yolanda Orantos-Aguilera
- Department of Biochemistry and Molecular Biology, School of Life Sciences and Institute of Molecular Pathology Biomarkers, University of Extremadura, Badajoz, 06006, Spain
| | - Lola Rodriguez-Ruiz
- Department of Cell Biology and Histology, University of Murcia, IMIB-Arrixaca, Murcia, 30100, Spain
| | - Ana B Perez-Oliva
- Department of Cell Biology and Histology, University of Murcia, IMIB-Arrixaca, Murcia, 30100, Spain
| | - Victoriano Mulero
- Department of Cell Biology and Histology, University of Murcia, IMIB-Arrixaca, Murcia, 30100, Spain
| | - Eulalia Pozo-Guisado
- Department of Cell Biology, School of Medicine and Institute of Molecular Pathology Biomarkers, University of Extremadura, Badajoz, 06006, Spain.
| | - Francisco Javier Martin-Romero
- Department of Biochemistry and Molecular Biology, School of Life Sciences and Institute of Molecular Pathology Biomarkers, University of Extremadura, Badajoz, 06006, Spain.
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Clemente N, Argenziano M, Gigliotti CL, Ferrara B, Boggio E, Chiocchetti A, Caldera F, Trotta F, Benetti E, Annaratone L, Ribero S, Pizzimenti S, Barrera G, Dianzani U, Cavalli R, Dianzani C. Paclitaxel-Loaded Nanosponges Inhibit Growth and Angiogenesis in Melanoma Cell Models. Front Pharmacol 2019; 10:776. [PMID: 31354491 PMCID: PMC6639435 DOI: 10.3389/fphar.2019.00776] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2019] [Accepted: 06/17/2019] [Indexed: 12/25/2022] Open
Abstract
This study investigated the effects of free paclitaxel (PTX) and PTX-loaded in pyromellitic nanosponges (PTX-PNS) in reducing in vitro and in vivo melanoma cell growth and invasivity, and in inhibiting angiogenesis. To test the response of cells to the two PTX formulations, the cell viability was evaluated by MTT assay in seven continuous cell lines, in primary melanoma cells, both in 2D and 3D cultures, and in human umbilical vein endothelial cells (HUVECs) after exposure to different concentrations of PTX or PTX-PNS. Cell motility was assessed by a scratch assay or Boyden chamber assay, evaluating cell migration in presence or absence of diverse concentrations of PTX or PTX-PNS. The effect of PTX and PTX-PNS on angiogenesis was evaluated as endothelial tube formation assay, a test able to estimate the formation of three-dimensional vessels in vitro. To assess the anticancer effect of PTX and PTX-PNS in in vivo experiments, the two drug formulations were tested in a melanoma mouse model obtained by B16-BL6 cell implantation in C57/BL6 mice. Results obtained were as follows: 1) MTT analysis revealed that cell proliferation was more affected by PTX-PNS than by PTX in all tested cell lines, in both 2D and 3D cultures; 2) the analysis of the cell migration showed that PTX-PNS acted at very lower concentrations than PTX; 3) tube formation assay showed that PTX-PNS were more effective in inhibiting tube formation than free PTX; and 4) in vivo experiments demonstrated that tumor weights, volumes, and growth were significantly reduced by PTX-PNS treatment with respect to PTX; the angiogenesis and the cell proliferation, detected in the tumor samples with CD31 and Ki-67 antibodies, respectively, indicated that, in the PTX-PNS-treated tumors, the tube formation was inhibited, and a low amount of proliferating cells was present. Taken together, our data demonstrated that our new PTX nanoformulation can respond to some important issues related to PTX treatment, lowering the anti-tumor effective doses and increasing the effectiveness in inhibiting melanoma growth in vivo.
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Affiliation(s)
- Nausicaa Clemente
- Department of Health Sciences and Interdisciplinary Research Center of Autoimmune Diseases (IRCAD), UPO, Novara, Italy
| | - Monica Argenziano
- Dipartimento di Scienza e Tecnologia del Farmaco, University of Torino, Torino, Italy
| | - Casimiro Luca Gigliotti
- Department of Health Sciences and Interdisciplinary Research Center of Autoimmune Diseases (IRCAD), UPO, Novara, Italy
| | - Benedetta Ferrara
- Dipartimento di Scienza e Tecnologia del Farmaco, University of Torino, Torino, Italy
| | - Elena Boggio
- Department of Health Sciences and Interdisciplinary Research Center of Autoimmune Diseases (IRCAD), UPO, Novara, Italy
| | - Annalisa Chiocchetti
- Department of Health Sciences and Interdisciplinary Research Center of Autoimmune Diseases (IRCAD), UPO, Novara, Italy
| | | | | | - Elisa Benetti
- Dipartimento di Scienza e Tecnologia del Farmaco, University of Torino, Torino, Italy
| | - Laura Annaratone
- Department of Medical Sciences, University of Torino, Torino, Italy
| | - Simone Ribero
- Department of Medical Sciences, University of Torino, Torino, Italy
| | - Stefania Pizzimenti
- Department of Clinical and Biological Sciences, University of Torino, Torino, Italy
| | - Giuseppina Barrera
- Department of Clinical and Biological Sciences, University of Torino, Torino, Italy
| | - Umberto Dianzani
- Department of Health Sciences and Interdisciplinary Research Center of Autoimmune Diseases (IRCAD), UPO, Novara, Italy
| | - Roberta Cavalli
- Dipartimento di Scienza e Tecnologia del Farmaco, University of Torino, Torino, Italy
| | - Chiara Dianzani
- Dipartimento di Scienza e Tecnologia del Farmaco, University of Torino, Torino, Italy
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27
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Frank D, Cser A, Kolarovszki B, Farkas N, Miseta A, Nagy T. Mechanical stress alters protein O-GlcNAc in human periodontal ligament cells. J Cell Mol Med 2019; 23:6251-6259. [PMID: 31237748 PMCID: PMC6714205 DOI: 10.1111/jcmm.14509] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2018] [Revised: 05/10/2019] [Accepted: 06/04/2019] [Indexed: 12/27/2022] Open
Abstract
Protein O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of intracellular proteins that regulates several physiological and pathophysiological process, including response to various stressors. However, O-GlcNAc's response to mechanical stress has not been investigated yet. As human periodontal ligament (PDL) cells are stimulated by compression force during orthodontic tooth movement that results in structural remodelling, in this study we investigated whether mechanical stress induces any alteration in protein O-GlcNAc in PDL cells. In this study, PDL cells isolated from premolars extracted for orthodontic indications were exposed to 0, 1.5, 3, 7 and 14 g/cm2 compression forces for 12 hours. Cell viability was measured by flow cytometry, and protein O-GlcNAc was analysed by Western blot. Cellular structure and intracellular distribution of O-GlcNAc was studied by immunofluorescence microscopy. We found that between 1.5 and 3 g/cm2 mechanical compression, O-GlcNAc significantly elevated; however, at higher forces O-GlcNAc level was not increased. We also found that intracellular localization of O-GlcNAc proteins became more centralized under 2 g/cm2 compression force. Our results suggest that structural changes stimulated by compression forces have a significant effect on the regulation of O-GlcNAc; thus, it might play a role in the mechanical stress adaptation of PDL cells.
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Affiliation(s)
- Dorottya Frank
- Department of Dentistry, Oral and Maxillofacial Surgery, Medical School, University of Pécs, Pécs, Hungary
| | - Annamária Cser
- Department of Dentistry, Oral and Maxillofacial Surgery, Medical School, University of Pécs, Pécs, Hungary
| | - Béla Kolarovszki
- Department of Dentistry, Oral and Maxillofacial Surgery, Medical School, University of Pécs, Pécs, Hungary
| | - Nelli Farkas
- Institute of Bioanalysis, Medical School, University of Pécs, Pécs, Hungary
| | - Attila Miseta
- Department of Laboratory Medicine, Medical School, University of Pécs, Pécs, Hungary
| | - Tamás Nagy
- Department of Laboratory Medicine, Medical School, University of Pécs, Pécs, Hungary
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28
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Jou JL, Liu SC, I L. Tail shape evolution dynamics of MDCK cells on fibronectin substrates. Biomed Phys Eng Express 2019. [DOI: 10.1088/2057-1976/ab1e11] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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29
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Kruger TM, Bell KJ, Lansakara TI, Tivanski AV, Doorn JA, Stevens LL. Reduced Extracellular Matrix Stiffness Prompts SH-SY5Y Cell Softening and Actin Turnover To Selectively Increase Aβ(1-42) Endocytosis. ACS Chem Neurosci 2019; 10:1284-1293. [PMID: 30499651 DOI: 10.1021/acschemneuro.8b00366] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Alzheimer's disease (AD), the most common neurodegenerative disorder, is characterized by the extracellular deposition of dense amyloid beta plaques. Emerging evidence suggests that the production of these plaques is initiated by the intracellular uptake and lysosomal preconcentration of the amyloid-beta (Aβ) peptide. All previous endocytosis studies assess Aβ uptake with cells plated on traditional tissue culture plastic; however, brain tissue is distinctly soft with a low-kPa stiffness. Use of an ultrastiff plastic/glass substrate prompts a mechanosensitive response (increased cell spreading, cell stiffness, and membrane tension) that potentially distorts a cell's endocytic behavior from that observed in vivo or in a more physiologically relevant mechanical environment. Our studies demonstrate substrate stiffness significantly modifies the behavior of undifferentiated SH-SY5Y neuroblastoma, where cells plated on soft (∼1 kPa) substrates display a rounded morphology, decreased actin polymerization, reduced adhesion (decreased β1 integrin expression), and reduced cell stiffness compared to cells plated on tissue culture plastic. Moreover, these neuroblastoma on softer substrates display a preferential increase in the uptake of the Aβ(1-42) compared to Aβ(1-40), while both isoforms display a clear stiffness-dependent increase of uptake relative to cells plated on plastic. Considering the brain is a soft tissue that continues to soften with age, this mechanosensitive endocytosis of Aβ has significant implications for understanding age-related neurodegeneration and the mechanism behind Aβ uptake and fibril production. Overall, identifying these physical factors that contribute to the pathology of AD may offer novel avenues of therapeutic intervention.
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Affiliation(s)
- Terra M. Kruger
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, The University of Iowa, Iowa City, Iowa 52242, United States
| | - Kendra J. Bell
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, The University of Iowa, Iowa City, Iowa 52242, United States
| | | | - Alexei V. Tivanski
- Department of Chemistry, The University of Iowa, Iowa City, Iowa 52242, United States
| | - Jonathan A. Doorn
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, The University of Iowa, Iowa City, Iowa 52242, United States
| | - Lewis L. Stevens
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, The University of Iowa, Iowa City, Iowa 52242, United States
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30
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Breuzard G, Pagano A, Bastonero S, Malesinski S, Parat F, Barbier P, Peyrot V, Kovacic H. Tau regulates the microtubule-dependent migration of glioblastoma cells via the Rho-ROCK signaling pathway. J Cell Sci 2019; 132:jcs.222851. [PMID: 30659115 DOI: 10.1242/jcs.222851] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2018] [Accepted: 01/08/2019] [Indexed: 12/24/2022] Open
Abstract
The pathological significance of Tau (encoded by MAPT) in mechanisms driving cell migration in glioblastoma is unclear. By using an shRNA approach to deplete microtubule-stabilizing Tau in U87 cells, we determined its impact on cytoskeletal coordination during migration. We demonstrated here that the motility of these Tau-knockdown cells (shTau cells) was significantly (36%) lower than that of control cells. The shTau cells displayed a slightly changed motility in the presence of nocodazole, which inhibits microtubule formation. Such reduced motility of shTau cells was characterized by a 28% lower number of microtubule bundles at the non-adhesive edges of the tails. In accordance with Tau-stabilized microtubules being required for cell movement, measurements of the front, body and rear section displacements of cells showed inefficient tail retraction in shTau cells. The tail retraction was restored by treatment with Y27632, an inhibitor of Rho-ROCK signaling. Moreover, we clearly identified that shTau cells displayed relocation of the active phosphorylated form of p190-RhoGAP (also known as ARHGAP35), which inhibits Rho-ROCK signaling, and focal adhesion kinase (FAK, also known as PTK2) in cell bodies. In conclusion, our findings indicate that Tau governs the remodeling of microtubule and actin networks for the retraction of the tail of cells, which is necessary for effective migration.
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Affiliation(s)
- Gilles Breuzard
- Aix-Marseille University, CNRS, Institute of Neurophysiopathology (INP), 13385 Marseille, France
| | - Alessandra Pagano
- Aix-Marseille University, CNRS, Institute of Neurophysiopathology (INP), 13385 Marseille, France
| | - Sonia Bastonero
- Aix-Marseille University, CNRS, Institute of Neurophysiopathology (INP), 13385 Marseille, France
| | - Soazig Malesinski
- Aix-Marseille University, CNRS, Institute of Neurophysiopathology (INP), 13385 Marseille, France
| | - Fabrice Parat
- Aix-Marseille University, CNRS, Institute of Neurophysiopathology (INP), 13385 Marseille, France
| | - Pascale Barbier
- Aix-Marseille University, CNRS, Institute of Neurophysiopathology (INP), 13385 Marseille, France
| | - Vincent Peyrot
- Aix-Marseille University, CNRS, Institute of Neurophysiopathology (INP), 13385 Marseille, France
| | - Hervé Kovacic
- Aix-Marseille University, CNRS, Institute of Neurophysiopathology (INP), 13385 Marseille, France
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31
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Kim JK, Shin YJ, Ha LJ, Kim DH, Kim DH. Unraveling the Mechanobiology of the Immune System. Adv Healthc Mater 2019; 8:e1801332. [PMID: 30614636 PMCID: PMC7700013 DOI: 10.1002/adhm.201801332] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2018] [Revised: 12/01/2018] [Indexed: 12/20/2022]
Abstract
Cells respond and actively adapt to environmental cues in the form of mechanical stimuli. This extends to immune cells and their critical role in the maintenance of tissue homeostasis. Multiple recent studies have begun illuminating underlying mechanisms of mechanosensation in modulating immune cell phenotypes. Since the extracellular microenvironment is critical to modify cellular physiology that ultimately determines the functionality of the cell, understanding the interactions between immune cells and their microenvironment is necessary. This review focuses on mechanoregulation of immune responses mediated by macrophages, dendritic cells, and T cells, in the context of modern mechanobiology.
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Affiliation(s)
- Jeong-Ki Kim
- KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul 02841, Republic of Korea
| | - Yu Jung Shin
- Department of Bioengineering, University of Washington, Seattle, WA 98109, USA
| | - Leslie Jaesun Ha
- KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul 02841, Republic of Korea
| | - Deok-Ho Kim
- Department of Bioengineering, University of Washington, Seattle, WA 98109, USA
| | - Dong-Hwee Kim
- KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul 02841, Republic of Korea
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32
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Wang X, Decker CC, Zechner L, Krstin S, Wink M. In vitro wound healing of tumor cells: inhibition of cell migration by selected cytotoxic alkaloids. BMC Pharmacol Toxicol 2019; 20:4. [PMID: 30626448 PMCID: PMC6327619 DOI: 10.1186/s40360-018-0284-4] [Citation(s) in RCA: 78] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2018] [Accepted: 12/27/2018] [Indexed: 12/30/2022] Open
Abstract
Background Cell migration is involved in several pathological processes such as tumor invasion, neoangiogenesis and metastasis. Microtubules are needed in directional migration. Methods To investigate the effects of microtubule-binding agents (paclitaxel, vinblastine, colchicine, podophyllotoxin), benzophenanthridine alkaloids (sanguinarine, chelerythrine, chelidonine) and other anti-tumor drugs (homoharringtonine, doxorubicin) on cell migration, we performed the in vitro wound healing assay. The interactions between selected alkaloids and microtubules were studied via U2OS cells expressing microtubule-GFP markers. Results The microtubule-binding natural products paclitaxel, vinblastine, colchicine and podophyllotoxin significantly altered microtubule dynamics in living cells and inhibited cell migration at concentrations below apparent cytotoxicity. The benzophenanthridine alkaloid sanguinarine, chelerythrine and chelidonine which affected microtubules in living cells, did not inhibit cell migration. Homoharringtonine (protein biosynthesis inhibitor) and doxorubicin significantly inhibited cell migration, however, they did not exert obvious effects on microtubules. Conclusion In this study, we demonstrated that microtubule-binding agents are effective anti-migrating agents; moreover, homoharringtonine and doxorubicin can be referred as anti-migrating agents, but direct microtubule dynamics are not involved in their mode of action. Our study provides evidence that some alkaloids and other microtubule-binding natural products may be interesting candidates for the development of novel agents against metastasis. Electronic supplementary material The online version of this article (10.1186/s40360-018-0284-4) contains supplementary material, which is available to authorized users.
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33
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Cao J, Schnittler H. Putting VE-cadherin into JAIL for junction remodeling. J Cell Sci 2019; 132:132/1/jcs222893. [DOI: 10.1242/jcs.222893] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
ABSTRACT
Junction dynamics of endothelial cells are based on the integration of signal transduction, cytoskeletal remodeling and contraction, which are necessary for the formation and maintenance of monolayer integrity, but also enable repair and regeneration. The VE-cadherin–catenin complex forms the molecular basis of the adherence junctions and cooperates closely with actin filaments. Several groups have recently described small actin-driven protrusions at the cell junctions that are controlled by the Arp2/3 complex, contributing to cell junction regulation. We identified these protrusions as the driving force for VE-cadherin dynamics, as they directly induce new VE-cadherin-mediated adhesion sites, and have accordingly referred to these structures as junction-associated intermittent lamellipodia (JAIL). JAIL extend over only a few microns and thus provide the basis for a subcellular regulation of adhesion. The local (subcellular) VE-cadherin concentration and JAIL formation are directly interdependent, which enables autoregulation. Therefore, this mechanism can contribute a subcellularly regulated adaptation of cell contact dynamics, and is therefore of great importance for monolayer integrity and relative cell migration during wound healing and angiogenesis, as well as for inflammatory responses. In this Review, we discuss the mechanisms and functions underlying these actin-driven protrusions and consider their contribution to the dynamic regulation of endothelial cell junctions.
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Affiliation(s)
- Jiahui Cao
- Institute of Anatomy and Vascular Biology, Westfälische Wilhelms-Universität Münster, Münster Germany
| | - Hans Schnittler
- Institute of Anatomy and Vascular Biology, Westfälische Wilhelms-Universität Münster, Münster Germany
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Chen Z, Chen G, Zhou K, Zhang P, Ren X, Mei X. Toxicity of food sweetener-sodium cyclamate on osteoblasts cells. Biochem Biophys Res Commun 2018; 508:507-511. [PMID: 30509495 DOI: 10.1016/j.bbrc.2018.11.172] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2018] [Accepted: 11/27/2018] [Indexed: 11/30/2022]
Abstract
In this paper, the effect of commonly used food sweetener (sodium cyclamate) on the proliferation and differentiation of osteoblasts has been researched. The morophology change of osteoblasts was investigated by confocal laser scanning microscopy. Cell viability was studied by MTT analysis. BMP2 expression was analyzed by western blot and immunofluorescence. Mineralization ability of osteoblasts was researched by using alizarin red staining method. The results indicate that a very low concentration (0.06 μM) of sodium cyclamate can curle and fold microfilament and microtubule of osteoblasts. The increase addition of sodium cyclamate resulted significantly decrease of cells viability. The expression of bone morphogenetic protein-2 (BMP2) was seriously suppressed by sodium cyclamate. Alizarin Red staining experiment revealed that sodium cyclamate decreased the mineralization ability of osteoblasts. The present results suggest that sodium cyclamate can seriously inhibit the proliferation and differentiation of osteoblasts.
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Affiliation(s)
- Zhenhua Chen
- Jinzhou Medical University, Jinzhou, 121001, PR China.
| | - Guanyu Chen
- Jinzhou Medical University, Jinzhou, 121001, PR China
| | - Kang Zhou
- Jinzhou Medical University, Jinzhou, 121001, PR China
| | - Peng Zhang
- Jinzhou Medical University, Jinzhou, 121001, PR China
| | - Xiuli Ren
- Jinzhou Medical University, Jinzhou, 121001, PR China.
| | - Xifan Mei
- Jinzhou Medical University, Jinzhou, 121001, PR China.
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Natarajan A, Smith AST, Berry B, Lambert S, Molnar P, Hickman JJ. Temporal Characterization of Neuronal Migration Behavior on Chemically Patterned Neuronal Circuits in a Defined in Vitro Environment. ACS Biomater Sci Eng 2018; 4:3460-3470. [PMID: 31475239 PMCID: PMC6713422 DOI: 10.1021/acsbiomaterials.8b00610] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2018] [Accepted: 08/27/2018] [Indexed: 02/07/2023]
Abstract
Directed control of neuronal migration, facilitating the correct spatial positioning of neurons, is crucial to the development of a functional nervous system. An understanding of neuronal migration and positioning on patterned surfaces in vitro would also be beneficial for investigators seeking to design culture platforms capable of mimicking the complex functional architectures of neuronal tissues for drug development as well as basic biomedical research applications. This study used coplanar self-assembled monolayer patterns of cytophilic, N-1[3-(trimethoxysilyly)propyl] diethylenetriamine (DETA) and cytophobic, tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (13F) to assess the migratory behavior and physiological characteristics of cultured neurons. Analysis of time-lapse microscopy data revealed a dynamic procedure underlying the controlled migration of neurons, in response to extrinsic geometric and chemical cues, to promote the formation of distinct two-neuron circuits. Immunocytochemical characterization of the neurons highlights the organization of actin filaments (phalloidin) and microtubules (β-tubulin) at each migration stage. These data have applications in the development of precise artificial neuronal networks and provide a platform for investigating neuronal migration as well as neurite identification in differentiating cultured neurons. Importantly, the cytoskeletal arrangement of these cells identifies a specific mode of neuronal migration on these in vitro surfaces characterized by a single process determining the direction of cell migration and mimicking somal translocation behavior in vivo. Such information provides valuable additional insight into the mechanisms controlling neuronal development and maturation in vitro and validates the biochemical mechanisms underlying this behavior as representative of neuronal positioning phenomena in vivo.
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Affiliation(s)
- Anupama Natarajan
- NanoScience
Technology Center, University of Central
Florida, 12424 Research Parkway, Suite 400, Orlando, Florida 32826, United States
- Burnett
School of Biomedical Sciences, University
of Central Florida, 6900
Lake Nona Boulevard, Orlando, Florida 32827, United
States
| | - Alec S. T. Smith
- NanoScience
Technology Center, University of Central
Florida, 12424 Research Parkway, Suite 400, Orlando, Florida 32826, United States
| | - Bonnie Berry
- NanoScience
Technology Center, University of Central
Florida, 12424 Research Parkway, Suite 400, Orlando, Florida 32826, United States
- Burnett
School of Biomedical Sciences, University
of Central Florida, 6900
Lake Nona Boulevard, Orlando, Florida 32827, United
States
| | - Stephen Lambert
- College
of Medicine, University of Central Florida, 6900 Lake Nona Boulevard, Suite
101, Orlando, Florida 32827, United States
| | - Peter Molnar
- College
of Medicine, University of Central Florida, 6900 Lake Nona Boulevard, Suite
101, Orlando, Florida 32827, United States
- Department
of Zoology, Institute of Biology, Savaria Campus, University of West Hungary, H-9700 Szombathely, Hungary
| | - James J. Hickman
- NanoScience
Technology Center, University of Central
Florida, 12424 Research Parkway, Suite 400, Orlando, Florida 32826, United States
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Huang C, Fu C, Wren JD, Wang X, Zhang F, Zhang YH, Connel SA, Chen T, Zhang XA. Tetraspanin-enriched microdomains regulate digitation junctions. Cell Mol Life Sci 2018; 75:3423-3439. [PMID: 29589089 PMCID: PMC6615572 DOI: 10.1007/s00018-018-2803-2] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2017] [Revised: 02/18/2018] [Accepted: 03/21/2018] [Indexed: 12/22/2022]
Abstract
Tetraspanins co-emerged with multi-cellular organisms during evolution and are typically localized at the cell–cell interface, [corrected] and form tetraspanin-enriched microdomains (TEMs) by associating with each other and other membrane molecules. Tetraspanins affect various biological functions, but how tetraspanins engage in multi-faceted functions at the cellular level is largely unknown. When cells interact, the membrane microextrusions at the cell-cell interfaces form dynamic, digit-like structures between cells, which we term digitation junctions (DJs). We found that (1) tetraspanins CD9, CD81, and CD82 and (2) TEM-associated molecules integrin α3β1, CD44, EWI2/PGRL, and PI-4P are present in DJs of epithelial, endothelial, and cancer cells. Tetraspanins and their associated molecules also regulate the formation and development of DJs. Moreover, (1) actin cytoskeleton, RhoA, and actomyosin activities and (2) growth factor receptor-Src-MAP kinase signaling, but not PI-3 kinase, regulate DJs. Finally, we showed that DJs consist of various forms in different cells. Thus, DJs are common, interactive structures between cells, and likely affect cell adhesion, migration, and communication. TEMs probably modulate various cell functions through DJs. Our findings highlight that DJ morphogenesis reflects the transition between cell-matrix adhesion and cell-cell adhesion and involves both cell-cell and cell-matrix adhesion molecules.
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Affiliation(s)
- Chao Huang
- Stephenson Cancer Center and Department of Physiology, University of Oklahoma Health Sciences Center, BRC Building West Room 1474, 975 N.E. 10th Street, Oklahoma City, OK, 73104, USA
| | - Chenying Fu
- Stephenson Cancer Center and Department of Physiology, University of Oklahoma Health Sciences Center, BRC Building West Room 1474, 975 N.E. 10th Street, Oklahoma City, OK, 73104, USA
| | - Jonathan D Wren
- Oklahoma Medical Research Foundation, Oklahoma City, OK, USA
| | - Xuejun Wang
- Stephenson Cancer Center and Department of Physiology, University of Oklahoma Health Sciences Center, BRC Building West Room 1474, 975 N.E. 10th Street, Oklahoma City, OK, 73104, USA
| | - Feng Zhang
- Stephenson Cancer Center and Department of Physiology, University of Oklahoma Health Sciences Center, BRC Building West Room 1474, 975 N.E. 10th Street, Oklahoma City, OK, 73104, USA
| | - Yanhui H Zhang
- University of Tennessee Health Science Center, Memphis, TN, USA
| | | | - Taosheng Chen
- St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Xin A Zhang
- Stephenson Cancer Center and Department of Physiology, University of Oklahoma Health Sciences Center, BRC Building West Room 1474, 975 N.E. 10th Street, Oklahoma City, OK, 73104, USA.
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Zhang D, Wu S, Feng J, Duan Y, Xing D, Gao C. Micropatterned biodegradable polyesters clicked with CQAASIKVAV promote cell alignment, directional migration, and neurite outgrowth. Acta Biomater 2018; 74:143-155. [PMID: 29768188 DOI: 10.1016/j.actbio.2018.05.018] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2018] [Revised: 03/28/2018] [Accepted: 05/11/2018] [Indexed: 12/31/2022]
Abstract
The interplay of microstructures and biological cues is critical to regulate the behaviors of Schwann cells (SCs) in terms of cellular spatial arrangement and directional migration as well as neurite orientation for bridging the proximal and distal stumps of the injured peripheral nervous system. In this study, stripe micropatterns having ridges/grooves of width 20/20 and 20/40 μm were fabricated on the surface of maleimide-functionalized biodegradable poly(ester carbonate) (P(LLA-MTMC)) films by the polydimethylsiloxane mold-pressing method, respectively. The laminin-derived CQAASIKVAV peptides end-capped with an SH group were then grafted by the thiol-ene click reaction under mild conditions to obtain micropatterned and peptide-grafted films. SCs cultured on these films, especially on the 20/40-μm film, displayed faster and aligned adhesion as well as a larger number of elongated cells with a higher length-to-width (L/W) ratio along the stripe direction than those on the flat-pep film. The migration rate of SCs was significantly enhanced in parallel to the stripe direction with a large net displacement. The micropatterned and peptide-grafted films, especially the 20/40-μm film, could promote SC proliferation and nerve growth factor (NGF) secretion in a manner similar to that of the peptide-grafted planar film. Moreover, the neurites of rat pheochromocytoma 12 (PC12) cells sprouted along the ridges with a longer average length on the micropatterned and peptide-grafted films. The synergistic effect of physical patterns and biological cues was evaluated by considering the results of cell adhesion force; immunofluorescence staining of vinculin; fluorescence staining of F-actin and the nucleus; as well as gene expression of neural cadherin (NCAD), neurocan (NCAN), and myelin protein zero (P0). STATEMENT OF SIGNIFICANCE The interplay of microstructures and biological cues is critical to regulate the behaviors of Schwann cells (SCs) and nerve cells, and thereby the regeneration of peripheral nerve system. In this study, the combined micropatterning and CQAASIKVAV grafting endowed the modified P(LLA-MTMC) films with both contact guidance and bioactive chemical cues to enhance cell proliferation, directional alignment and migration, longer net displacement and larger NGF secretion, and stronger neurite outgrowth of SCs and PC12 cells. Hence, the integration of physical micropatterns and bioactive molecules is an effective way to obtain featured biomaterials for the regeneration of nerves and other types of tissues.
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Proietti S, Catizone A, Masiello MG, Dinicola S, Fabrizi G, Minini M, Ricci G, Verna R, Reiter RJ, Cucina A, Bizzarri M. Increase in motility and invasiveness of MCF7 cancer cells induced by nicotine is abolished by melatonin through inhibition of ERK phosphorylation. J Pineal Res 2018; 64:e12467. [PMID: 29338098 DOI: 10.1111/jpi.12467] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/22/2017] [Accepted: 01/04/2018] [Indexed: 01/18/2023]
Abstract
Through activation of the ERK pathway, nicotine, in both normal MCF-10A and low-malignant breast cancer cells (MCF7), promotes increased motility and invasiveness. Melatonin antagonizes both these effects by inhibiting almost completely ERK phosphorylation. As melatonin has no effect on nonstimulated cells, it is likely that melatonin can counteract ERK activation only downstream of nicotine-induced activation. This finding suggests that melatonin hampers ERK phosphorylation presumably by targeting a still unknown intermediate factor that connects nicotine stimulation to ERK phosphorylation. Furthermore, downstream of ERK activation, melatonin significantly reduces fascin and calpain activation while restoring normal vinculin levels. Melatonin also counteracts nicotine effects by reshaping the overall cytoskeleton architecture and abolishing invasive membrane protrusion. In addition, melatonin decreases nicotine-dependent ROCK1/ROCK2 activation, thus further inhibiting cell contractility and motility. Melatonin actions are most likely attributable to ERK inhibition, although melatonin could display other ERK-independent effects, namely through a direct modulation of additional molecular and structural factors, including coronin, cofilin, and cytoskeleton components.
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Affiliation(s)
- Sara Proietti
- Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Rome, Italy
- Systems Biology Group, Rome, Italy
| | - Angela Catizone
- Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University of Rome, Rome, Italy
| | - Maria Grazia Masiello
- Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Rome, Italy
- Systems Biology Group, Rome, Italy
- Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy
| | - Simona Dinicola
- Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Rome, Italy
- Systems Biology Group, Rome, Italy
- Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy
| | - Gianmarco Fabrizi
- Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Rome, Italy
- Systems Biology Group, Rome, Italy
| | - Mirko Minini
- Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Rome, Italy
- Systems Biology Group, Rome, Italy
- Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy
| | - Giulia Ricci
- Department of Experimental Medicine, Second University of Naples, Naples, Italy
| | - Roberto Verna
- Systems Biology Group, Rome, Italy
- Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy
| | - Russel J Reiter
- Department of Cellular and Structural Biology, The University of Texas Health Science Center, San Antonio, TX, USA
| | - Alessandra Cucina
- Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Rome, Italy
- Systems Biology Group, Rome, Italy
- Azienda Policlinico Umberto I, Rome, Italy
| | - Mariano Bizzarri
- Systems Biology Group, Rome, Italy
- Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy
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Zheng G, Guan B, Hu P, Qi X, Wang P, Kong Y, Liu Z, Gao P, Li R, Zhang X, Wu X, Sui L. Topographical cues of direct metal laser sintering titanium surfaces facilitate osteogenic differentiation of bone marrow mesenchymal stem cells through epigenetic regulation. Cell Prolif 2018; 51:e12460. [PMID: 29701270 DOI: 10.1111/cpr.12460] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2018] [Accepted: 03/03/2018] [Indexed: 12/25/2022] Open
Abstract
OBJECTIVES To investigate the role of hierarchical micro/nanoscale topography of direct metal laser sintering (DMLS) titanium surfaces in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), as well as the possible underlying epigenetic mechanism. MATERIALS AND METHODS Three groups of titanium specimens were prepared, including DMLS group, sandblasted, large-grit, acid-etched (SLA) group and smooth titanium (Ti) group. BMSCs were cultured on discs followed by surface characterization. Cell adhesion and proliferation were examined by SEM and CCK-8 assay, while osteogenic-related gene expression was detected by real-time RT-PCR. Immunofluorescence, western blotting and in vivo study were also performed to evaluate the potential for osteogenic induction of materials. In addition, to investigate the underlying epigenetic mechanisms, immunofluorescence and western blotting were performed to evaluate the global level of H3K4me3 during osteogenesis. The H3K4me3 and H3K27me3 levels at the promoter area of the osteogenic gene Runx2 were detected by ChIP assay. RESULTS The DMLS surface exhibits greater protein adsorption ability and shows better cell adhesion performance than SLA and Ti surfaces. Moreover, both in vitro and in vivo studies demonstrated that the DMLS surface is more favourable for the osteogenic differentiation of BMSCs than SLA and Ti surfaces. Accordingly, osteogenesis-associated gene expression in BMSCs is efficiently induced by a rapid H3K27 demethylation and increase in H3K4me3 levels at gene promoters upon osteogenic differentiation on DMLS titanium surface. CONCLUSIONS Topographical cues of DMLS surfaces have greater potential for the induction of osteogenic differentiation of BMSCs than SLA and Ti surfaces both in vitro and in vivo. A potential epigenetic mechanism is that the appropriate topography allows rapid H3K27 demethylation and an increased H3K4me3 level at the promoter region of osteogenesis-associated genes during the osteogenic differentiation of BMSCs.
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Affiliation(s)
- Guoying Zheng
- Department of Prosthodontics, Tianjin Medical University School and Hospital of Stomatology, Tianjin, China.,Department of Cell Biology, 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Tianjin Medical University, Tianjin, China
| | - Binbin Guan
- Department of Stomatology, Tianjin Medical University General Hospital, Tianjin, China
| | - Penghui Hu
- Department of Prosthodontics, Tianjin Medical University School and Hospital of Stomatology, Tianjin, China
| | - Xingying Qi
- Department of Prosthodontics, Tianjin Medical University School and Hospital of Stomatology, Tianjin, China
| | - Pingting Wang
- Department of Endodontics, Tianjin Medical University School and Hospital of Stomatology, Tianjin, China
| | - Yu Kong
- Department of Cell Biology, 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Tianjin Medical University, Tianjin, China
| | - Zihao Liu
- Department of Prosthodontics, Tianjin Medical University School and Hospital of Stomatology, Tianjin, China
| | - Ping Gao
- Department of Prosthodontics, Tianjin Medical University School and Hospital of Stomatology, Tianjin, China
| | - Rui Li
- Department of Cell Biology, 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Tianjin Medical University, Tianjin, China
| | - Xu Zhang
- Department of Endodontics, Tianjin Medical University School and Hospital of Stomatology, Tianjin, China
| | - Xudong Wu
- Department of Cell Biology, 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Tianjin Medical University, Tianjin, China
| | - Lei Sui
- Department of Prosthodontics, Tianjin Medical University School and Hospital of Stomatology, Tianjin, China
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40
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Lopata A, Hughes R, Tiede C, Heissler SM, Sellers JR, Knight PJ, Tomlinson D, Peckham M. Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells. Sci Rep 2018; 8:6572. [PMID: 29700342 PMCID: PMC5920084 DOI: 10.1038/s41598-018-24953-4] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2017] [Accepted: 04/10/2018] [Indexed: 01/21/2023] Open
Abstract
Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.
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Affiliation(s)
- Anna Lopata
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.,Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.,Laboratory of Molecular Physiology, National Heart, Lung, and Blood Institute, Bethesda, MD, 20892, USA
| | - Ruth Hughes
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.,Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK
| | - Christian Tiede
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.,Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK
| | - Sarah M Heissler
- Laboratory of Molecular Physiology, National Heart, Lung, and Blood Institute, Bethesda, MD, 20892, USA
| | - James R Sellers
- Laboratory of Molecular Physiology, National Heart, Lung, and Blood Institute, Bethesda, MD, 20892, USA
| | - Peter J Knight
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.,Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK
| | - Darren Tomlinson
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.,Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK
| | - Michelle Peckham
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK. .,Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
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41
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Zhang D, Xu S, Wu S, Gao C. Micropatterned poly(d,l-lactide-co-caprolactone) films entrapped with gelatin for promoting the alignment and directional migration of Schwann cells. J Mater Chem B 2018; 6:1226-1237. [DOI: 10.1039/c7tb03073h] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Gelatin entrapped and micropatterned poly(d,l-lactide-co-caprolactone) (PLCL) film promotes the alignment and directional migration of Schwann cells.
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Affiliation(s)
- Deteng Zhang
- MOE Key Laboratory of Macromolecular Synthesis and Functionalization
- Department of Polymer Science and Engineering
- Zhejiang University
- Hangzhou 310027
- China
| | - Shengjun Xu
- MOE Key Laboratory of Macromolecular Synthesis and Functionalization
- Department of Polymer Science and Engineering
- Zhejiang University
- Hangzhou 310027
- China
| | - Sai Wu
- MOE Key Laboratory of Macromolecular Synthesis and Functionalization
- Department of Polymer Science and Engineering
- Zhejiang University
- Hangzhou 310027
- China
| | - Changyou Gao
- MOE Key Laboratory of Macromolecular Synthesis and Functionalization
- Department of Polymer Science and Engineering
- Zhejiang University
- Hangzhou 310027
- China
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Cao J, Ehling M, März S, Seebach J, Tarbashevich K, Sixta T, Pitulescu ME, Werner AC, Flach B, Montanez E, Raz E, Adams RH, Schnittler H. Polarized actin and VE-cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis. Nat Commun 2017; 8:2210. [PMID: 29263363 PMCID: PMC5738342 DOI: 10.1038/s41467-017-02373-8] [Citation(s) in RCA: 112] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2016] [Accepted: 11/24/2017] [Indexed: 02/07/2023] Open
Abstract
VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis. The formation of new blood vessels requires both polarized cell migration and coordinated control of endothelial cell contacts. Here, Cao and colleagues describe at the sub-cellular level the cytoskeletal and cell junction dynamics regulating these processes upon VEGF-induced cell elongation.
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Affiliation(s)
- Jiahui Cao
- Institute of Anatomy and Vascular Biology, Westfälische Wilhelms University of Münster, Faculty of Medicine, D-48149, Münster, Germany
| | - Manuel Ehling
- Max Planck Institute for Molecular Biomedicine and Westfälische Wilhelms University of Münster, Faculty of Medicine, D-48149, Münster, Germany
| | - Sigrid März
- Institute of Anatomy and Vascular Biology, Westfälische Wilhelms University of Münster, Faculty of Medicine, D-48149, Münster, Germany
| | - Jochen Seebach
- Institute of Anatomy and Vascular Biology, Westfälische Wilhelms University of Münster, Faculty of Medicine, D-48149, Münster, Germany
| | - Katsiaryna Tarbashevich
- Institute of Cell Biology, Center for Molecular Biology of Inflammation, D-48149, Münster, Germany
| | - Tomas Sixta
- Department of Cybernetics, Czech Technical University, 16627, Prague 6, Czech Republic
| | - Mara E Pitulescu
- Max Planck Institute for Molecular Biomedicine and Westfälische Wilhelms University of Münster, Faculty of Medicine, D-48149, Münster, Germany
| | - Ann-Cathrin Werner
- Walter-Brendel-Centre of Experimental Medicine, University Hospital, LMU Munich, D-81377 Munich, Germany
| | - Boris Flach
- Department of Cybernetics, Czech Technical University, 16627, Prague 6, Czech Republic
| | - Eloi Montanez
- Walter-Brendel-Centre of Experimental Medicine, University Hospital, LMU Munich, D-81377 Munich, Germany
| | - Erez Raz
- Institute of Cell Biology, Center for Molecular Biology of Inflammation, D-48149, Münster, Germany
| | - Ralf H Adams
- Max Planck Institute for Molecular Biomedicine and Westfälische Wilhelms University of Münster, Faculty of Medicine, D-48149, Münster, Germany
| | - Hans Schnittler
- Institute of Anatomy and Vascular Biology, Westfälische Wilhelms University of Münster, Faculty of Medicine, D-48149, Münster, Germany.
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Reimer M, Denby E, Zustiak SP, Schober JM. Ras GAP-related and C-terminal domain-dependent localization and tumorigenic activities of IQGAP1 in melanoma cells. PLoS One 2017; 12:e0189589. [PMID: 29240845 PMCID: PMC5730206 DOI: 10.1371/journal.pone.0189589] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2017] [Accepted: 11/29/2017] [Indexed: 12/19/2022] Open
Abstract
IQGAP1 interacts with a number of binding partners through a calponin homology domain (CHD), a WW motif, IQ repeats, a Ras GAP-related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 is known to play a role in actin cytoskeleton dynamics during membrane ruffling and lamellipodium protrusion. In addition, phosphorylation near the CT domain is thought to control IQGAP1 activity through regulation of intramolecular interaction. In a previous study, we discovered that IQGAP1 preferentially localizes to retracting areas in B16F10 mouse melanoma cells, not areas of membrane ruffling and lamellipodium protrusion. Nothing is known of the domains needed for retraction localization and very little is known of IQGAP1 function in the actin cytoskeleton of melanoma cells. Thus, we examined localization of IQGAP1 mutants to retracting areas, and characterized knock down phenotypes on tissue culture plastic and physiologic-stiffness hydrogels. Localization of IQGAP1 mutants (S1441E/S1443D, S1441A/S1443A, ΔCHD, ΔGRD or ΔCT) to retracting and protruding cell edges were measured. In retracting areas there was a decrease in S1441A/S1443A, ΔGRD and ΔCT localization, a minor decrease in ΔCHD localization, and normal localization of the S1441E/S1443D mutant. In areas of cell protrusion just behind the lamellipodium leading edge, we surprisingly observed both ΔGRD and ΔCT localization, and increased number of microtubules. IQGAP1 knock down caused loss of cell polarity on laminin-coated glass, decreased proliferation on tissue culture polystyrene, and abnormal spheroid growth on laminin-coated hydrogels. We propose that the GRD and CT domains regulate IQGAP1 localization to retracting actin networks to promote a tumorigenic role in melanoma cells.
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Affiliation(s)
- Michael Reimer
- Department of Pharmaceutical Sciences, Southern Illinois University Edwardsville, Edwardsville, Illinois, United States of America
| | - Elisabeth Denby
- Department of Pharmaceutical Sciences, Southern Illinois University Edwardsville, Edwardsville, Illinois, United States of America
| | - Silviya P. Zustiak
- Department of Biomedical Engineering, Saint Louis University, Saint Louis, Missouri, United States of America
| | - Joseph M. Schober
- Department of Pharmaceutical Sciences, Southern Illinois University Edwardsville, Edwardsville, Illinois, United States of America
- * E-mail:
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Antcin-H Isolated from Antrodia cinnamomea Inhibits Renal Cancer Cell Invasion Partly through Inactivation of FAK-ERK-C/EBP- β/c-Fos-MMP-7 Pathways. EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE 2017; 2017:5052870. [PMID: 29234409 PMCID: PMC5688354 DOI: 10.1155/2017/5052870] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/01/2017] [Revised: 09/29/2017] [Accepted: 10/09/2017] [Indexed: 12/19/2022]
Abstract
Antcin-H, a natural triterpene, is purified from a famous anticancer medicinal mushroom, Antrodia cinnamomea, in Taiwan. This study showed that antcin-H inhibited the growth of human renal carcinoma 786-0 cells; the IC50 value (for 48 h) was 170 μM. Besides, the migration and invasion of 786-0 cells were suppressed by antcin-H under noncytotoxic concentrations (<100 μM); these events were accompanied by inhibition of FAK and Src kinase activities, decrease of paxillin phosphorylation, impairment of lamellipodium formation, and upregulation of TIMPs and downregulation of MMPs, especially MMP-7 expression. Luciferase reporter assay showed that antcin-H repressed the MMP-7 promoter activity, in parallel to inhibiting c-Fos/AP-1 and C/EBP-β transactivation abilities. Moreover, antcin-H suppressed the activity of ERK1/2 and decreased the binding ability of C/EBP-β and c-Fos on the upstream/enhancer region of MMP-7 promoter. Overall, this study demonstrated that the anti-invasive effect of antcin-H in human renal carcinoma 786-0 cells might be at least in part by abrogating focal adhesion complex and lamellipodium formation through inhibiting the Src/FAK-paxillin signaling pathways and decreasing MMP-7 expression through suppressing the ERK1/2-AP-1/c-Fos and C/EBP-β signaling axis. Our findings provide the evidence that antcin-H may be an active component existing in A. cinnamomea with anticancer effect.
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Autocrine hGH stimulates oncogenicity, epithelial-mesenchymal transition and cancer stem cell-like behavior in human colorectal carcinoma. Oncotarget 2017; 8:103900-103918. [PMID: 29262609 PMCID: PMC5732775 DOI: 10.18632/oncotarget.21812] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2017] [Accepted: 09/08/2017] [Indexed: 11/25/2022] Open
Abstract
Tumor derived human growth hormone (hGH) has been implicated in cancer development and progression. However, the specific functional role of autocrine/paracrine hGH in colorectal cancer (CRC) remains largely to be determined. Herein, we demonstrated a crucial oncogenic role of autocrine hGH in CRC progression. Elevated hGH expression was detected in CRC compared to normal colorectal tissue, and hGH expression in CRC was positively associated with tumor size and lymph node metastasis. Forced expression of hGH stimulated cell proliferation, survival, oncogenicity and epithelial to mesenchymal transition (EMT) of CRC cells, and promoted xenograft growth and local invasion in vivo. Autocrine hGH expression in CRC cells stimulated the activation of the ERK1/2 pathway, which in turn resulted in increased transcription of the mesenchymal marker FIBRONECTIN 1 and transcriptional repression of the epithelial marker E-CADHERIN. The autocrine hGH-stimulated increase in CRC cell proliferation, cell survival and EMT was abrogated upon ERK1/2 inhibition. Furthermore, autocrine hGH-stimulated CRC cell migration and invasion was dependent on the ERK1/2-mediated increase in FIBRONECTIN 1 expression and decrease in E-CADHERIN expression. Forced expression of hGH also enhanced CSC-like behavior of CRC cells, as characterized by increased colonosphere formation, ALDH-positive population and CSC marker expression. Autocrine hGH-enhanced cancer stem cell (CSC)-like behavior in CRC cells was also observed to be E-CADHERIN-dependent. Thus, autocrine hGH plays a critical role in CRC progression, and inhibition of hGH could be a promising targeted therapeutic approach to limit disease progression in metastatic CRC patients.
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Regulation of focal adhesion turnover in SDF-1α-stimulated migration of mesenchymal stem cells in neural differentiation. Sci Rep 2017; 7:10013. [PMID: 28855566 PMCID: PMC5577153 DOI: 10.1038/s41598-017-09736-7] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2017] [Accepted: 07/28/2017] [Indexed: 12/13/2022] Open
Abstract
Directed migration of the transplanted mesenchymal stem cells (MSCs) to the lesion sites plays a pivotal role in the efficacy of cell-based therapy. Our previous study demonstrates that MSCs under varying neural differentiation states possess different migratory capacities in response to chemoattractants. However, the underlying mechanism has not been fully addressed. Herein, we show that the assembly and turnover of focal adhesions, the phosphorylation of FAK and paxillin, and the reorganisation of F-actin in MSCs are closely related to their differentiation states in response to SDF-1α. Upon SDF-1α stimulation, FAs turnover more rapidly with the most obvious reduction in the existing time of FAs in MSCs of 24-h preinduction that exhibit the most effective migration towards SDF-1α. Further, we confirm that PI3K/Akt and MAPK pathways participate in the regulation of SDF-1α-induced cell migration and FA assembly, and moreover, that the regulatory effects vary greatly depending on the differentiation states. Collectively, these results demonstrate that FA assembly and turnover, which is accompanied with F-actin reorganisation in response to SDF-1α, correlates closely with the differentiation states of MSCs, which might contribute to the different chemotactic responses of these cells, and thus help develop new strategy to improve the efficacy of MSCs-based therapy.
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Li X, Lu J, Kan Q, Li X, Fan Q, Li Y, Huang R, Slipicevic A, Dong HP, Eide L, Wang J, Zhang H, Berge V, Goscinski MA, Kvalheim G, Nesland JM, Suo Z. Metabolic reprogramming is associated with flavopiridol resistance in prostate cancer DU145 cells. Sci Rep 2017; 7:5081. [PMID: 28698547 PMCID: PMC5506068 DOI: 10.1038/s41598-017-05086-6] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2016] [Accepted: 05/24/2017] [Indexed: 01/19/2023] Open
Abstract
Flavopiridol (FP) is a pan-cyclin dependent kinase inhibitor, which shows strong efficacy in inducing cancer cell apoptosis. Although FP is potent against most cancer cells in vitro, unfortunately it proved less efficacious in clinical trials in various aggressive cancers. To date, the molecular mechanisms of the FP resistance are mostly unknown. Here, we report that a small fraction human prostate cancer DU145 cells can survive long-term FP treatment and emerge as FP-resistant cells (DU145FP). These DU145FP cells show accumulated mitochondrial lesions with stronger glycolytic features, and they proliferate in slow-cycling and behave highly migratory with strong anti-apoptotic potential. In addition, the cells are less sensitive to cisplatin and docetaxel-induced apoptotic pressure, and over-express multiple stem cell associated biomarkers. Our studies collectively uncover for the first time that FP-resistant prostate cancer cells show metabolic remodeling, and the metabolic plasticity might be required for the FP resistance-associated cancer cell stemness up-regulation.
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Affiliation(s)
- Xiaoran Li
- Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway
- Department of Pathology, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, 0318, Norway
| | - Jie Lu
- Department of Epidemiology and Biostatistics, College of Public Health, Zhengzhou University, Zhengzhou, Henan, 450001, China
| | - Quancheng Kan
- Department of Clinical Pharmacology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, 450001, China
| | - Xiaoli Li
- Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou, Henan, 450052, China
| | - Qiong Fan
- Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo, 0316, Norway
| | - Yaqing Li
- Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou, Henan, 450052, China
| | - Ruixia Huang
- Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, 0379, Norway
| | - Ana Slipicevic
- Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway
| | - Hiep Phuc Dong
- Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway
| | - Lars Eide
- Department of Medical Biochemistry, University of Oslo and Oslo University Hospital, Oslo, 0372, Norway
| | - Junbai Wang
- Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway
| | - Hongquan Zhang
- Laboratory of Molecular Cell Biology and Tumor Biology, Department of Anatomy, Histology and Embryology, Peking University Health Science Center, Beijing, 100191, China
| | - Viktor Berge
- Department of Urology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway
| | - Mariusz Adam Goscinski
- Departments of Surgery, The Norwegian Radium Hospital, Oslo University Hospital, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, 0379, Norway
| | - Gunnar Kvalheim
- Department of Cell Therapy, Cancer Institute, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway
| | - Jahn M Nesland
- Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway
- Department of Pathology, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, 0318, Norway
| | - Zhenhe Suo
- Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway.
- Department of Pathology, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, 0318, Norway.
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Atherton P, Lausecker F, Harrison A, Ballestrem C. Low-intensity pulsed ultrasound promotes cell motility through vinculin-controlled Rac1 GTPase activity. J Cell Sci 2017; 130:2277-2291. [PMID: 28576970 DOI: 10.1242/jcs.192781] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2016] [Accepted: 05/29/2017] [Indexed: 12/16/2022] Open
Abstract
Low-intensity pulsed ultrasound (LIPUS) is a therapy used clinically to promote healing. Using live-cell imaging we show that LIPUS stimulation, acting through integrin-mediated cell-matrix adhesions, rapidly induces Rac1 activation associated with dramatic actin cytoskeleton rearrangements. Our study demonstrates that the mechanosensitive focal adhesion (FA) protein vinculin, and both focal adhesion kinase (FAK, also known as PTK2) and Rab5 (both the Rab5a and Rab5b isoforms) have key roles in regulating these effects. Inhibiting the link of vinculin to the actin-cytoskeleton abolished LIPUS sensing. We show that this vinculin-mediated link was not only critical for Rac1 induction and actin rearrangements, but was also important for the induction of a Rab5-dependent increase in the number of early endosomes. Expression of dominant-negative Rab5, or inhibition of endocytosis with dynasore, also blocked LIPUS-induced Rac1 signalling events. Taken together, our data show that LIPUS is sensed by cell matrix adhesions through vinculin, which in turn modulates a Rab5-Rac1 pathway to control ultrasound-mediated endocytosis and cell motility. Finally, we demonstrate that a similar FAK-Rab5-Rac1 pathway acts to control cell spreading upon fibronectin.
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Affiliation(s)
- Paul Atherton
- Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester M13 9PT, USA
| | - Franziska Lausecker
- Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester M13 9PT, USA
| | - Andrew Harrison
- Bioventus Cooperatief, Taurusavenue 31, 2132 LS Hoofddorp, The Netherlands
| | - Christoph Ballestrem
- Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester M13 9PT, USA
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Intermediate filament reorganization dynamically influences cancer cell alignment and migration. Sci Rep 2017; 7:45152. [PMID: 28338091 PMCID: PMC5364536 DOI: 10.1038/srep45152] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2015] [Accepted: 02/20/2017] [Indexed: 01/24/2023] Open
Abstract
The interactions between a cancer cell and its extracellular matrix (ECM) have been the focus of an increasing amount of investigation. The role of the intermediate filament keratin in cancer has also been coming into focus of late, but more research is needed to understand how this piece fits in the puzzle of cytoskeleton-mediated invasion and metastasis. In Panc-1 invasive pancreatic cancer cells, keratin phosphorylation in conjunction with actin inhibition was found to be sufficient to reduce cell area below either treatment alone. We then analyzed intersecting keratin and actin fibers in the cytoskeleton of cyclically stretched cells and found no directional correlation. The role of keratin organization in Panc-1 cellular morphological adaptation and directed migration was then analyzed by culturing cells on cyclically stretched polydimethylsiloxane (PDMS) substrates, nanoscale grates, and rigid pillars. In general, the reorganization of the keratin cytoskeleton allows the cell to become more ‘mobile’- exhibiting faster and more directed migration and orientation in response to external stimuli. By combining keratin network perturbation with a variety of physical ECM signals, we demonstrate the interconnected nature of the architecture inside the cell and the scaffolding outside of it, and highlight the key elements facilitating cancer cell-ECM interactions.
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Jokhadar ŠZ, Derganc J. Structural Rearrangements in CHO Cells After Disruption of Individual Cytoskeletal Elements and Plasma Membrane. Cell Biochem Biophys 2016; 71:1605-13. [PMID: 25395197 DOI: 10.1007/s12013-014-0383-9] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Cellular structural integrity is provided primarily by the cytoskeleton, which comprises microtubules, actin filaments, and intermediate filaments. The plasma membrane has been also recognized as a mediator of physical forces, yet its contribution to the structural integrity of the cell as a whole is less clear. In order to investigate the relationship between the plasma membrane and the cytoskeleton, we selectively disrupted the plasma membrane and each of the cytoskeletal elements in Chinese hamster ovary cells and assessed subsequent changes in cellular structural integrity. Confocal microscopy was used to visualize cytoskeletal rearrangements, and optical tweezers were utilized to quantify membrane tether extraction. We found that cholesterol depletion from the plasma membrane resulted in rearrangements of all cytoskeletal elements. Conversely, the state of the plasma membrane, as assessed by tether extraction, was affected by disruption of any of the cytoskeletal elements, including microtubules and intermediate filaments, which are located mainly in the cell interior. The results demonstrate that, besides the cytoskeleton, the plasma membrane is an important contributor to cellular integrity, possibly by acting as an essential framework for cytoskeletal anchoring. In agreement with the tensegrity model of cell mechanics, our results support the notion of the cell as a prestressed structure.
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Affiliation(s)
- Špela Zemljič Jokhadar
- Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000, Ljubljana, Slovenia.
| | - Jure Derganc
- Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000, Ljubljana, Slovenia
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