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1
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Pudlowski R, Xu L, Milenkovic L, Kumar C, Hemsworth K, Aqrabawi Z, Stearns T, Wang JT. A delta-tubulin/epsilon-tubulin/Ted protein complex is required for centriole architecture. eLife 2025; 13:RP98704. [PMID: 40067174 PMCID: PMC11896610 DOI: 10.7554/elife.98704] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/15/2025] Open
Abstract
Centrioles have a unique, conserved architecture formed by three linked, 'triplet', microtubules arranged in ninefold symmetry. The mechanisms by which these triplet microtubules are formed remain unclear but likely involve the noncanonical tubulins delta-tubulin and epsilon-tubulin. Previously, we found that human cells lacking delta-tubulin or epsilon-tubulin form abnormal centrioles, characterized by an absence of triplet microtubules, lack of central core protein POC5, and a futile cycle of centriole formation and disintegration (Wang et al., 2017). Here, we show that human cells lacking either TEDC1 or TEDC2 have similar abnormalities. Using ultrastructure expansion microscopy, we observed that mutant centrioles elongate to the same length as control centrioles in G2 phase and fail to recruit central core scaffold proteins. Remarkably, mutant centrioles also have an expanded proximal region. During mitosis, these mutant centrioles further elongate before fragmenting and disintegrating. All four proteins physically interact and TEDC1 and TEDC2 can form a subcomplex in the absence of the tubulins, supporting an AlphaFold Multimer model of the tetramer. TEDC1 and TEDC2 localize to centrosomes and are mutually dependent on each other and on delta-tubulin and epsilon-tubulin for localization. Our results demonstrate that delta-tubulin, epsilon-tubulin, TEDC1, and TEDC2 function together to promote robust centriole architecture, laying the foundation for future studies on the mechanisms underlying the assembly of triplet microtubules and their interactions with centriole structure.
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Affiliation(s)
- Rachel Pudlowski
- Department of Biology, Washington University in St. LouisSt. LouisUnited States
| | - Lingyi Xu
- Department of Biology, Washington University in St. LouisSt. LouisUnited States
| | | | - Chandan Kumar
- Department of Biology, Washington University in St. LouisSt. LouisUnited States
| | - Katherine Hemsworth
- Department of Biology, Washington University in St. LouisSt. LouisUnited States
| | - Zayd Aqrabawi
- Department of Biology, Washington University in St. LouisSt. LouisUnited States
| | - Tim Stearns
- Department of Biology, Stanford UniversityStanfordUnited States
- Rockefeller UniversityNew York CityUnited States
| | - Jennifer T Wang
- Department of Biology, Washington University in St. LouisSt. LouisUnited States
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2
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Langner E, Puapatanakul P, Pudlowski R, Alsabbagh DY, Miner JH, Horani A, Dutcher SK, Brody SL, Wang JT, Suleiman HY, Mahjoub MR. Ultrastructure expansion microscopy (U-ExM) of mouse and human kidneys for analysis of subcellular structures. Cytoskeleton (Hoboken) 2024; 81:618-638. [PMID: 38715433 PMCID: PMC11540979 DOI: 10.1002/cm.21870] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2024] [Revised: 04/11/2024] [Accepted: 04/22/2024] [Indexed: 05/21/2024]
Abstract
Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and embedding conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both preclinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.
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Affiliation(s)
- Ewa Langner
- Department of Medicine, Washington University, St. Louis, Missouri, USA
| | - Pongpratch Puapatanakul
- Department of Medicine, Washington University, St. Louis, Missouri, USA
- Division of Nephrology, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
| | - Rachel Pudlowski
- Department of Biology, Washington University, St. Louis, Missouri, USA
| | | | - Jeffrey H Miner
- Department of Medicine, Washington University, St. Louis, Missouri, USA
| | - Amjad Horani
- Department of Pediatrics, Washington University, St. Louis, Missouri, USA
| | - Susan K Dutcher
- Department of Genetics, Washington University, St. Louis, Missouri, USA
| | - Steven L Brody
- Department of Medicine, Washington University, St. Louis, Missouri, USA
| | - Jennifer T Wang
- Division of Nephrology, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
| | - Hani Y Suleiman
- Department of Medicine, Washington University, St. Louis, Missouri, USA
| | - Moe R Mahjoub
- Department of Medicine, Washington University, St. Louis, Missouri, USA
- Department of Cell Biology and Physiology, Washington University, St. Louis, Missouri, USA
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3
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Langner E, Puapatanakul P, Pudlowski R, Alsabbagh DY, Miner JH, Horani A, Dutcher SK, Brody SL, Wang JT, Suleiman HY, Mahjoub MR. Ultrastructure expansion microscopy (U-ExM) of mouse and human kidneys for analysis of subcellular structures. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.02.16.580708. [PMID: 38405695 PMCID: PMC10889020 DOI: 10.1101/2024.02.16.580708] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/27/2024]
Abstract
Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and storage conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both pre-clinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.
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4
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Langner E, Cheng T, Kefaloyianni E, Gluck C, Wang B, Mahjoub MR. Cep120 is essential for kidney stromal progenitor cell growth and differentiation. EMBO Rep 2024; 25:428-454. [PMID: 38177914 PMCID: PMC10897188 DOI: 10.1038/s44319-023-00019-z] [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: 07/07/2023] [Revised: 11/15/2023] [Accepted: 11/22/2023] [Indexed: 01/06/2024] Open
Abstract
Mutations in genes that disrupt centrosome structure or function can cause congenital kidney developmental defects and lead to fibrocystic pathologies. Yet, it is unclear how defective centrosome biogenesis impacts renal progenitor cell physiology. Here, we examined the consequences of impaired centrosome duplication on kidney stromal progenitor cell growth, differentiation, and fate. Conditional deletion of the ciliopathy gene Cep120, which is essential for centrosome duplication, in the stromal mesenchyme resulted in reduced abundance of interstitial lineages including pericytes, fibroblasts and mesangial cells. These phenotypes were caused by a combination of delayed mitosis, activation of the mitotic surveillance pathway leading to apoptosis, and changes in both Wnt and Hedgehog signaling that are key for differentiation of stromal cells. Cep120 ablation resulted in small hypoplastic kidneys with medullary atrophy and delayed nephron maturation. Finally, Cep120 and centrosome loss in the interstitium sensitized kidneys of adult mice, causing rapid fibrosis after renal injury via enhanced TGF-β/Smad3-Gli2 signaling. Our study defines the cellular and developmental defects caused by loss of Cep120 and aberrant centrosome biogenesis in the embryonic kidney stroma.
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Affiliation(s)
- Ewa Langner
- Department of Medicine (Nephrology Division), Washington University, St Louis, MO, USA
| | - Tao Cheng
- Department of Medicine (Nephrology Division), Washington University, St Louis, MO, USA
| | - Eirini Kefaloyianni
- Department of Medicine (Rheumatology Division), Washington University, St Louis, MO, USA
| | - Charles Gluck
- Department of Medicine (Nephrology Division), Washington University, St Louis, MO, USA
| | - Baolin Wang
- Department of Genetic Medicine, Weill Medical College of Cornell University, New York, NY, USA
| | - Moe R Mahjoub
- Department of Medicine (Nephrology Division), Washington University, St Louis, MO, USA.
- Department of Cell Biology and Physiology, Washington University, St Louis, MO, USA.
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5
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Cheng T, Agwu C, Shim K, Wang B, Jain S, Mahjoub MR. Aberrant centrosome biogenesis disrupts nephron and collecting duct progenitor growth and fate resulting in fibrocystic kidney disease. Development 2023; 150:dev201976. [PMID: 37982452 PMCID: PMC10753588 DOI: 10.1242/dev.201976] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2023] [Accepted: 11/13/2023] [Indexed: 11/21/2023]
Abstract
Mutations that disrupt centrosome biogenesis or function cause congenital kidney developmental defects and fibrocystic pathologies. Yet how centrosome dysfunction results in the kidney disease phenotypes remains unknown. Here, we examined the consequences of conditional knockout of the ciliopathy gene Cep120, essential for centrosome duplication, in the nephron and collecting duct progenitor niches of the mouse embryonic kidney. Cep120 loss led to reduced abundance of both cap mesenchyme and ureteric bud populations, due to a combination of delayed mitosis, increased apoptosis and premature differentiation of progenitor cells. These defects resulted in dysplastic kidneys at birth, which rapidly formed cysts, displayed increased interstitial fibrosis and decline in kidney function. RNA sequencing of embryonic and postnatal kidneys from Cep120-null mice identified changes in the pathways essential for development, fibrosis and cystogenesis. Our study defines the cellular and developmental defects caused by centrosome dysfunction during kidney morphogenesis and identifies new therapeutic targets for patients with renal centrosomopathies.
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Affiliation(s)
- Tao Cheng
- Department of Medicine, Division of Nephrology, Washington University in St Louis, St. Louis, MO 63110, USA
| | - Chidera Agwu
- Department of Medicine, Division of Nephrology, Washington University in St Louis, St. Louis, MO 63110, USA
| | - Kyuhwan Shim
- Department of Medicine, Division of Nephrology, Washington University in St Louis, St. Louis, MO 63110, USA
| | - Baolin Wang
- Department of Genetic Medicine, Weill Medical College of Cornell University, New York, NY 10065, USA
| | - Sanjay Jain
- Department of Medicine, Division of Nephrology, Washington University in St Louis, St. Louis, MO 63110, USA
| | - Moe R. Mahjoub
- Department of Medicine, Division of Nephrology, Washington University in St Louis, St. Louis, MO 63110, USA
- Department of Cell Biology and Physiology, Washington University in St Louis, St. Louis, MO 63110, USA
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6
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Atmakuru PS, Dhawan J. The cilium-centrosome axis in coupling cell cycle exit and cell fate. J Cell Sci 2023; 136:308872. [PMID: 37144419 DOI: 10.1242/jcs.260454] [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] [Indexed: 05/06/2023] Open
Abstract
The centrosome is an evolutionarily conserved, ancient organelle whose role in cell division was first described over a century ago. The structure and function of the centrosome as a microtubule-organizing center, and of its extracellular extension - the primary cilium - as a sensory antenna, have since been extensively studied, but the role of the cilium-centrosome axis in cell fate is still emerging. In this Opinion piece, we view cellular quiescence and tissue homeostasis from the vantage point of the cilium-centrosome axis. We focus on a less explored role in the choice between distinct forms of mitotic arrest - reversible quiescence and terminal differentiation, which play distinct roles in tissue homeostasis. We outline evidence implicating the centrosome-basal body switch in stem cell function, including how the cilium-centrosome complex regulates reversible versus irreversible arrest in adult skeletal muscle progenitors. We then highlight exciting new findings in other quiescent cell types that suggest signal-dependent coupling of nuclear and cytoplasmic events to the centrosome-basal body switch. Finally, we propose a framework for involvement of this axis in mitotically inactive cells and identify future avenues for understanding how the cilium-centrosome axis impacts central decisions in tissue homeostasis.
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Affiliation(s)
- Priti S Atmakuru
- CSIR Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
| | - Jyotsna Dhawan
- CSIR Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
- Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
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7
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Streubel JMS, Pereira G. Control of centrosome distal appendages assembly and disassembly. Cells Dev 2023; 174:203839. [PMID: 37062431 DOI: 10.1016/j.cdev.2023.203839] [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: 02/18/2023] [Revised: 03/29/2023] [Accepted: 04/08/2023] [Indexed: 04/18/2023]
Abstract
Centrosomes are microtubule organizing centers involved in chromosome segregation, spindle orientation, cell motility and cilia formation. In recent years, they have also emerged as key modulators of asymmetric cell division. Centrosomes are composed of two centrioles that initiate duplication in S phase. The conservative nature of centriole duplication means that the two centrioles of a G1 cell are of different ages. They are also structurally different as only the older centriole carry appendages, an assembly of a subset of proteins primarily required for cilia formation. In a growing tissue, the non-motile, primary cilium acts as a mechano- and sensory organelle that influences cell behavior via modulation of signaling pathways. Here, we discuss the most recent findings about distal appendage composition and function, as well as cell cycle-specific regulation and their implications in various diseases.
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Affiliation(s)
- Johanna M S Streubel
- Centre for Organismal Studies (COS), University of Heidelberg, Heidelberg, Germany; German Cancer Research Centre (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany; Centre for Molecular Biology (ZMBH), University of Heidelberg, Heidelberg, Germany
| | - Gislene Pereira
- Centre for Organismal Studies (COS), University of Heidelberg, Heidelberg, Germany; German Cancer Research Centre (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany; Centre for Molecular Biology (ZMBH), University of Heidelberg, Heidelberg, Germany.
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8
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Cheng T, Agwu C, Shim K, Wang B, Jain S, Mahjoub MR. Aberrant centrosome biogenesis disrupts nephron progenitor cell renewal and fate resulting in fibrocystic kidney disease. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.04.04.535568. [PMID: 37066373 PMCID: PMC10104032 DOI: 10.1101/2023.04.04.535568] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/18/2023]
Abstract
Mutations that disrupt centrosome structure or function cause congenital kidney developmental defects and fibrocystic pathologies. Yet, it remains unclear how mutations in proteins essential for centrosome biogenesis impact embryonic kidney development. Here, we examined the consequences of conditional deletion of a ciliopathy gene, Cep120 , in the two nephron progenitor niches of the embryonic kidney. Cep120 loss led to reduced abundance of both metanephric mesenchyme and ureteric bud progenitor populations. This was due to a combination of delayed mitosis, increased apoptosis, and premature differentiation of progenitor cells. These defects resulted in dysplastic kidneys at birth, which rapidly formed cysts, displayed increased interstitial fibrosis, and decline in filtration function. RNA sequencing of embryonic and postnatal kidneys from Cep120-null mice identified changes in pathways essential for branching morphogenesis, cystogenesis and fibrosis. Our study defines the cellular and developmental defects caused by centrosome dysfunction during kidney development, and identifies new therapeutic targets for renal centrosomopathies. Highlights Defective centrosome biogenesis in nephron progenitors causes:Reduced abundance of metanephric mesenchyme and premature differentiation into tubular structuresAbnormal branching morphogenesis leading to reduced nephron endowment and smaller kidneysChanges in cell-autonomous and paracrine signaling that drive cystogenesis and fibrosisUnique cellular and developmental defects when compared to Pkd1 knockout models.
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9
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Langner E, Cheng T, Kefaloyianni E, Gluck C, Wang B, Mahjoub MR. Impaired centrosome biogenesis in kidney stromal progenitors reduces abundance of interstitial lineages and accelerates injury-induced fibrosis. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.04.04.535583. [PMID: 37066241 PMCID: PMC10104024 DOI: 10.1101/2023.04.04.535583] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/18/2023]
Abstract
Defective centrosome function can disrupt embryonic kidney development, by causing changes to the renal interstitium that leads to fibrocystic disease pathologies. Yet, it remains unknown how mutations in centrosome genes impact kidney interstitial cells. Here, we examined the consequences of defective centrosome biogenesis on stromal progenitor cell growth, differentiation and fate. Conditional deletion of Cep120 , a ciliopathy gene essential for centrosome duplication, in the stromal mesenchyme resulted in reduced abundance of pericytes, interstitial fibroblasts and mesangial cells. This was due to delayed mitosis, increased apoptosis, and changes in Wnt and Hedgehog signaling essential for differentiation of stromal lineages. Cep120 ablation resulted in hypoplastic kidneys with medullary atrophy and delayed nephron maturation. Finally, centrosome loss in the interstitium sensitized kidneys of adult mice, causing rapid fibrosis via enhanced TGF-β/Smad3-Gli2 signaling after renal injury. Our study defines the cellular and developmental defects caused by centrosome dysfunction in embryonic kidney stroma. Highlights Defective centrosome biogenesis in kidney stroma causes:Reduced abundance of stromal progenitors, interstitial and mesangial cell populationsDefects in cell-autonomous and paracrine signalingAbnormal/delayed nephrogenesis and tubular dilationsAccelerates injury-induced fibrosis via defective TGF-β/Smad3-Gli2 signaling axis.
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10
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Feng Z, Zhang L, Liu Y, Zhang W. NCAPG2 contributes to the progression of malignant melanoma through regulating proliferation and metastasis. Biochem Cell Biol 2022; 100:473-484. [PMID: 36265182 DOI: 10.1139/bcb-2022-0048] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Malignant melanoma is a highly aggressive cutaneous neoplasm with increasing incidence worldwide. Non-SMC condensin II complex subunit G2 (NCAPG2) exerts import biological function in the pathogenesis of several tumors. In this study, the functional roles of NCAPG2 knockdown in malignant melanoma were revealed in in vitro and in vivo experiments. In vitro study demonstrated that NCAPG2 depletion could inhibit proliferation and migration and promote apoptosis of malignant melanoma cells. Our in vivo date further confirmed that NCAPG2 knockdown attenuated tumor growth of malignant melanoma. Interestingly, NCAPG2 drove tumor development of malignant melanoma through activating the signal transducer and activator of transcription 3 (STAT3). In conclusion, this study elaborated the tumor-promoting effects of NCAPG2 on malignant melanoma, and NCAPG2 may be a potential therapeutic target for malignant melanoma therapy.
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Affiliation(s)
- Zhang Feng
- Department of Burn and Plastic Surgery, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong 250021, China
| | - Linfeng Zhang
- Department of Burn and Plastic Surgery, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong 250021, China
| | - Yanxin Liu
- Department of Burn and Plastic Surgery, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong 250021, China
| | - Wei Zhang
- Department of Burn and Plastic Surgery, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong 250021, China
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11
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Meka DP, Kobler O, Hong S, Friedrich CM, Wuesthoff S, Henis M, Schwanke B, Krisp C, Schmuelling N, Rueter R, Ruecker T, Betleja E, Cheng T, Mahjoub MR, Soba P, Schlüter H, Fornasiero EF, Calderon de Anda F. Centrosome-dependent microtubule modifications set the conditions for axon formation. Cell Rep 2022; 39:110686. [PMID: 35443171 PMCID: PMC10150443 DOI: 10.1016/j.celrep.2022.110686] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2021] [Revised: 12/27/2021] [Accepted: 03/24/2022] [Indexed: 11/29/2022] Open
Abstract
Microtubule (MT) modifications are critical during axon development, with stable MTs populating the axon. How these modifications are spatially coordinated is unclear. Here, via high-resolution microscopy, we show that early developing neurons have fewer somatic acetylated MTs restricted near the centrosome. At later stages, however, acetylated MTs spread out in soma and concentrate in growing axon. Live imaging in early plated neurons of the MT plus-end protein, EB3, show increased displacement and growth rate near the MTOC, suggesting local differences that might support axon selection. Moreover, F-actin disruption in early developing neurons, which show fewer somatic acetylated MTs, does not induce multiple axons, unlike later stages. Overexpression of centrosomal protein 120 (Cep120), which promotes MT acetylation/stabilization, induces multiple axons, while its knockdown downregulates proteins modulating MT dynamics and stability, hampering axon formation. Collectively, we show how centrosome-dependent MT modifications contribute to axon formation.
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Affiliation(s)
- Durga Praveen Meka
- Institute of Developmental Neurophysiology, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany
| | - Oliver Kobler
- Combinatorial Neuroimaging Core Facility, Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany
| | - Shuai Hong
- Institute of Developmental Neurophysiology, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany
| | - Carina Meta Friedrich
- Institute of Developmental Neurophysiology, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany
| | - Souhaila Wuesthoff
- Institute of Developmental Neurophysiology, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany
| | - Melad Henis
- Institute of Developmental Neurophysiology, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany; Department of Anatomy and Histology, Faculty of Veterinary Medicine, New Valley University, 72511 El-Kharga, Egypt
| | - Birgit Schwanke
- Institute of Developmental Neurophysiology, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany
| | - Christoph Krisp
- Institute for Clinical Chemistry and Laboratory Medicine, Mass Spectrometric Proteomics Group, Campus Forschung, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Nessa Schmuelling
- Institute of Developmental Neurophysiology, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany
| | - René Rueter
- Institute of Developmental Neurophysiology, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany
| | - Tabitha Ruecker
- Institute of Developmental Neurophysiology, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany
| | - Ewelina Betleja
- Department of Medicine (Nephrology Division), Washington University, St. Louis, MO 63110, USA
| | - Tao Cheng
- Department of Medicine (Nephrology Division), Washington University, St. Louis, MO 63110, USA
| | - Moe R Mahjoub
- Department of Medicine (Nephrology Division), Washington University, St. Louis, MO 63110, USA
| | - Peter Soba
- LIMES Institute, Department of Molecular Brain Physiology and Behavior, University of Bonn, 53115 Bonn, Germany; Institute of Physiology and Pathophysiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany
| | - Hartmut Schlüter
- Institute for Clinical Chemistry and Laboratory Medicine, Mass Spectrometric Proteomics Group, Campus Forschung, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Eugenio F Fornasiero
- Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, 37073 Göttingen, Germany
| | - Froylan Calderon de Anda
- Institute of Developmental Neurophysiology, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany.
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12
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Goutas A, Trachana V. Stem cells' centrosomes: How can organelles identified 130 years ago contribute to the future of regenerative medicine? World J Stem Cells 2021; 13:1177-1196. [PMID: 34630857 PMCID: PMC8474719 DOI: 10.4252/wjsc.v13.i9.1177] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/01/2021] [Revised: 05/03/2021] [Accepted: 08/09/2021] [Indexed: 02/06/2023] Open
Abstract
At the core of regenerative medicine lies the expectation of repair or replacement of damaged tissues or whole organs. Donor scarcity and transplant rejection are major obstacles, and exactly the obstacles that stem cell-based therapy promises to overcome. These therapies demand a comprehensive understanding of the asymmetric division of stem cells, i.e. their ability to produce cells with identical potency or differentiated cells. It is believed that with better understanding, researchers will be able to direct stem cell differentiation. Here, we describe extraordinary advances in manipulating stem cell fate that show that we need to focus on the centrosome and the centrosome-derived primary cilium. This belief comes from the fact that this organelle is the vehicle that coordinates the asymmetric division of stem cells. This is supported by studies that report the significant role of the centrosome/cilium in orchestrating signaling pathways that dictate stem cell fate. We anticipate that there is sufficient evidence to place this organelle at the center of efforts that will shape the future of regenerative medicine.
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Affiliation(s)
- Andreas Goutas
- Department of Biology, Faculty of Medicine, University of Thessaly, Larisa 41500, Biopolis, Greece
| | - Varvara Trachana
- Department of Biology, Faculty of Medicine, University of Thessaly, Larisa 41500, Biopolis, Greece.
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13
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Gurkaslar HK, Culfa E, Arslanhan MD, Lince-Faria M, Firat-Karalar EN. CCDC57 Cooperates with Microtubules and Microcephaly Protein CEP63 and Regulates Centriole Duplication and Mitotic Progression. Cell Rep 2021; 31:107630. [PMID: 32402286 DOI: 10.1016/j.celrep.2020.107630] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2019] [Revised: 03/08/2020] [Accepted: 04/20/2020] [Indexed: 12/21/2022] Open
Abstract
Centrosomes function in key cellular processes ranging from cell division to cellular signaling. Their dysfunction is linked to cancer and developmental disorders. Here, we identify CCDC57 as a pleiotropic regulator of centriole duplication, mitosis, and ciliogenesis. Combining proximity mapping with superresolution imaging, we show that CCDC57 localizes to the proximal end of centrioles and interacts with the microcephaly protein CEP63, centriolar satellite proteins, and microtubules. Loss of CCDC57 causes defects in centriole duplication and results in a failure to localize CEP63 and CEP152 to the centrosome. Additionally, CCDC57 depletion perturbs mitotic progression both in wild-type and centriole-less cells. Importantly, its centrosome-targeting region is required for its interaction with CEP63 and functions during centriole duplication and cilium assembly, whereas the microtubule-targeting region is required for its mitotic functions. Together, our results identify CCDC57 as a critical interface between centrosome and microtubule-mediated cellular processes that are deregulated in microcephaly.
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Affiliation(s)
- H Kubra Gurkaslar
- Department of Molecular Biology and Genetics, Koç University, Sarıyer, İstanbul 34450, Turkey
| | - Efraim Culfa
- Department of Molecular Biology and Genetics, Koç University, Sarıyer, İstanbul 34450, Turkey
| | - Melis D Arslanhan
- Department of Molecular Biology and Genetics, Koç University, Sarıyer, İstanbul 34450, Turkey
| | - Mariana Lince-Faria
- Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, Oeiras 2780-156, Portugal
| | - Elif Nur Firat-Karalar
- Department of Molecular Biology and Genetics, Koç University, Sarıyer, İstanbul 34450, Turkey.
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14
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Sharma A, Olieric N, Steinmetz MO. Centriole length control. Curr Opin Struct Biol 2020; 66:89-95. [PMID: 33220554 DOI: 10.1016/j.sbi.2020.10.011] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2020] [Revised: 10/10/2020] [Accepted: 10/13/2020] [Indexed: 10/22/2022]
Abstract
Centrioles are microtubule-based structures involved in cell division and ciliogenesis. Centriole formation is a highly regulated cellular process and aberrations in centriole structure, size or numbers have implications in multiple human pathologies. In this review, we propose that the proteins that control centriole length can be subdivided into two classes based on their antagonistic activities on centriolar microtubules, which we refer to as 'centriole elongation activators' (CEAs) and 'centriole elongation inhibitors' (CEIs). We discuss and illustrate the structure-function relationship of CEAs and CEIs as well as their interaction networks. Based on our current knowledge, we formulate some outstanding open questions in the field and present possible routes for future studies.
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Affiliation(s)
- Ashwani Sharma
- Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, CH-5232 Villigen, Switzerland.
| | - Natacha Olieric
- Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, CH-5232 Villigen, Switzerland.
| | - Michel O Steinmetz
- Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, CH-5232 Villigen, Switzerland; University of Basel, Biozentrum, CH-4056 Basel, Switzerland.
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15
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Tona Y, Wu DK. Live imaging of hair bundle polarity acquisition demonstrates a critical timeline for transcription factor Emx2. eLife 2020; 9:e59282. [PMID: 32965215 PMCID: PMC7535933 DOI: 10.7554/elife.59282] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2020] [Accepted: 09/17/2020] [Indexed: 12/22/2022] Open
Abstract
Directional sensitivity of hair cells (HCs) is conferred by the aymmetric apical hair bundle, comprised of a kinocilium and stereocilia staircase. The mother centriole (MC) forms the base of the kinocilium and the stereocilia develop adjacent to it. Previously, we showed that transcription factor Emx2 reverses hair bundle orientation and its expression in the mouse vestibular utricle is restricted, resulting in two regions of opposite bundle orientation (Jiang et al., 2017). Here, we investigated establishment of opposite bundle orientation in embryonic utricles by live-imaging GFP-labeled centrioles in HCs. The daughter centriole invariably migrated ahead of the MC from the center to their respective peripheral locations in HCs. Comparing HCs between utricular regions, centriole trajectories were similar but they migrated toward opposite directions, suggesting that Emx2 pre-patterned HCs prior to centriole migration. Ectopic Emx2, however, reversed centriole trajectory within hours during a critical time-window when centriole trajectory was responsive to Emx2.
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Affiliation(s)
- Yosuke Tona
- National Institute on Deafness and Other Communication Disorders, National Institutes of HealthBethesdaUnited States
| | - Doris K Wu
- National Institute on Deafness and Other Communication Disorders, National Institutes of HealthBethesdaUnited States
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16
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Gallaud E, Ramdas Nair A, Horsley N, Monnard A, Singh P, Pham TT, Salvador Garcia D, Ferrand A, Cabernard C. Dynamic centriolar localization of Polo and Centrobin in early mitosis primes centrosome asymmetry. PLoS Biol 2020; 18:e3000762. [PMID: 32760088 PMCID: PMC7433902 DOI: 10.1371/journal.pbio.3000762] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Revised: 08/18/2020] [Accepted: 07/13/2020] [Indexed: 01/30/2023] Open
Abstract
Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older "mother" and a younger "daughter" centriole. Stem cells either inherit the mother or daughter-centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-structured illumination microscopy (3D-SIM) and live-cell imaging, we show in fly neural stem cells (neuroblasts) that the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) accumulate on the daughter centriole during mitosis, thereby generating molecularly distinct mother and daughter centrioles before interphase. Cnb's asymmetric localization, potentially involving a direct relocalization mechanism, is regulated by Polo-mediated phosphorylation, whereas Polo's daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments, we propose that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation.
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Affiliation(s)
- Emmanuel Gallaud
- Biozentrum, University of Basel, Klingelbergstrasse, Basel, Switzerland
| | | | - Nicole Horsley
- Department of Biology, University of Washington, Life Science Building, Seattle, Washington State, United States of America
| | - Arnaud Monnard
- Biozentrum, University of Basel, Klingelbergstrasse, Basel, Switzerland
- Department of Biology, University of Washington, Life Science Building, Seattle, Washington State, United States of America
| | - Priyanka Singh
- Biozentrum, University of Basel, Klingelbergstrasse, Basel, Switzerland
| | - Tri Thanh Pham
- Department of Biology, University of Washington, Life Science Building, Seattle, Washington State, United States of America
| | | | - Alexia Ferrand
- Biozentrum, University of Basel, Klingelbergstrasse, Basel, Switzerland
| | - Clemens Cabernard
- Department of Biology, University of Washington, Life Science Building, Seattle, Washington State, United States of America
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17
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Acute inhibition of centriolar satellite function and positioning reveals their functions at the primary cilium. PLoS Biol 2020; 18:e3000679. [PMID: 32555591 PMCID: PMC7326281 DOI: 10.1371/journal.pbio.3000679] [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: 01/30/2020] [Revised: 06/30/2020] [Accepted: 06/04/2020] [Indexed: 12/12/2022] Open
Abstract
Centriolar satellites are dynamic, membraneless granules composed of over 200 proteins. They store, modify, and traffic centrosome and primary cilium proteins, and help to regulate both the biogenesis and some functions of centrosomes and cilium. In most cell types, satellites cluster around the perinuclear centrosome, but their integrity and cellular distribution are dynamically remodeled in response to different stimuli, such as cell cycle cues. Dissecting the specific and temporal functions and mechanisms of satellites and how these are influenced by their cellular positioning and dynamics has been challenging using genetic approaches, particularly in ciliated and proliferating cells. To address this, we developed a chemical-based trafficking assay to rapidly and efficiently redistribute satellites to either the cell periphery or center, and fuse them into stable clusters in a temporally controlled way. Induced satellite clustering at either the periphery or center resulted in antagonistic changes in the pericentrosomal levels of a subset of proteins, revealing a direct and selective role for their positioning in protein targeting and sequestration. Systematic analysis of the interactome of peripheral satellite clusters revealed enrichment of proteins implicated in cilium biogenesis and mitosis. Importantly, induction of peripheral satellite targeting in ciliated cells revealed a function for satellites not just for efficient cilium assembly but also in the maintenance of steady-state cilia and in cilia disassembly by regulating the structural integrity of the ciliary axoneme. Finally, perturbing satellite distribution and dynamics inhibited their mitotic dissolution, and mitotic progression was perturbed only in cells with centrosomal satellite clustering. Collectively, our results for the first time showed a direct link between satellite functions and their pericentrosomal clustering, suggested new mechanisms underlying satellite functions during cilium assembly, and provided a new tool for probing temporal satellite functions in different contexts What happens when centriolar satellites are not in the right place at the right time? By redistributing satellites to the periphery or center of the cell and assessing the consequences of their mispositioning, this study reveals novel functions for satellites during mitosis, cilium maintenance, and cilium disassembly and suggests new mechanisms.
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18
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Katsurahara K, Shiozaki A, Kosuga T, Kudou M, Shoda K, Arita T, Konishi H, Komatsu S, Kubota T, Fujiwara H, Okamoto K, Kishimoto M, Konishi E, Marunaka Y, Otsuji E. ANO9 Regulated Cell Cycle in Human Esophageal Squamous Cell Carcinoma. Ann Surg Oncol 2020; 27:3218-3230. [PMID: 32227267 DOI: 10.1245/s10434-020-08368-y] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2019] [Indexed: 12/14/2022]
Abstract
BACKGROUND Few studies have reported the function and activation mechanism of ANO9 in esophageal squamous cell carcinoma (ESCC). The current study aimed to investigate the role of ANO9 in the regulation of tumor progression. METHODS Knockdown experiments with human ESCC cell lines were performed using ANO9 siRNA, and the effects on cell proliferation, the cell cycle, apoptosis, and cellular movement were analyzed. Immunohistochemistry (IHC) analysis was performed on 57 primary tumor samples obtained from ESCC patients. RESULTS In an in vitro study, depletion of ANO9 reduced cell proliferation, invasion, and migration in KYSE150 and KYSE 790 cells. In the cell cycle analysis, depletion of ANO9 increased the number of cells in G0/G1 arrest. In addition, the knockdown of ANO9 increased apoptosis. The results of the microarray analysis indicated that various centrosome-related genes such as CEP120, CNTRL, and SPAST were up- or downregulated in ANO9-depleted KYSE150 cells. The IHC results showed that high expression of ANO9 was associated with poor prognosis. CONCLUSIONS The results of the current study suggest that ANO9 regulates the cell cycle via centrosome-related genes in ESCC.
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Affiliation(s)
- Keita Katsurahara
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Atsushi Shiozaki
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan.
| | - Toshiyuki Kosuga
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Michihiro Kudou
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Katsutoshi Shoda
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Tomohiro Arita
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Hirotaka Konishi
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Shuhei Komatsu
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Takeshi Kubota
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Hitoshi Fujiwara
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Kazuma Okamoto
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Mitsuo Kishimoto
- Department of Pathology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Eiichi Konishi
- Department of Pathology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Yoshinori Marunaka
- Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Kyoto, Japan.,Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan.,Research Center for Drug Discovery and Pharmaceutical Development Science, Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Japan.,International Research Center for Food Nutrition and Safety, College of Food and Biological Engineering, Jiangsu University, Zhenjiang, China
| | - Eigo Otsuji
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
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19
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Naslavsky N, Caplan S. Endocytic membrane trafficking in the control of centrosome function. Curr Opin Cell Biol 2020; 65:150-155. [PMID: 32143977 DOI: 10.1016/j.ceb.2020.01.009] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2019] [Revised: 01/15/2020] [Accepted: 01/17/2020] [Indexed: 12/15/2022]
Abstract
Until recently, endocytic trafficking and its regulators were thought to function almost exclusively on membrane-bound organelles and/or vesicles containing a lipid bilayer. Recent studies have demonstrated that endocytic regulatory proteins play much wider roles in trafficking regulation and influence a variety of nonendocytic pathways, including trafficking to/from mitochondria and peroxisomes. Moreover, new studies also suggest that endocytic regulators also control trafficking to and from cellular organelles that lack membranes, such as the centrosome. Although endocytic membrane trafficking (EMT) clearly impacts pathways downstream of the centrosome, such as ciliogenesis (including transport to and from cilia), mitotic spindle formation, and cytokinesis, relatively few studies have focused on the growing role for EMT more directly on centrosome biogenesis, maintenance and control throughout cell cycle, and centrosome duplication. Indeed, a growing number of endocytic regulatory proteins have been implicated in centrosome regulation, including various Rab proteins (among them Rab11) and the leucine-rich repeat kinase 2. In this review, we will examine the relationship between centrosomes and EMT, focusing primarily on how EMT directly influences the centrosome.
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Affiliation(s)
- Naava Naslavsky
- The Department of Biochemistry and Molecular Biology and Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, 68198-5870, United States
| | - Steve Caplan
- The Department of Biochemistry and Molecular Biology and Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, 68198-5870, United States.
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20
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Nanjundappa R, Kong D, Shim K, Stearns T, Brody SL, Loncarek J, Mahjoub MR. Regulation of cilia abundance in multiciliated cells. eLife 2019; 8:e44039. [PMID: 31025935 PMCID: PMC6504233 DOI: 10.7554/elife.44039] [Citation(s) in RCA: 47] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2018] [Accepted: 04/25/2019] [Indexed: 12/14/2022] Open
Abstract
Multiciliated cells (MCC) contain hundreds of motile cilia used to propel fluid over their surface. To template these cilia, each MCC produces between 100-600 centrioles by a process termed centriole amplification. Yet, how MCC regulate the precise number of centrioles and cilia remains unknown. Airway progenitor cells contain two parental centrioles (PC) and form structures called deuterosomes that nucleate centrioles during amplification. Using an ex vivo airway culture model, we show that ablation of PC does not perturb deuterosome formation and centriole amplification. In contrast, loss of PC caused an increase in deuterosome and centriole abundance, highlighting the presence of a compensatory mechanism. Quantification of centriole abundance in vitro and in vivo identified a linear relationship between surface area and centriole number. By manipulating cell size, we discovered that centriole number scales with surface area. Our results demonstrate that a cell-intrinsic surface area-dependent mechanism controls centriole and cilia abundance in multiciliated cells.
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Affiliation(s)
- Rashmi Nanjundappa
- Nephrology Division, Department of MedicineWashington UniversitySt LouisUnited States
| | - Dong Kong
- Center for Cancer Research, National Cancer InstituteFrederickUnited States
| | - Kyuhwan Shim
- Nephrology Division, Department of MedicineWashington UniversitySt LouisUnited States
| | - Tim Stearns
- Department of BiologyStanford UniversityStanfordUnited States
| | - Steven L Brody
- Pulmonary Division, Department of MedicineWashington UniversitySt LouisUnited States
| | - Jadranka Loncarek
- Center for Cancer Research, National Cancer InstituteFrederickUnited States
| | - Moe R Mahjoub
- Nephrology Division, Department of MedicineWashington UniversitySt LouisUnited States
- Department of Cell Biology and PhysiologyWashington UniversitySt LouisUnited States
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21
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Tsai JJ, Hsu WB, Liu JH, Chang CW, Tang TK. CEP120 interacts with C2CD3 and Talpid3 and is required for centriole appendage assembly and ciliogenesis. Sci Rep 2019; 9:6037. [PMID: 30988386 PMCID: PMC6465297 DOI: 10.1038/s41598-019-42577-0] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2018] [Accepted: 04/03/2019] [Indexed: 12/19/2022] Open
Abstract
Centrosomal protein 120 (CEP120) was originally identified as a daughter centriole-enriched protein that participates in centriole elongation. Recent studies showed that CEP120 gene mutations cause complex ciliopathy phenotypes in humans, including Joubert syndrome and Jeune asphyxiating thoracic dystrophy, suggesting that CEP120 plays an additional role in ciliogenesis. To investigate the potential roles of CEP120 in centriole elongation and cilia formation, we knocked out the CEP120 gene in p53-deficient RPE1 cells using the CRISPR/Cas9 editing system, and performed various analyses. We herein report that loss of CEP120 produces short centrioles with no apparent distal and subdistal appendages. CEP120 knockout was also associated with defective centriole elongation, impaired recruitment of C2CD3 and Talpid3 to the distal ends of centrioles, and consequent defects in centriole appendage assembly and cilia formation. Interestingly, wild-type CEP120 interacts with C2CD3 and Talpid3, whereas a disease-associated CEP120 mutant (I975S) has a low affinity for C2CD3 binding and perturbs cilia assembly. Together, our findings reveal a novel role of CEP120 in ciliogenesis by showing that it interacts with C2CD3 and Talpid3 to assemble centriole appendages and by illuminating the molecular mechanism through which the CEP120 (I975S) mutation causes complex ciliopathies.
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Affiliation(s)
- Jhih-Jie Tsai
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Wen-Bin Hsu
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Jia-Hua Liu
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Ching-Wen Chang
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Tang K Tang
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
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22
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Galati DF, Sullivan KD, Pham AT, Espinosa JM, Pearson CG. Trisomy 21 Represses Cilia Formation and Function. Dev Cell 2018; 46:641-650.e6. [PMID: 30100262 PMCID: PMC6557141 DOI: 10.1016/j.devcel.2018.07.008] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2018] [Revised: 06/15/2018] [Accepted: 07/10/2018] [Indexed: 12/22/2022]
Abstract
Trisomy 21 (T21) is the most prevalent human chromosomal disorder, causing a range of cardiovascular, musculoskeletal, and neurological abnormalities. However, the cellular processes disrupted by T21 are poorly understood. Consistent with the clinical overlap between T21 and ciliopathies, we discovered that T21 disrupts cilia formation and signaling. Cilia defects arise from increased expression of Pericentrin, a centrosome scaffold and trafficking protein encoded on chromosome 21. Elevated Pericentrin is necessary and sufficient for T21 cilia defects. Pericentrin accumulates at centrosomes and dramatically in the cytoplasm surrounding centrosomes. Centrosome Pericentrin recruits more γ-tubulin and enhances microtubules, whereas cytoplasmic Pericentrin assembles into large foci that do not efficiently traffic. Moreover, the Pericentrin-associated cilia assembly factor IFT20 and the ciliary signaling molecule Smoothened do not efficiently traffic to centrosomes and cilia. Thus, increased centrosome protein dosage produces ciliopathy-like outcomes in T21 cells by decreasing trafficking between the cytoplasm, centrosomes, and cilia.
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Affiliation(s)
- Domenico F Galati
- Department of Cell and Developmental Biology, University of Colorado-Anschutz Medical Campus, Aurora, CO 80045, USA; Linda Crnic Institute for Down Syndrome, University of Colorado-Anschutz Medical Campus, Aurora, CO 80045, USA.
| | - Kelly D Sullivan
- Department of Pharmacology, University of Colorado-Anschutz Medical Campus, Aurora, CO 80045, USA; Linda Crnic Institute for Down Syndrome, University of Colorado-Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Andrew T Pham
- Department of Cell and Developmental Biology, University of Colorado-Anschutz Medical Campus, Aurora, CO 80045, USA; Linda Crnic Institute for Down Syndrome, University of Colorado-Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Joaquin M Espinosa
- Department of Pharmacology, University of Colorado-Anschutz Medical Campus, Aurora, CO 80045, USA; Linda Crnic Institute for Down Syndrome, University of Colorado-Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Chad G Pearson
- Department of Cell and Developmental Biology, University of Colorado-Anschutz Medical Campus, Aurora, CO 80045, USA; Linda Crnic Institute for Down Syndrome, University of Colorado-Anschutz Medical Campus, Aurora, CO 80045, USA.
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