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Zhang L, Zhang X, Hsieh LS, Lin TV, Bordey A. Rab27a-Dependent Paracrine Communication Controls Dendritic Spine Formation and Sensory Responses in the Barrel Cortex. Cells 2021; 10:cells10030622. [PMID: 33799820 PMCID: PMC8000154 DOI: 10.3390/cells10030622] [Citation(s) in RCA: 4] [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] [Received: 02/25/2021] [Revised: 03/04/2021] [Accepted: 03/05/2021] [Indexed: 11/20/2022] Open
Abstract
Rab27a is an evolutionarily conserved small GTPase that regulates vesicle trafficking, and copy number variants of RAB27a are associated with increased risk of autism. However, the function of Rab27a on brain development is unknown. Here, we identified a form of paracrine communication that regulates spine development between distinct populations of developing cortical neurons. In the developing somatosensory cortex of mice, we show that decreasing Rab27a levels in late-born pyramidal neurons destined for layer (L) 2/3 had no cell-autonomous effect on their synaptic integration but increased excitatory synaptic transmission onto L4 neurons that receive somatosensory information. This effect resulted in an increased number of L4 neurons activated by whisker stimulation in juvenile mice. In addition, we found that Rab27a, the level of which decreases as neurons mature, regulates the release of small extracellular vesicles (sEVs) in developing neurons in vitro and decreasing Rab27a levels led to the accumulation of CD63-positive vesicular compartments in L2/3 neurons in vivo. Together, our study reveals that Rab27a-mediated paracrine communication regulates the development of synaptic connectivity, ultimately tuning responses to sensory stimulation, possibly via controlling the release of sEVs.
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Affiliation(s)
- Longbo Zhang
- Departments of Neurosurgery, and Cellular & Molecular Physiology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT 06520-8082, USA; (L.Z.); (L.S.H.); (T.V.L.)
| | - Xiaobing Zhang
- Department of Psychology, Florida State University, Tallahassee, FL 32306, USA;
| | - Lawrence S. Hsieh
- Departments of Neurosurgery, and Cellular & Molecular Physiology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT 06520-8082, USA; (L.Z.); (L.S.H.); (T.V.L.)
| | - Tiffany V. Lin
- Departments of Neurosurgery, and Cellular & Molecular Physiology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT 06520-8082, USA; (L.Z.); (L.S.H.); (T.V.L.)
| | - Angélique Bordey
- Departments of Neurosurgery, and Cellular & Molecular Physiology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT 06520-8082, USA; (L.Z.); (L.S.H.); (T.V.L.)
- Correspondence: ; Tel.: +1-203-737-2515; Fax: +1-203-737-2159
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2
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Fu R, Edman MC, Hamm-Alvarez SF. Rab27a Contributes to Cathepsin S Secretion in Lacrimal Gland Acinar Cells. Int J Mol Sci 2021; 22:1630. [PMID: 33562815 PMCID: PMC7914720 DOI: 10.3390/ijms22041630] [Citation(s) in RCA: 4] [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] [Received: 01/07/2021] [Revised: 01/30/2021] [Accepted: 02/02/2021] [Indexed: 02/06/2023] Open
Abstract
Altered lacrimal gland (LG) secretion is a feature of autoimmune dacryoadenitis in Sjögren's syndrome (SS). Cathepsin S (CTSS) is increased in tears of SS patients, which may contribute to disease. Rab3D and Rab27a/b isoforms are effectors of exocytosis in LG, but Rab27a is poorly studied. To investigate whether Rab27a mediates CTSS secretion, we utilized quantitative confocal fluorescence microscopy of LG from SS-model male NOD and control male BALB/c mice, showing that Rab27a-enriched vesicles containing CTSS were increased in NOD mouse LG. Live-cell imaging of cultured lacrimal gland acinar cells (LGAC) transduced with adenovirus encoding wild-type (WT) mCFP-Rab27a revealed carbachol-stimulated fusion and depletion of mCFP-Rab27a-enriched vesicles. LGAC transduced with dominant-negative (DN) mCFP-Rab27a exhibited significantly reduced carbachol-stimulated CTSS secretion by 0.5-fold and β-hexosaminidase by 0.3-fold, relative to stimulated LGAC transduced with WT mCFP-Rab27a. Colocalization of Rab27a and endolysosomal markers (Rab7, Lamp2) with the apical membrane was increased in both stimulated BALB/c and NOD mouse LG, but the extent of colocalization was much greater in NOD mouse LG. Following stimulation, Rab27a colocalization with endolysosomal membranes was decreased. In conclusion, Rab27a participates in CTSS secretion in LGAC though the major regulated pathway, and through a novel endolysosomal pathway that is increased in SS.
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Affiliation(s)
- Runzhong Fu
- Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA;
- Department of Ophthalmology, Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA;
| | - Maria C. Edman
- Department of Ophthalmology, Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA;
| | - Sarah F. Hamm-Alvarez
- Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA;
- Department of Ophthalmology, Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA;
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3
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Eguizabal C, Herrera L, Inglés-Ferrándiz M, Izpisua Belmonte JC. Treating primary immunodeficiencies with defects in NK cells: from stem cell therapy to gene editing. Stem Cell Res Ther 2020; 11:453. [PMID: 33109263 PMCID: PMC7590703 DOI: 10.1186/s13287-020-01964-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2020] [Accepted: 10/05/2020] [Indexed: 12/29/2022] Open
Abstract
Primary immunodeficiency diseases (PIDs) are rare diseases that are characterized by genetic mutations that damage immunological function, defense, or both. Some of these rare diseases are caused by aberrations in the normal development of natural killer cells (NKs) or affect their lytic synapse. The pathogenesis of these types of diseases as well as the processes underlying target recognition by human NK cells is not well understood. Utilizing induced pluripotent stem cells (iPSCs) will aid in the study of human disorders, especially in the PIDs with defects in NK cells for PID disease modeling. This, together with genome editing technology, makes it possible for us to facilitate the discovery of future therapeutics and/or cell therapy treatments for these patients, because, to date, the only curative treatment available in the most severe cases is hematopoietic stem cell transplantation (HSCT). Recent progress in gene editing technology using CRISPR/Cas9 has significantly increased our capability to precisely modify target sites in the human genome. Among the many tools available for us to study human PIDs, disease- and patient-specific iPSCs together with gene editing offer unique and exceptional methodologies to gain deeper and more thorough understanding of these diseases as well as develop possible alternative treatment strategies. In this review, we will discuss some immunodeficiency disorders affecting NK cell function, such as classical NK deficiencies (CNKD), functional NK deficiencies (FNKD), and PIDs with involving NK cells as well as strategies to model and correct these diseases for further study and possible avenues for future therapies.
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Affiliation(s)
- C Eguizabal
- Cell Therapy, Stem Cells and Tissues Group, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain.
- Research Unit, Basque Center for Blood Transfusion and Human Tissues, Osakidetza, Galdakao, Spain.
| | - L Herrera
- Cell Therapy, Stem Cells and Tissues Group, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain
- Research Unit, Basque Center for Blood Transfusion and Human Tissues, Osakidetza, Galdakao, Spain
| | - M Inglés-Ferrándiz
- Cell Therapy, Stem Cells and Tissues Group, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain
- Research Unit, Basque Center for Blood Transfusion and Human Tissues, Osakidetza, Galdakao, Spain
| | - J C Izpisua Belmonte
- Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA, 93027, USA
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Zhou W, Zheng X, Cheng C, Guo G, Zhong Y, Liu W, Liu K, Chen Y, Liu S, Liu S. Rab27a deletion impairs the therapeutic potential of endothelial progenitor cells for myocardial infarction. Mol Cell Biochem 2020; 476:797-807. [PMID: 33095380 DOI: 10.1007/s11010-020-03945-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2020] [Accepted: 10/10/2020] [Indexed: 12/14/2022]
Abstract
Endothelial progenitor cell (EPC) transplantation has shown advantages in the treatment of myocardial infarction (MI) in animal models and clinical trials through mechanisms of direct intercellular contacts, autocrine, and paracrine. However, the effects of EPC transplantation for MI treatment remain controversial and the underlying mechanisms have not been fully elucidated. Here, we explored the role of Rab27a in the therapeutic potential of EPC transplantation in MI. We found that Rab27a knockout impaired the viability, and reduced the proliferation and tube formation function of ECPs. The recovery of cardiac function and improvement of ventricular remodeling from EPCs transplantation were significantly damaged by Rab27a deletion in vivo. Rab27a deletion inhibited the protein expression of phosphoinositide 3-kinase (PI3K) and cyclin D1 and the phosphorylation levels of Akt and FoxO3a. Therefore, Rab27a knockout suppressed the PI3K-Akt-FoxO3a/cyclin D1 signaling pathway. Furthermore, Rab27a ablation dramatically reduced exosome release in EPCs. These results demonstrated that Rab27a plays an essential role in EPC functions. The elucidation of this mechanism provides novel insights into EPC transplantation as a promising treatment for post-MI injuries.
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Affiliation(s)
- Wenyi Zhou
- Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, State Key Laboratory of Respiratory Disease, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, People's Republic of China
| | - Xuefei Zheng
- Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, State Key Laboratory of Respiratory Disease, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, People's Republic of China
| | - Chuanfang Cheng
- Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, State Key Laboratory of Respiratory Disease, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, People's Republic of China
| | - Guixian Guo
- Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, State Key Laboratory of Respiratory Disease, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, People's Republic of China
| | - Yun Zhong
- Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, State Key Laboratory of Respiratory Disease, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, People's Republic of China
| | - Weihua Liu
- Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, State Key Laboratory of Respiratory Disease, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, People's Republic of China
| | - Kefeng Liu
- Department of Histology and Embryology, School of Basic Medical Sciences, Xiangnan University, Chenzhou, Hunan, 423000, People's Republic of China
| | - Yanfang Chen
- Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH, 45435, USA
| | - Shiming Liu
- Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, State Key Laboratory of Respiratory Disease, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, People's Republic of China.
| | - Shaojun Liu
- Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, State Key Laboratory of Respiratory Disease, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, People's Republic of China.
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5
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Rab27a Contributes to the Processing of Inflammatory Pain in Mice. Cells 2020; 9:cells9061488. [PMID: 32570938 PMCID: PMC7349490 DOI: 10.3390/cells9061488] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 06/13/2020] [Accepted: 06/15/2020] [Indexed: 12/26/2022] Open
Abstract
Tissue injury and inflammation may result in chronic pain, a severe debilitating disease that is associated with great impairment of quality of life. An increasing body of evidence indicates that members of the Rab family of small GTPases contribute to pain processing; however, their specific functions remain poorly understood. Here, we found using immunofluorescence staining and in situ hybridization that the small GTPase Rab27a is highly expressed in sensory neurons and in the superficial dorsal horn of the spinal cord of mice. Rab27a mutant mice, which carry a single-nucleotide missense mutation of Rab27a leading to the expression of a nonfunctional protein, show reduced mechanical hyperalgesia and spontaneous pain behavior in inflammatory pain models, while their responses to acute noxious mechanical and thermal stimuli is not affected. Our study uncovers a previously unrecognized function of Rab27a in the processing of persistent inflammatory pain in mice.
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6
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Nishioka S, Wu PH, Yakabe T, Giaccia AJ, Le QT, Aoyama H, Shimizu S, Shirato H, Onodera Y, Nam JM. Rab27b contributes to radioresistance and exerts a paracrine effect via epiregulin in glioblastoma. Neurooncol Adv 2020; 2:vdaa091. [PMID: 33409495 PMCID: PMC7770522 DOI: 10.1093/noajnl/vdaa091] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
BACKGROUND Radiotherapy is the standard treatment for glioblastoma (GBM). However, radioresistance of GBM cells leads to recurrence and poor patient prognosis. Recent studies suggest that secretion factors have important roles in radioresistance of tumor cells. This study aims to determine whether Rab27b, a small GTPase involved in secretory vesicle trafficking, plays a role in radioresistance of GBM. METHODS Microarray analysis, cell viability analysis, apoptosis assay, immunostaining, and in vivo experiments were performed to assess the effect of Rab27b on radioresistance of GBM. We further investigated paracrine effects mediated by Rab27b after X-ray irradiation using coculture systems of glioma cell lines. RESULTS Rab27b was specifically upregulated in irradiated U87MG cells. Furthermore, Rab27b knockdown decreased the proliferation of GBM cells after irradiation. Knockdown of Rab27b in U87MG cells combined with radiation treatment suppressed orthotopic tumor growth in the mouse brain and prolonged the survival of recipient mice. Interestingly, the co-upregulation of Rab27b and epiregulin (EREG), a member of the epidermal growth factor (EGF) family, correlated with radioresistance in glioma cell lines. Additionally, EREG, which was secreted from U87MG cells via Rab27b-mediated mechanism, activated EGF receptor and contributed to H4 cell proliferation in a paracrine manner. CONCLUSIONS Our results show that Rab27b mediates the radioresistance of highly malignant GBM cells. Rab27b promotes the proliferation of adjacent cells through EREG-mediated paracrine signaling after irradiation. Thus, the Rab27b-EREG pathway is a novel potential target to improve the efficacy of radiotherapy in GBM.
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Affiliation(s)
- Soichiro Nishioka
- Molecular and Cellular Dynamics Research, Graduate School of Biomedical Science and Engineering, Hokkaido University, Sapporo, Japan
- Global Center for Biomedical Science and Engineering, Faculty of Medicine, Hokkaido University, Sapporo, Japan
| | - Ping-Hsiu Wu
- Global Center for Biomedical Science and Engineering, Faculty of Medicine, Hokkaido University, Sapporo, Japan
| | | | - Amato J Giaccia
- Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California, USA
| | - Quynh-Thu Le
- Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California, USA
| | - Hidefumi Aoyama
- Department of Radiation Oncology, Faculty of Medicine, Hokkaido University, Sapporo, Japan
| | - Shinichi Shimizu
- Global Center for Biomedical Science and Engineering, Faculty of Medicine, Hokkaido University, Sapporo, Japan
- Department of Radiation Medical Science and Engineering, Faculty of Medicine, Hokkaido University, Sapporo, Japan
| | - Hiroki Shirato
- Global Center for Biomedical Science and Engineering, Faculty of Medicine, Hokkaido University, Sapporo, Japan
| | - Yasuhito Onodera
- Global Center for Biomedical Science and Engineering, Faculty of Medicine, Hokkaido University, Sapporo, Japan
- Department of Molecular Biology, Faculty of Medicine, Hokkaido University, Sapporo, Japan
| | - Jin-Min Nam
- Global Center for Biomedical Science and Engineering, Faculty of Medicine, Hokkaido University, Sapporo, Japan
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Dynamins 2 and 3 control the migration of human megakaryocytes by regulating CXCR4 surface expression and ITGB1 activity. Blood Adv 2019; 2:3540-3552. [PMID: 30538113 DOI: 10.1182/bloodadvances.2018021923] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Accepted: 11/04/2018] [Indexed: 12/14/2022] Open
Abstract
Megakaryocyte (MK) migration from the bone marrow periosteal niche toward the vascular niche is a prerequisite for proplatelet extension and release into the circulation. The mechanism for this highly coordinated process is poorly understood. Here we show that dynasore (DNSR), a small-molecule inhibitor of dynamins (DNMs), or short hairpin RNA knockdown of DNM2 and DNM3 impairs directional migration in a human MK cell line or MKs derived from cultured CD34+ cells. Because cell migration requires actin cytoskeletal rearrangements, we measured actin polymerization and the activity of cytoskeleton regulator RhoA and found them to be decreased after inhibition of DNM2 and DNM3. Because SDF-1α is important for hematopoiesis, we studied the expression of its receptor CXCR4 in DNSR-treated cells. CXCR4 expression on the cell surface was increased, at least partially because of slower endocytosis and internalization after SDF-1α treatment. Combined inhibition of DNM2 and DNM3 or forced expression of dominant-negative Dnm2-K44A or GTPase-defective DNM3 diminished β1 integrin (ITGB1) activity. DNSR-treated MKs showed an abnormally clustered staining pattern of Rab11, a marker of recycling endosomes. This suggests decreased recruitment of the recycling pathway in DNSR-treated cells. Altogether, we show that the GTPase activity of DNMs, which governs endocytosis and regulates cell receptor trafficking, exerts control on MK migration toward SDF-1α gradients, such as those originating from the vascular niche. DNMs play a critical role in MKs by triggering membrane-cytoskeleton rearrangements downstream of CXCR4 and integrins.
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KIBRA controls exosome secretion via inhibiting the proteasomal degradation of Rab27a. Nat Commun 2019; 10:1639. [PMID: 30967557 PMCID: PMC6456494 DOI: 10.1038/s41467-019-09720-x] [Citation(s) in RCA: 160] [Impact Index Per Article: 26.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2018] [Accepted: 03/22/2019] [Indexed: 02/07/2023] Open
Abstract
Exosomes are nanosized membrane vesicles released from cells after fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) and play important roles in intercellular communication and numerous biological processes. However, the molecular mechanisms regulating exosome secretion remain poorly understood. Here we identify KIBRA as an adaptor-like protein that stabilizes Rab27a, which in turn controls exosome secretion both in vitro and in vivo. Knockdown or overexpression of KIBRA in neuronal and podocyte cell lines leads to a decrease or increase of exosome secretion, respectively, and KIBRA depletion increases MVB size and number. Comparing protein profiles between KIBRA knockout and wild-type mouse brain showed significantly decreased Rab27a, a small GTPase that regulates MVB-PM docking. Rab27a is stabilized by interacting with KIBRA, which prevents ubiquitination and degradation via the ubiquitin-proteasome pathway. In conclusion, we show that KIBRA controls exosome secretion via inhibiting the proteasomal degradation of Rab27a. Exosomes are intercellular signaling vesicles created by fusion of multivesicular bodies (MVBs) and the plasma membrane (PM), but secretory regulation is ill-defined. Song et al. show that KIBRA controls exosome secretion by protecting Rab27a from proteasomal degradation, promoting MVB-PM docking.
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Abstract
Our understanding of fundamental biological processes within platelets is continually evolving. A critical feature of platelet biology relates to the intricate uptake, packaging and release of bioactive cargo from storage vesicles, essential in mediating a range of classical (haemostasis/thrombosis) and non-classical (regeneration/inflammation/metastasis) roles platelets assume. Pivotal to the molecular control of these vesicle trafficking events are the small GTPases of the Ras superfamily, which function as spatially distinct, molecular switches controlling essential cellular processes. Herein, we specifically focus on members of the Rab, Arf and Ras subfamilies, which comprise over 130 members and platelet proteomic datasets suggest that more than half of these are expressed in human platelets. We provide an update of current literature relating to trafficking roles for these GTPases in platelets, particularly regarding endocytic and exocytic events, but also vesicle biogenesis and provide speculative argument for roles that other related GTPases and regulatory proteins may adopt in platelets. Advances in our understanding of small GTPase function in the anucleate platelet has been hampered by the lack of specific molecular tools, but it is anticipated that this will be greatly accelerated in the years ahead and will be crucial to the identification of novel therapeutic targets controlling different platelet processes.
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Affiliation(s)
- Tony G Walsh
- a From the School of Physiology, Pharmacology and Neuroscience, Biomedical Sciences Building , University of Bristol , Bristol , UK
| | - Yong Li
- a From the School of Physiology, Pharmacology and Neuroscience, Biomedical Sciences Building , University of Bristol , Bristol , UK
| | - Andreas Wersäll
- a From the School of Physiology, Pharmacology and Neuroscience, Biomedical Sciences Building , University of Bristol , Bristol , UK
| | - Alastair W Poole
- a From the School of Physiology, Pharmacology and Neuroscience, Biomedical Sciences Building , University of Bristol , Bristol , UK
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10
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Li Z, Fang R, Fang J, He S, Liu T. Functional implications of Rab27 GTPases in Cancer. Cell Commun Signal 2018; 16:44. [PMID: 30081925 PMCID: PMC6080553 DOI: 10.1186/s12964-018-0255-9] [Citation(s) in RCA: 56] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2018] [Accepted: 07/27/2018] [Indexed: 12/14/2022] Open
Abstract
Background The Rab27 family of small GTPases promotes the progression of breast cancer, melanoma, and other human cancers. In this review, we discuss the role of Rab27 GTPases in cancer progression and the potential applications of these targets in cancer treatment. Main body Elevated expression of Rab27 GTPases is associated with poor prognosis and cancer metastasis. Moreover, these GTPases govern a variety of oncogenic functions, including cell proliferation, cell motility, and chemosensitivity. In addition, small GTPases promote tumor growth and metastasis by enhancing exosome secretion, which alters intracellular microRNA levels, signaling molecule expression, and the tumor microenvironment. Conclusion Rab27 GTPases may have applications as prognostic markers and therapeutic targets in cancer treatment.
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Affiliation(s)
- Zhihong Li
- Department of Orthopaedics, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Rui Fang
- Department of Orthopaedics, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Jia Fang
- Department of Neurology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Shasha He
- Department of Oncology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, People's Republic of China.
| | - Tang Liu
- Department of Orthopaedics, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China.
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11
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Banworth MJ, Li G. Consequences of Rab GTPase dysfunction in genetic or acquired human diseases. Small GTPases 2017; 9:158-181. [PMID: 29239692 DOI: 10.1080/21541248.2017.1397833] [Citation(s) in RCA: 43] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Rab GTPases are important regulators of intracellular membrane trafficking in eukaryotes. Both activating and inactivating mutations in Rab genes have been identified and implicated in human diseases ranging from neurological disorders to cancer. In addition, altered Rab expression is often associated with disease prognosis. As such, the study of diseases associated with Rabs or Rab-interacting proteins has shed light on the important role of intracellular membrane trafficking in disease etiology. In this review, we cover recent advances in the field with an emphasis on cellular mechanisms.
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Affiliation(s)
- Marcellus J Banworth
- a Department of Biochemistry and Molecular Biology , University of Oklahoma Health Sciences Center , Oklahoma City , OK , USA
| | - Guangpu Li
- a Department of Biochemistry and Molecular Biology , University of Oklahoma Health Sciences Center , Oklahoma City , OK , USA
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12
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Ramadass M, Catz SD. Molecular mechanisms regulating secretory organelles and endosomes in neutrophils and their implications for inflammation. Immunol Rev 2017; 273:249-65. [PMID: 27558339 DOI: 10.1111/imr.12452] [Citation(s) in RCA: 53] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Neutrophils constitute the first line of cellular defense against invading microorganisms and modulate the subsequent innate and adaptive immune responses. In order to execute a rapid and precise response to infections, neutrophils rely on preformed effector molecules stored in a variety of intracellular granules. Neutrophil granules contain microbicidal factors, the membrane-bound components of the respiratory burst oxidase, membrane-bound adhesion molecules, and receptors that facilitate the execution of all neutrophil functions including adhesion, transmigration, phagocytosis, degranulation, and neutrophil extracellular trap formation. The rapid mobilization of intracellular organelles is regulated by vesicular trafficking mechanisms controlled by effector molecules that include small GTPases and their interacting proteins. In this review, we focus on recent discoveries of mechanistic processes that are at center stage of the regulation of neutrophil function, highlighting the discrete and selective pathways controlled by trafficking modulators. In particular, we describe novel pathways controlled by the Rab27a effectors JFC1 and Munc13-4 in the regulation of degranulation, reactive oxygen species and neutrophil extracellular trap production, and endolysosomal signaling. Finally, we discuss the importance of understanding these molecular mechanisms in order to design novel approaches to modulate neutrophil-mediated inflammatory processes in a targeted fashion.
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Affiliation(s)
- Mahalakshmi Ramadass
- Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA
| | - Sergio D Catz
- Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA
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13
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Kurz ARM, Pruenster M, Rohwedder I, Ramadass M, Schäfer K, Harrison U, Gouveia G, Nussbaum C, Immler R, Wiessner JR, Margraf A, Lim DS, Walzog B, Dietzel S, Moser M, Klein C, Vestweber D, Haas R, Catz SD, Sperandio M. MST1-dependent vesicle trafficking regulates neutrophil transmigration through the vascular basement membrane. J Clin Invest 2016; 126:4125-4139. [PMID: 27701149 DOI: 10.1172/jci87043] [Citation(s) in RCA: 48] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2016] [Accepted: 08/25/2016] [Indexed: 12/20/2022] Open
Abstract
Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20-like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1-/-) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1-/- neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1-/- neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency.
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Wankel B, Ouyang J, Guo X, Hadjiolova K, Miller J, Liao Y, Tham DKL, Romih R, Andrade LR, Gumper I, Simon JP, Sachdeva R, Tolmachova T, Seabra MC, Fukuda M, Schaeren-Wiemers N, Hong WJ, Sabatini DD, Wu XR, Kong X, Kreibich G, Rindler MJ, Sun TT. Sequential and compartmentalized action of Rabs, SNAREs, and MAL in the apical delivery of fusiform vesicles in urothelial umbrella cells. Mol Biol Cell 2016; 27:1621-34. [PMID: 27009205 PMCID: PMC4865319 DOI: 10.1091/mbc.e15-04-0230] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2015] [Accepted: 03/17/2016] [Indexed: 01/28/2023] Open
Abstract
As major urothelial differentiation products, uroplakins are targeted to the apical surface of umbrella cells. Via the sequential actions of Rabs 11, 8, and 27b and their effectors, uroplakin vesicles are transported to a subapical zone above a K20 network and fuse, via a SNARE-mediated and MAL-facilitated step, with the urothelial apical membrane. Uroplakins (UPs) are major differentiation products of urothelial umbrella cells and play important roles in forming the permeability barrier and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic Escherichia coli receptor. Although it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immunomicroscopy of normal and mutant mouse urothelia show that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently a Rab27b/Slp2-a complex mediated FV–membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also show that keratin 20 (K20), which forms a chicken-wire network ∼200 nm below the apical membrane and has hole sizes allowing FV passage, defines a subapical compartment containing FVs primed and strategically located for fusion. Finally, we show that Rab8/11 and Rab27b function in the same pathway, Rab27b knockout leads to uroplakin and Slp2-a destabilization, and Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking.
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Affiliation(s)
- Bret Wankel
- Department of Cell Biology, New York University School of Medicine, New York, NY10016
| | - Jiangyong Ouyang
- Department of Cell Biology, New York University School of Medicine, New York, NY10016
| | - Xuemei Guo
- Department of Cell Biology, New York University School of Medicine, New York, NY10016
| | - Krassimira Hadjiolova
- Department of Cell Biology, New York University School of Medicine, New York, NY10016
| | - Jeremy Miller
- Department of Cell Biology, New York University School of Medicine, New York, NY10016
| | - Yi Liao
- Department of Cell Biology, New York University School of Medicine, New York, NY10016
| | - Daniel Kai Long Tham
- Department of Cell Biology, New York University School of Medicine, New York, NY10016
| | - Rok Romih
- Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia
| | - Leonardo R Andrade
- Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil
| | - Iwona Gumper
- Department of Cell Biology, New York University School of Medicine, New York, NY10016
| | - Jean-Pierre Simon
- Department of Cell Biology, New York University School of Medicine, New York, NY10016
| | - Rakhee Sachdeva
- Department of Cell Biology, New York University School of Medicine, New York, NY10016
| | - Tanya Tolmachova
- Molecular and Cellular Medicine, Imperial College, London SW7 2AZ, United Kingdom
| | - Miguel C Seabra
- Molecular and Cellular Medicine, Imperial College, London SW7 2AZ, United Kingdom
| | - Mitsunori Fukuda
- Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578, Japan
| | - Nicole Schaeren-Wiemers
- Neurobiology Laboratory, Department of Biomedicine, University Hospital Basel, University of Basel, CH-4031 Basel, Switzerland
| | - Wan Jin Hong
- Cancer and Developmental Cell Biology Division, Institute of Molecular and Cell Biology, A*STAR, Biopolis, Singapore 138673
| | - David D Sabatini
- Department of Cell Biology, New York University School of Medicine, New York, NY10016
| | - Xue-Ru Wu
- Department of Urology, New York University School of Medicine, New York, NY10016
| | - Xiangpeng Kong
- Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY10016
| | - Gert Kreibich
- Department of Cell Biology, New York University School of Medicine, New York, NY10016
| | - Michael J Rindler
- Department of Cell Biology, New York University School of Medicine, New York, NY10016
| | - Tung-Tien Sun
- Department of Cell Biology, New York University School of Medicine, New York, NY10016 Department of Urology, New York University School of Medicine, New York, NY10016 Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY10016 Department of Dermatology, New York University School of Medicine, New York, NY10016
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15
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Higashio H, Satoh YI, Saino T. Mast cell degranulation is negatively regulated by the Munc13-4-binding small-guanosine triphosphatase Rab37. Sci Rep 2016; 6:22539. [PMID: 26931073 PMCID: PMC4773767 DOI: 10.1038/srep22539] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2015] [Accepted: 02/17/2016] [Indexed: 02/07/2023] Open
Abstract
Mast cell degranulation is regulated by the small guanosine triphosphatases (GTPases) Rab27a and Rab27b, which have distinct and opposing roles: Rab27b acts as a positive regulator through its effector protein Munc13-4, a non-neuronal isoform of the vesicle-priming Munc13 family of proteins, whereas Rab27a acts as a negative regulator through its effector protein melanophilin, by maintaining integrity of cortical filamentous actin (F-actin), a barrier to degranulation. Here we investigated the role of Rab37, one of the Rab GTPases assumed to be implicated in regulated secretion during mast cell degranulation. Using the RBL-2H3 mast cell line, we detected Rab37 on the secretory granules and found that antigen-induced degranulation was extensively increased by either knockdown of Rab37 or overexpression of a dominant-active Rab37 mutant. This hypersecretion phenotype in the Rab37-knockdown cells was suppressed by simultaneous knockdown of Rab27a and Rab27b or of Munc13-4, but not by disruption of cortical F-actin. We further found that Rab37 interacted with Munc13-4 in a GTP-independent manner and formed a Rab27-Munc13-4-Rab37 complex. These results suggest that Rab37 is a Munc13-4-binding protein that inhibits mast cell degranulation through its effector protein, by counteracting the vesicle-priming activity of the Rab27-Munc13-4 system.
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Affiliation(s)
- Hironori Higashio
- Department of Chemistry, Center for Liberal Arts and Sciences, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan
| | - Yoh-ichi Satoh
- Division of Cell Biology, Department of Anatomy, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan.,Department of Medical Education, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan
| | - Tomoyuki Saino
- Division of Cell Biology, Department of Anatomy, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan
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16
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Hou Y, Ernst SA, Lentz SI, Williams JA. Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion. Biochem Biophys Res Commun 2016; 471:610-5. [PMID: 26845357 DOI: 10.1016/j.bbrc.2016.01.180] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2016] [Accepted: 01/29/2016] [Indexed: 11/26/2022]
Abstract
Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D.
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Affiliation(s)
- Yanan Hou
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Stephen A Ernst
- Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Stephen I Lentz
- Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA
| | - John A Williams
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA; Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.
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Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion. PLoS One 2015; 10:e0125596. [PMID: 25951179 PMCID: PMC4423933 DOI: 10.1371/journal.pone.0125596] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2014] [Accepted: 03/16/2015] [Indexed: 01/24/2023] Open
Abstract
The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.
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Hemophagocytic lymphohistiocytosis caused by dominant-negative mutations in STXBP2 that inhibit SNARE-mediated membrane fusion. Blood 2015; 125:1566-77. [PMID: 25564401 DOI: 10.1182/blood-2014-11-610816] [Citation(s) in RCA: 91] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Familial hemophagocytic lymphohistiocytosis (F-HLH) and Griscelli syndrome type 2 (GS) are life-threatening immunodeficiencies characterized by impaired cytotoxic T lymphocyte (CTL) and natural killer (NK) cell lytic activity. In the majority of cases, these disorders are caused by biallelic inactivating germline mutations in genes such as RAB27A (GS) and PRF1, UNC13D, STX11, and STXBP2 (F-HLH). Although monoallelic (ie, heterozygous) mutations have been identified in certain patients, the clinical significance and molecular mechanisms by which these mutations influence CTL and NK cell function remain poorly understood. Here, we characterize 2 novel monoallelic hemophagocytic lymphohistiocytosis (HLH)-associated mutations affecting codon 65 of STXPB2, the gene encoding Munc18-2, a member of the SEC/MUNC18 family. Unlike previously described Munc18-2 mutants, Munc18-2(R65Q) and Munc18-2(R65W) retain the ability to interact with and stabilize syntaxin 11. However, presence of Munc18-2(R65Q/W) in patient-derived lymphocytes and forced expression in control CTLs and NK cells diminishes degranulation and cytotoxic activity. Mechanistic studies reveal that mutations affecting R65 hinder membrane fusion in vitro by arresting the late steps of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-complex assembly. Collectively, these results reveal a direct role for SEC/MUNC18 proteins in promoting SNARE-complex assembly in vivo and suggest that STXBP2 R65 mutations operate in a novel dominant-negative fashion to impair lytic granule fusion and contribute to HLH.
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19
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Chua CEL, Tang BL. The role of the small GTPase Rab31 in cancer. J Cell Mol Med 2014; 19:1-10. [PMID: 25472813 PMCID: PMC4288343 DOI: 10.1111/jcmm.12403] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2014] [Accepted: 07/18/2014] [Indexed: 12/28/2022] Open
Abstract
Members of the small GTPase family Rab are emerging as potentially important factors in cancer development and progression. A good number of Rabs have been implicated or associated with various human cancers, and much recent excitement has been associated with the roles of the Rab11 subfamily member Rab25 and its effector, the Rab coupling protein (RCP), in tumourigenesis and metastasis. In this review, we focus on a Rab5 subfamily member, Rab31, and its implicated role in cancer. Well recognized as a breast cancer marker with good prognostic value, recent findings have provided some insights as to the mechanism underlying Rab31's influence on oncogenesis. Levels of Oestrogen Receptor α (ERα)- responsive Rab31 could be elevated through stabilization of its transcript by the RNA binding protein HuR, or though activation by the oncoprotein mucin1-C (MUC1-C), which forms a transcriptional complex with ERα. Elevated Rab31 stabilizes MUC1-C levels in an auto-inductive loop that could lead to aberrant signalling and gene expression associated with cancer progression. Rab31 and its guanine nucleotide exchange factor GAPex-5 have, however, also been shown to enhance early endosome-late endosome transport and degradation of the epidermal growth factor receptor (EGFR). The multifaceted action and influences of Rab31 in cancer is discussed in the light of its new interacting partners and pathways.
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Affiliation(s)
- Christelle En Lin Chua
- Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore
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20
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A role for Rab27 in neutrophil chemotaxis and lung recruitment. BMC Cell Biol 2014; 15:39. [PMID: 25359237 PMCID: PMC4221698 DOI: 10.1186/s12860-014-0039-z] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2014] [Accepted: 10/16/2014] [Indexed: 01/08/2023] Open
Abstract
Background Neutrophils are a critical part of the innate immune system. Their ability to migrate into infected or injured tissues precedes their role in microbial killing and clearance. We have previously shown that Rab27a can promote neutrophil migration by facilitating uropod release through protease secretion from primary granule exocytosis at the cell rear. Rab27b has been implicated in primary granule exocytosis but its role in neutrophil migration has not been investigated. Results Here we found Rab27b to be expressed in bone marrow derived neutrophils and Rab27b knockout (Rab27b KO) along with Rab27a/b double knockout (Rab27DKO) neutrophils exhibited impaired transwell migration in vitro in response to chemokines MIP-2 and LTB4. Interestingly, no additional defect in migration was observed in Rab27DKO neutrophils compared with Rab27b KO neutrophils. In vivo, Rab27DKO mice displayed severe impairment in neutrophil recruitment to the lungs in a MIP-2 dependent model but not in an LPS dependent model. Conclusions These data taken together implicate Rab27b in the regulation of neutrophil chemotaxis, likely through the regulation of primary granule exocytosis.
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21
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Brinton LT, Sloane HS, Kester M, Kelly KA. Formation and role of exosomes in cancer. Cell Mol Life Sci 2014; 72:659-71. [PMID: 25336151 DOI: 10.1007/s00018-014-1764-3] [Citation(s) in RCA: 189] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2014] [Revised: 10/06/2014] [Accepted: 10/13/2014] [Indexed: 12/19/2022]
Abstract
Exosomes offer new insight into cancer biology with both diagnostic and therapeutic implications. Because of their cell-to-cell communication, exosomes influence tumor progression, metastasis, and therapeutic efficacy. They can be isolated from blood and other bodily fluids to reveal disease processes occurring within the body, including cancerous growth. In addition to being a reservoir of cancer biomarkers, they can be re-engineered to reinstate tumor immunity. Tumor exosomes interact with various cells of the microenvironment to confer tumor-advantageous changes that are responsible for stromal activation, induction of the angiogenic switch, increased vascular permeability, and immune escape. Exosomes also contribute to metastasis by aiding in the epithelial-to-mesenchymal transition and formation of the pre-metastatic niche. Furthermore, exosomes protect tumor cells from the cytotoxic effects of chemotherapy drugs and transfer chemoresistance properties to nearby cells. Thus, exosomes are essential to many lethal elements of cancer and it is important to understand their biogenesis and role in cancer.
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Affiliation(s)
- Lindsey T Brinton
- Department of Biomedical Engineering, University of Virginia, PO Box 800759 Health System, Charlottesville, VA, 22908, USA,
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22
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Honma M, Ikebuchi Y, Kariya Y, Suzuki H. Regulatory mechanisms of RANKL presentation to osteoclast precursors. Curr Osteoporos Rep 2014; 12:115-20. [PMID: 24477414 DOI: 10.1007/s11914-014-0189-0] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
It is important to understand the molecular mechanisms regulating osteoclast formation, as excess activation of osteoclasts is associated with various osteopenic disorders. Receptor activator of nuclear factor kappa B (RANKL) is a central player in osteoclastogenesis. Recent findings suggest that osteocytes are the major supplier of RANKL to osteoclast precursors. It has also been suggested that osteocyte cell death upregulates the RANKL/osteoprotegerin (OPG) ratio in viable osteocytes adjacent to apoptotic osteocytes in areas of bone microdamage, thus, contributing to localized osteoclast formation. Indeed, viable osteocytes can provide RANKL through direct interactions with osteoclast precursors at osteocyte dendritic processes. In addition, OPG tightly regulates RANKL cell surface presentation in osteocytes, which contributes to the inhibition of RANKL signaling, as well as the decoy receptor function of OPG. By contrast, the physiological role of RANKL in osteoblasts is yet to be clarified, although similar mechanisms of regulation are observed in both osteocytes and osteoblasts.
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Affiliation(s)
- Masashi Honma
- Department of Pharmacy, the University of Tokyo Hospital, Faculty of Medicine, the University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan,
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23
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Kim JD, Willetts L, Ochkur S, Srivastava N, Hamburg R, Shayeganpour A, Seabra MC, Lee JJ, Moqbel R, Lacy P. An essential role for Rab27a GTPase in eosinophil exocytosis. J Leukoc Biol 2013; 94:1265-74. [PMID: 23986549 DOI: 10.1189/jlb.0812431] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Eosinophil degranulation has been implicated in inflammatory processes associated with allergic asthma. Rab27a, a Rab-related GTPase, is a regulatory intracellular signaling molecule expressed in human eosinophils. We postulated that Rab27a regulates eosinophil degranulation. We investigated the role of Rab27a in eosinophil degranulation within the context of airway inflammation. Rab27a expression and localization in eosinophils were investigated by using subcellular fractionation combined with Western blot analysis, and the results were confirmed by immunofluorescence analysis of Rab27a and the granule membrane marker CD63. To determine the function of eosinophil Rab27a, we used Ashen mice, a strain of Rab27a-deficient animals. Ashen eosinophils were tested for degranulation in response to PAF and calcium ionophore by measuring released EPX activity. Airway EPX release was also determined by intratracheal injection of eosinophils into mice lacking EPX. Rab27a immunoreactivity colocalized with eosinophil crystalloid granules, as determined by subcellular fractionation and immunofluorescence analysis. PAF induced eosinophil degranulation in correlation with redistribution of Rab27a(+) structures, some of which colocalized with CD63(+) crystalloid granules at the cell membrane. Eosinophils from mice had significantly reduced EPX release compared with normal WT eosinophils, both in vitro and in vivo. In mouse models, Ashen mice demonstrated reduced EPX release in BAL fluid. These findings suggest that Rab27a has a key role in eosinophil degranulation. Furthermore, these findings have implications for Rab27a-dependent eosinophil degranulation in airway inflammation.
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Affiliation(s)
- John Dongil Kim
- 2.559 HMRC, Department of Medicine, University of Alberta, Edmonton, AB, T6G 2S2, Canada.
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24
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Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo. J Mol Med (Berl) 2013; 91:825-37. [PMID: 23756766 PMCID: PMC3695676 DOI: 10.1007/s00109-013-1006-4] [Citation(s) in RCA: 70] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2012] [Revised: 01/23/2013] [Accepted: 01/31/2013] [Indexed: 01/10/2023]
Abstract
Choroideremia (CHM) is an X-linked retinal degeneration of photoreceptors, the retinal pigment epithelium (RPE) and choroid caused by loss of function mutations in the CHM/REP1 gene that encodes Rab escort protein 1. As a slowly progressing monogenic retinal degeneration with a clearly identifiable phenotype and a reliable diagnosis, CHM is an ideal candidate for gene therapy. We developed a serotype 2 adeno-associated viral vector AAV2/2-CBA-REP1, which expresses REP1 under control of CMV-enhanced chicken β-actin promoter (CBA) augmented by a Woodchuck hepatitis virus post-transcriptional regulatory element. We show that the AAV2/2-CBA-REP1 vector provides strong and functional transgene expression in the D17 dog osteosarcoma cell line, CHM patient fibroblasts and CHM mouse RPE cells in vitro and in vivo. The ability to transduce human photoreceptors highly effectively with this expression cassette was confirmed in AAV2/2-CBA-GFP transduced human retinal explants ex vivo. Electroretinogram (ERG) analysis of AAV2/2-CBA-REP1 and AAV2/2-CBA-GFP-injected wild-type mouse eyes did not show toxic effects resulting from REP1 overexpression. Subretinal injections of AAV2/2-CBA-REP1 into CHM mouse retinas led to a significant increase in a- and b-wave of ERG responses in comparison to sham-injected eyes confirming that AAV2/2-CBA-REP1 is a promising vector suitable for choroideremia gene therapy in human clinical trials.
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25
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Hou Y, Chen X, Tolmachova T, Ernst SA, Williams JA. EPI64B acts as a GTPase-activating protein for Rab27B in pancreatic acinar cells. J Biol Chem 2013; 288:19548-57. [PMID: 23671284 DOI: 10.1074/jbc.m113.472134] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells.
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Affiliation(s)
- Yanan Hou
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109, USA
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26
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Park HH. Structural basis of membrane trafficking by Rab family small G protein. Int J Mol Sci 2013; 14:8912-23. [PMID: 23698755 PMCID: PMC3676764 DOI: 10.3390/ijms14058912] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2013] [Revised: 04/01/2013] [Accepted: 04/10/2013] [Indexed: 12/18/2022] Open
Abstract
The Ras-superfamily of small G proteins is a family of GTP hydrolases that is regulated by GTP/GDP binding states. One member of the Ras-superfamily, Rab, is involved in the regulation of vesicle trafficking, which is critical to endocytosis, biosynthesis, secretion, cell differentiation and cell growth. The active form of the Rab proteins, which contains GTP, can recruit specific binding partners, such as sorting adaptors, tethering factors, kinases, phosphatases and motor proteins, thereby influencing vesicle formation, transport, and tethering. Many Rab proteins share the same interacting partners and perform unique roles in specific locations. Because functional loss of the Rab pathways has been implicated in a variety of diseases, the Rab GTPase family has been extensively investigated. In this review, we summarize Rab GTPase- mediated membrane trafficking while focusing on the structures of Rab protein and Rab-effector complexes. This review provides detailed information that helps explain how the Rab GTPase family is involved in membrane trafficking.
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Affiliation(s)
- Hyun Ho Park
- School of Biotechnology and Graduate School of Biochemistry, Yeungnam University, Gyeongsan 712-749, Korea.
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Harper MT, van den Bosch MTJ, Hers I, Poole AW. Absence of platelet phenotype in mice lacking the motor protein myosin Va. PLoS One 2013; 8:e53239. [PMID: 23349704 PMCID: PMC3548825 DOI: 10.1371/journal.pone.0053239] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2012] [Accepted: 11/27/2012] [Indexed: 12/22/2022] Open
Abstract
Background The motor protein myosin Va plays an important role in the trafficking of intracellular vesicles. Mutation of the Myo5a gene causes Griscelli syndrome type 1 in humans and the dilute phenotype in mice, which are both characterised by pigment dilution and neurological defects as a result of impaired vesicle transport in melanocytes and neuroendocrine cells. The role of myosin Va in platelets is currently unknown. Rab27 has been shown to be associated with myosin Va cargo vesicles and is known to be important in platelet dense granule biogenesis and secretion, a crucial event in thrombus formation. Therefore, we hypothesised that myosin Va may regulate granule secretion or formation in platelets. Methodology/Principal Findings Platelet function was studied in vitro using a novel Myo5a gene deletion mouse model. Myo5a−/− platelets were devoid of myosin Va, as determined by immunoblotting, and exhibited normal expression of surface markers. We assessed dense granule, α-granule and lysosomal secretion, integrin αIIbβ3 activation, Ca2+ signalling, and spreading on fibrinogen in response to collagen-related peptide or the PAR4 agonist, AYPGKF in washed mouse platelets lacking myosin Va or wild-type platelets. Surprisingly, Myo5a−/− platelets showed no significant functional defects in these responses, or in the numbers of dense and α-granules expressed. Conclusion Despite the importance of myosin Va in vesicle transport in other cells, our data demonstrate this motor protein has no non-redundant role in the secretion of dense and α-granules or other functional responses in platelets.
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Affiliation(s)
- Matthew T. Harper
- School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom
| | | | - Ingeborg Hers
- School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom
| | - Alastair W. Poole
- School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom
- * E-mail:
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Bello-Morales R, Crespillo AJ, Fraile-Ramos A, Tabarés E, Alcina A, López-Guerrero JA. Role of the small GTPase Rab27a during herpes simplex virus infection of oligodendrocytic cells. BMC Microbiol 2012; 12:265. [PMID: 23164453 PMCID: PMC3554593 DOI: 10.1186/1471-2180-12-265] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2012] [Accepted: 11/01/2012] [Indexed: 12/28/2022] Open
Abstract
BACKGROUND The morphogenesis of herpes simplex virus type 1 (HSV-1) comprises several events, of which some are not completely understood. It has been shown that HSV-1 glycoproteins accumulate in the trans-Golgi network (TGN) and in TGN-derived vesicles. It is also accepted that HSV-1 acquires its final morphology through a secondary envelopment by budding into TGN-derived vesicles coated with viral glycoproteins and tegument proteins. Nevertheless, several aspects of this process remain elusive. The small GTPase Rab27a has been implicated in regulated exocytosis, and it seems to play a key role in certain membrane trafficking events. Rab27a also seems to be required for human cytomegalovirus assembly. However, despite the involvement of various Rab GTPases in HSV-1 envelopment, there is, to date, no data reported on the role of Rab27a in HSV-1 infection. RESULTS Herein, we show that Rab27a colocalized with GHSV-UL46, a tegument-tagged green fluorescent protein-HSV-1, in the TGN. In fact, this small GTPase colocalized with viral glycoproteins gH and gD in that compartment. Functional analysis through Rab27a depletion showed a significant decrease in the number of infected cells and viral production in Rab27a-silenced cells. CONCLUSIONS Altogether, our results indicate that Rab27a plays an important role in HSV-1 infection of oligodendrocytic cells.
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Affiliation(s)
- Raquel Bello-Morales
- Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Nicolás Cabrera 5, Cantoblanco, 28049, Madrid, Spain
| | - Antonio Jesús Crespillo
- Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Nicolás Cabrera 5, Cantoblanco, 28049, Madrid, Spain
| | - Alberto Fraile-Ramos
- Universidad Complutense de Madrid, Facultad de Medicina, Ciudad Universitaria, 28040, Madrid, Spain
| | - Enrique Tabarés
- Universidad Autónoma de Madrid, Facultad de Medicina, Arzobispo Morcillo 4, 28029, Madrid, Spain
| | - Antonio Alcina
- Instituto de Parasitología y Biomedicina López Neyra, CSIC, Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento s/n, 18100, Armilla, Granada, Spain
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Nabavi N, Pustylnik S, Harrison RE. Rab GTPase mediated procollagen trafficking in ascorbic acid stimulated osteoblasts. PLoS One 2012; 7:e46265. [PMID: 23050002 PMCID: PMC3458846 DOI: 10.1371/journal.pone.0046265] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2012] [Accepted: 08/30/2012] [Indexed: 12/24/2022] Open
Abstract
Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly up-regulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and diseases involving defects in bone protein secretion and deposition. Protein trafficking along the exocytic and endocytic pathways is aided by many molecules, with Rab GTPases being master regulators of vesicle targeting. In this study, we used microarray analysis to identify the Rab GTPases that are up-regulated during a 5-day AA differentiation of OBs, namely Rab1, Rab3d, and Rab27b. Further, we investigated the role of identified Rabs in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane utilizing Rab dominant negative (DN) expression. We also observed that experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs partially alleviated this negative feedback mechanism and resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels was also observed implicating the functional importance of Rab1, Rab3d and Rab27b in these major collagen-producing cells.
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Affiliation(s)
- Noushin Nabavi
- Department of Cell and Systems Biology, University of Tronto Scarborought, Toronto, Ontario, Canada
- Department of Biological Sciences, University of Toronto Scarborough, Toronto, Ontario, Canada
| | - Sofia Pustylnik
- Department of Biological Sciences, University of Toronto Scarborough, Toronto, Ontario, Canada
| | - Rene E. Harrison
- Department of Cell and Systems Biology, University of Tronto Scarborought, Toronto, Ontario, Canada
- Department of Biological Sciences, University of Toronto Scarborough, Toronto, Ontario, Canada
- * E-mail:
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Dong WW, Mou Q, Chen J, Cui JT, Li WM, Xiao WH. Differential expression of Rab27A/B correlates with clinical outcome in hepatocellular carcinoma. World J Gastroenterol 2012; 18:1806-13. [PMID: 22553406 PMCID: PMC3332295 DOI: 10.3748/wjg.v18.i15.1806] [Citation(s) in RCA: 70] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/13/2012] [Revised: 02/28/2012] [Accepted: 03/20/2012] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the association of Rab27A and Rab27B expression with clinicopathological characteristics and prognosis of hepatocellular carcinoma (HCC).
METHODS: We used reverse transcription polymerase chain reaction (RT-PCR), real-time PCR, and Western blotting to detect Rab27A and Rab27B mRNA and protein expression in 5 human HCC lines and the immortalized hepatic HL-7702 cell line. We further examined 148 primary HCC samples matched with adjacent normal tissue and 80 non-HCC specimens by immunohistochemistry to evaluate the correlation of Rab27A and Rab27B expression with clinicopathological features and prognosis.
RESULTS: Our data showed that Rab27A and Rab27B were differentially expressed in cell lines and primary HCC tumors. Rab27A mRNA and protein were detected in 67% (4/6) of human cell lines and 80% (4/5) of HCC cell lines, while Rab27B was found in 50% (3/6) of human lines and 40% (2/5) of HCC lines. Rab27A expression was higher in primary HCC (46.2%, 66/143) than in matched adjacent tissue (24.3%, 33/136, P < 0.001), whereas immunopositivity for Rab27B was lower in primary HCC (57.4%, 81/141) than in matched adjacent tissue (87.5%, 119/136, P < 0.001). Analysis of clinicopathological characteristics of 148 HCC specimens revealed significant correlations between Rab27A and Rab27B expression and tumor tumor-node-metastasis (TNM) classification (P = 0.046 and P = 0.027, respectively), and between strong Rab27A expression and tumor differentiation grade (P = 0.008). Survival analyses revealed that patients with Rab27A+ or Rab27B+ tumors had significantly reduced overall survival compared with that of patients with Rab27A- or Rab27B- tumors (P = 0.015 and P = 0.005, respectively). Risk analyses revealed that Rab27B+ and TNM III-IV were independent poor prognosis factors associated with a 3.36- and 3.37- fold higher relative risk of death, respectively.
CONCLUSION: Rab27A and Rab27B expression were closely correlated with tumor progression and can be valuable prognostic indicators for HCC patients.
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Singh RK, Liao W, Tracey-White D, Recchi C, Tolmachova T, Rankin SM, Hume AN, Seabra MC. Rab27a-mediated protease release regulates neutrophil recruitment by allowing uropod detachment. J Cell Sci 2012; 125:1652-6. [PMID: 22375060 PMCID: PMC3346826 DOI: 10.1242/jcs.100438] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/02/2022] Open
Abstract
Neutrophil migration is vital for immunity and precedes effector functions such as pathogen killing. Here, we report that this process is regulated by the Rab27a GTPase, a protein known to control granule exocytosis. Rab27a-deficient (Rab27a KO) neutrophils exhibit migration defects in vitro and in vivo, and live-cell microscopy suggests that delayed uropod detachment causes the migratory defect. Surface expression of CD11b, a key adhesion molecule, is increased in chemokine-stimulated Rab27a KO neutrophils compared with the control, suggesting a turnover delay caused by a defect in elastase secretion from azurophilic granules at the rear of bone marrow polymorphonuclear leukocytes (BM-PMNs). We suggest that Rab27a-dependent protease secretion regulates neutrophil migration through proteolysis-dependent de-adhesion of uropods, a mechanism that could be conserved in cell migration and invasion.
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Affiliation(s)
- Rajesh K Singh
- Molecular Medicine, National Heart and Lung Institute (NHLI), Imperial College, London, UK
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32
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Li G. Rab GTPases, membrane trafficking and diseases. Curr Drug Targets 2012; 12:1188-93. [PMID: 21561417 DOI: 10.2174/138945011795906561] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2010] [Accepted: 02/12/2011] [Indexed: 11/22/2022]
Abstract
The Rab family of GTPases contains over 60 genes in the human genome and contributes to regulation of intracellular membrane trafficking along endocytic and exocytic pathways as well as specialized pathways in specific cell types. It has become increasingly clear that disruption of the intracellular membrane trafficking system at different stages can cause various diseases. In the past decade, altered expression levels and mutations of Rab GTPases have been associated with such diseases as cancer, Alzheimer's disease, and various genetic disorders. This review discusses the specific Rab GTPases and their involvement in the diseases.
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Affiliation(s)
- Guangpu Li
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
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33
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Diekmann Y, Seixas E, Gouw M, Tavares-Cadete F, Seabra MC, Pereira-Leal JB. Thousands of rab GTPases for the cell biologist. PLoS Comput Biol 2011; 7:e1002217. [PMID: 22022256 PMCID: PMC3192815 DOI: 10.1371/journal.pcbi.1002217] [Citation(s) in RCA: 150] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2011] [Accepted: 08/17/2011] [Indexed: 01/27/2023] Open
Abstract
Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform comparative analysis in cell biology. Intracellular compartmentalisation via membrane-delimited organelles is a fundamental feature of the eukaryotic cell. Understanding its origins and specialisation into functionally distinct compartments is a major challenge in evolutionary cell biology. We focus on the Rab enzymes, critical organisers of the trafficking pathways that link the endomembrane system. Rabs form a large family of evolutionarily related proteins, regulating distinct steps in vesicle transport. They mark pathways and organelles due to their specific subcellular and tissue localisation. We propose a solution to the problem of identifying and annotating Rabs in hundreds of sequenced genomes. We developed an accurate bioinformatics pipeline that is able to take into account pre-existing and often inconsistent, manual annotations. We made it available to the community in form of a web tool, as well as a database containing thousands of Rabs assigned to sub-families, which yields clear functional predictions. Thousands of Rabs allow for a new level of analysis. We illustrate this by characterising for the first time the global evolutionary dynamics of the Rab family. We dated the emergence of subfamilies and suggest that the Rab family expands by duplicates acquiring new functions.
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Affiliation(s)
- Yoan Diekmann
- Instituto Gulbenkian de Ciência, Oeiras, Portugal
- * E-mail: (YD); (JBPL)
| | - Elsa Seixas
- Instituto Gulbenkian de Ciência, Oeiras, Portugal
- Centro de Estudos de Doenças Crónicas (CEDOC), Faculdade de Ciências Médicas da Universidade Nova de Lisboa, Lisboa, Portugal
| | - Marc Gouw
- Instituto Gulbenkian de Ciência, Oeiras, Portugal
| | | | - Miguel C. Seabra
- Instituto Gulbenkian de Ciência, Oeiras, Portugal
- Centro de Estudos de Doenças Crónicas (CEDOC), Faculdade de Ciências Médicas da Universidade Nova de Lisboa, Lisboa, Portugal
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Westbroek W, Klar A, Cullinane AR, Ziegler SG, Hurvitz H, Ganem A, Wilson K, Dorward H, Huizing M, Tamimi H, Vainshtein I, Berkun Y, Lavie M, Gahl WA, Anikster Y. Cellular and clinical report of new Griscelli syndrome type III cases. Pigment Cell Melanoma Res 2011; 25:47-56. [PMID: 21883982 DOI: 10.1111/j.1755-148x.2011.00901.x] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The RAB27A/Melanophilin/Myosin-5a tripartite protein complex is required for capturing mature melanosomes in the peripheral actin network of melanocytes for subsequent transfer to keratinocytes. Mutations in any one member of this tripartite complex cause three forms of Griscelli syndrome (GS), each with distinct clinical features but with a similar cellular phenotype. To date, only one case of GS type III (GSIII), caused by mutations in the Melanophilin (MLPH) gene, has been reported. Here, we report seven new cases of GSIII in three distinct Arab pedigrees. All affected individuals carried a homozygous missense mutation (c.102C>T; p.R35W), located in the conserved Slp homology domain of MLPH, and had hypomelanosis of the skin and hair. We report the first cellular studies on GSIII melanocytes, which demonstrated that MLPH(R35W) causes perinuclear aggregation of melanosomes in melanocytes, typical for GS. Additionally, co-immunoprecipitation assays showed that MLPH(R35W) lost its interaction with RAB27A, indicating pathogenicity of the R35W mutation.
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Affiliation(s)
- Wendy Westbroek
- Section on Human Biochemical Genetics, Medical Genetics Branch, National Human Genome Research Institute, NIH, Bethesda MD, USA.
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35
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Hendrix A, Braems G, Bracke M, Seabra M, Gahl W, De Wever O, Westbroek W. The secretory small GTPase Rab27B as a marker for breast cancer progression. Oncotarget 2011; 1:304-8. [PMID: 21304180 DOI: 10.18632/oncotarget.100809] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
In contemporary oncology practice, an urgent need remains to refine the prognostic assessment of breast cancer. It is still difficult to identify patients with early breast cancer who are likely to benefit from adjuvant chemotherapy. Although invasion of cancer cells is the main prognostic denominator in tumor malignancy, our molecular understanding and diagnosis are often inadequate to cope with this activity. Therefore, deciphering molecular pathways of how tumors invade and metastasize may help in the identification of a useful prognostic marker. We recently discovered that the secretory small GTPase Rab27B, a regulator of vesicle exocytosis, delivers proinvasive signals for increased invasiveness, tumor size, and metastasis of various estrogen receptor (ER)-positive breast cancer cell lines, both in vitro and in vivo. In human breast cancer specimens, the presence of Rab27B protein proved to be associated with a low degree of differentiation and the presence of lymph node metastasis in ER-positive breast cancer.
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Affiliation(s)
- An Hendrix
- Department of Medical Oncology, Ghent University Hospital, De Pintelaan, Belgium.
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36
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Chiang L, Ngo J, Schechter JE, Karvar S, Tolmachova T, Seabra MC, Hume AN, Hamm-Alvarez SF. Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland. Am J Physiol Cell Physiol 2011; 301:C507-21. [PMID: 21525430 PMCID: PMC3154548 DOI: 10.1152/ajpcell.00355.2010] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2010] [Accepted: 04/22/2011] [Indexed: 11/22/2022]
Abstract
Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.
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Affiliation(s)
- Lilian Chiang
- School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA
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37
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Johnson JL, Hong H, Monfregola J, Kiosses WB, Catz SD. Munc13-4 restricts motility of Rab27a-expressing vesicles to facilitate lipopolysaccharide-induced priming of exocytosis in neutrophils. J Biol Chem 2010; 286:5647-56. [PMID: 21148308 DOI: 10.1074/jbc.m110.184762] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
LPS is an efficient sensitizer of the neutrophil exocytic response to a second stimulus. Although neutrophil exocytosis in response to pathogen-derived molecules plays an important role in the innate immune response to infections, the molecular mechanism underlying LPS-dependent regulation of neutrophil exocytosis is currently unknown. The small GTPase Rab27a and its effector Munc13-4 regulate exocytosis in hematopoietic cells. Whether Rab27a and Munc13-4 modulate discrete steps or the same steps during exocytosis also remains unknown. Here, using Munc13-4- and Rab27a-deficient neutrophils, we analyzed the mechanism of lipopolysaccharide-dependent vesicular priming to amplify exocytosis of azurophilic granules. We found that both Munc13-4 and Rab27a are necessary to mediate LPS-dependent priming of exocytosis. However, we show that LPS-induced mobilization of a small population of readily releasable vesicles is a Munc13-4-dependent but Rab27a-independent process. LPS-induced priming regulation could not be fully explained by secretory organelle maturation as the redistribution of the secretory proteins Rab27a or Munc13-4 in response to LPS treatment was minimal. Using total internal reflection fluorescence microscopy and a novel mouse model expressing EGFP-Rab27a under the endogenous Rab27a promoter but lacking Munc13-4, we demonstrate that Munc13-4 is essential for the mechanism of LPS-dependent exocytosis in neutrophils and unraveled a novel mechanism of vesicular dynamics in which Munc13-4 restricts motility of Rab27a-expressing vesicles to facilitate lipopolysaccharide-induced priming of exocytosis.
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Affiliation(s)
- Jennifer L Johnson
- Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA
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38
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Hendrix A, Maynard D, Pauwels P, Braems G, Denys H, Van den Broecke R, Lambert J, Van Belle S, Cocquyt V, Gespach C, Bracke M, Seabra MC, Gahl WA, De Wever O, Westbroek W. Effect of the secretory small GTPase Rab27B on breast cancer growth, invasion, and metastasis. J Natl Cancer Inst 2010; 102:866-80. [PMID: 20484105 DOI: 10.1093/jnci/djq153] [Citation(s) in RCA: 185] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Secretory GTPases like Rab27B control vesicle exocytosis and deliver critical proinvasive growth regulators into the tumor microenvironment. The expression and role of Rab27B in breast cancer were unknown. METHODS Expression of green fluorescent protein (GFP) fused with wild-type Rab3D, Rab27A, or Rab27B, or Rab27B point mutants defective in GTP/GDP binding or geranylgeranylation, or transient silencing RNA to the same proteins was used to study Rab27B in estrogen receptor (ER)-positive human breast cancer cell lines (MCF-7, T47D, and ZR75.1). Cell cycle progression was evaluated by flow cytometry, western blotting, and measurement of cell proliferation rates, and invasion was assessed using Matrigel and native type I collagen substrates. Orthotopic tumor growth, local invasion, and metastasis were analyzed in mouse xenograft models. Mass spectrometry identified proinvasive growth regulators that were secreted in the presence of Rab27B. Rab27B protein levels were evaluated by immunohistochemistry in 59 clinical breast cancer specimens, and Rab3D, Rab27A, and Rab27B mRNA levels were analyzed by quantitative real-time polymerase chain reaction in 20 specimens. Statistical tests were two-sided. RESULTS Increased expression of Rab27B promoted G(1) to S phase cell cycle transition, proliferation and invasiveness of cells in culture, and invasive tumor growth and hemorrhagic ascites production in a xenograft mouse model (n = 10; at 10 weeks, survival of MCF-7 GFP- vs GFP-Rab27B-injected mice was 100% vs 62.5%, hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.88, P = .03). Mass spectrometric analysis of purified Rab27B-secretory vesicles identified heat-shock protein 90alpha as key proinvasive growth regulator. Heat-shock protein 90alpha secretion was Rab27B-dependent and was required for matrix metalloproteinase-2 activation. All Rab27B-mediated functional responses were GTP- and geranylgeranyl-dependent. Presence of endogenous Rab27B mRNA and protein, but not of Rab3D or Rab27A mRNA, was associated with lymph node metastasis (P < .001) and differentiation grade (P = .001) in ER-positive human breast tumors. CONCLUSIONS Rab27B regulates invasive growth and metastasis in ER-positive breast cancer cell lines, and increased expression is associated with poor prognosis in humans.
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Affiliation(s)
- An Hendrix
- Medical Genetics Branch, National Human Genome Research Institute, 10 Center Dr, Bethesda, MD 20892, USA
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Johnson JL, Brzezinska AA, Tolmachova T, Munafo DB, Ellis BA, Seabra MC, Hong H, Catz SD. Rab27a and Rab27b regulate neutrophil azurophilic granule exocytosis and NADPH oxidase activity by independent mechanisms. Traffic 2009; 11:533-47. [PMID: 20028487 DOI: 10.1111/j.1600-0854.2009.01029.x] [Citation(s) in RCA: 85] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Neutrophils rely on exocytosis to mobilize receptors and adhesion molecules and to release microbicidal factors. This process should be strictly regulated because uncontrolled release of toxic proteins would be injurious to the host. In vivo studies showed that the small GTPase Rab27a regulates azurophilic granule exocytosis. Using mouse neutrophils deficient in Rab27a (Rab27a(ash/ash)), Rab27b [Rab27b knockout (KO)] or both [Rab27a/b double KO (DoKo)], we investigated the role of the Rab27 isoforms in neutrophils. We found that both Rab27a and Rab27b deficiencies impaired azurophilic granule exocytosis. Rab27a(ash/ash) neutrophils showed upregulation of Rab27b expression which did not compensate for the secretory defects observed in Rab27a-deficient cells, suggesting that Rab27 isoforms play independent roles in neutrophil exocytosis. Total internal reflection fluorescence microscopy analysis showed that Rab27a(ash/ash) and Rab27b KO neutrophils have a decreased number of azurophilic granules near the plasma membrane. The effect was exacerbated in Rab27a/b DoKo neutrophils. Rab27-deficient neutrophils showed impaired activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase at the plasma membrane although intraphagosomal reactive oxygen species (ROS) production was not affected. Exocytosis of secretory vesicles in Rab27-deficient neutrophils was functional, suggesting that Rab27 GTPases selectively control the exocytosis of neutrophil granules.
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Affiliation(s)
- Jennifer L Johnson
- Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
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Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat Cell Biol 2009; 12:19-30; sup pp 1-13. [PMID: 19966785 DOI: 10.1038/ncb2000] [Citation(s) in RCA: 1935] [Impact Index Per Article: 120.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2009] [Accepted: 11/10/2009] [Indexed: 11/08/2022]
Abstract
Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo.
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Guo X, Tu L, Gumper I, Plesken H, Novak EK, Chintala S, Swank RT, Pastores G, Torres P, Izumi T, Sun TT, Sabatini DD, Kreibich G. Involvement of vps33a in the fusion of uroplakin-degrading multivesicular bodies with lysosomes. Traffic 2009; 10:1350-61. [PMID: 19566896 PMCID: PMC4494113 DOI: 10.1111/j.1600-0854.2009.00950.x] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
The apical surface of the terminally differentiated mouse bladder urothelium is largely covered by urothelial plaques, consisting of hexagonally packed 16-nm uroplakin particles. These plaques are delivered to the cell surface by fusiform vesicles (FVs) that are the most abundant cytoplasmic organelles. We have analyzed the functional involvement of several proteins in the apical delivery and endocytic degradation of uroplakin proteins. Although FVs have an acidified lumen and Rab27b, which localizes to these organelles, is known to be involved in the targeting of lysosome-related organelles (LROs), FVs are CD63 negative and are therefore not typical LROs. Vps33a is a Sec1-related protein that plays a role in vesicular transport to the lysosomal compartment. A point mutation in mouse Vps33a (Buff mouse) causes albinism and bleeding (Hermansky-Pudlak syndrome) because of abnormalities in the trafficking of melanosomes and platelets. These Buff mice showed a novel phenotype observed in urothelial umbrella cells, where the uroplakin-delivering FVs were almost completely replaced by Rab27b-negative multivesicular bodies (MVBs) involved in uroplakin degradation. MVB accumulation leads to an increase in the amounts of uroplakins, Lysosomal-associated membrane protein (LAMP)-1/2, and the activities of beta-hexosaminidase and beta-glucocerebrosidase. These results suggest that FVs can be regarded as specialized secretory granules that deliver crystalline arrays of uroplakins to the cell surface, and that the Vps33a mutation interferes with the fusion of MVBs with mature lysosomes thus blocking uroplakin degradation.
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Affiliation(s)
- Xuemei Guo
- Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
| | - Liyu Tu
- Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
| | - Iwona Gumper
- Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
| | - Heide Plesken
- Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
| | - Edward K. Novak
- Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
| | - Sreenivasulu Chintala
- Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
| | - Richard T. Swank
- Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
| | - Gregory Pastores
- Department of Neurology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
| | - Paola Torres
- Department of Neurology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
| | - Tetsuro Izumi
- Department of Molecular Medicine, Gunma University, Maebashi, Japan
| | - Tung-Tien Sun
- Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
- Department of Pharmacology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
- Department of Urology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
- Department of Epithelial Biology Unit, The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
- Department of NYU Cancer Institute, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
| | - David D. Sabatini
- Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
- Department of NYU Cancer Institute, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
| | - Gert Kreibich
- Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
- Department of NYU Cancer Institute, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
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Abstract
alpha-Granules are essential to normal platelet activity. These unusual secretory granules derive their cargo from both regulated secretory and endocytotic pathways in megakaryocytes. Rare, inheritable defects of alpha-granule formation in mice and man have enabled identification of proteins that mediate cargo trafficking and alpha-granule formation. In platelets, alpha-granules fuse with the plasma membrane upon activation, releasing their cargo and increasing platelet surface area. The mechanisms that control alpha-granule membrane fusion have begun to be elucidated at the molecular level. SNAREs and SNARE accessory proteins that control alpha-granule secretion have been identified. Proteomic studies demonstrate that hundreds of bioactive proteins are released from alpha-granules. This breadth of proteins implies a versatile functionality. While initially known primarily for their participation in thrombosis and hemostasis, the role of alpha-granules in inflammation, atherosclerosis, antimicrobial host defense, wound healing, angiogenesis, and malignancy has become increasingly appreciated as the function of platelets in the pathophysiology of these processes has been defined. This review will consider the formation, release, and physiologic roles of alpha-granules with special emphasis on work performed over the last decade.
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Affiliation(s)
- Price Blair
- Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215
| | - Robert Flaumenhaft
- Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215
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Abstract
The natural killer (NK)-cell immunological synapse is the dynamic interface formed between an NK cell and its target cell. Formation of the NK-cell immunological synapse involves several distinct stages, from the initiation of contact with a target cell to the directed delivery of lytic-granule contents for target-cell lysis. Progression through the individual stages is regulated, and this tight regulation underlies the precision with which NK cells select and kill susceptible target cells (including virally infected cells and cancerous cells) that they encounter during their routine surveillance of the body.
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Affiliation(s)
- Jordan S Orange
- University of Pennsylvania School of Medicine, Joseph Stokes Jr Research Institute of The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, ARC 1016H, Philadelphia, Pennsylvania 19104, USA.
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Gomi H, Mori K, Itohara S, Izumi T. Rab27b is expressed in a wide range of exocytic cells and involved in the delivery of secretory granules near the plasma membrane. Mol Biol Cell 2007; 18:4377-86. [PMID: 17761531 PMCID: PMC2043558 DOI: 10.1091/mbc.e07-05-0409] [Citation(s) in RCA: 88] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Rab proteins regulate multiple, complex processes of membrane traffic. Among these proteins, Rab27a has been shown to function specifically in regulated exocytic pathways. However, the roles of Rab27b, another Rab27 subfamily member, have not been well characterized. We disrupted the Rab27b gene in mice. The targeting vector was designed to insert LacZ downstream of the initiation codon of the Rab27b gene so that the authentic promoter should drive this reporter gene. A comprehensive analysis of Rab27b expression using this mouse strain indicated that it is widely expressed not only in canonical secretory cells, but also in neurons and cells involved in surface protection and mechanical extension. To evaluate the function in pituitary endocrine cells where the isoform Rab27a is coexpressed, we generated Rab27a/Rab27b double knockout mice by crossing Rab27b knockout mice with Rab27a-mutated ashen mice. The polarized distribution of secretory granules close to the plasma membrane was markedly impaired in the pituitary of double knockout mice, indicating that the Rab27 subfamily is involved in the delivery of granules near the exocytic site. In conjunction with a phenotype having a pituitary devoid of the Rab27 effector granuphilin, we discuss the relationship between the residence and the releasable pool of granules.
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Affiliation(s)
- Hiroshi Gomi
- *Laboratory of Molecular Endocrinology and Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan; and
| | - Kenichi Mori
- *Laboratory of Molecular Endocrinology and Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan; and
| | - Shigeyoshi Itohara
- Laboratory for Behavioral Genetics, Brain Science Institute, Institute of Physical and Chemical Research, Wako 351-0198, Japan
| | - Tetsuro Izumi
- *Laboratory of Molecular Endocrinology and Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan; and
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Mizuno K, Tolmachova T, Ushakov DS, Romao M, Åbrink M, Ferenczi MA, Raposo G, Seabra MC. Rab27b regulates mast cell granule dynamics and secretion. Traffic 2007; 8:883-92. [PMID: 17587407 PMCID: PMC2063611 DOI: 10.1111/j.1600-0854.2007.00571.x] [Citation(s) in RCA: 89] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2007] [Revised: 03/28/2007] [Accepted: 03/23/2007] [Indexed: 11/30/2022]
Abstract
The Rab GTPase family regulates membrane domain organization and vesicular transport pathways. Recent studies indicate that one member of the family, Rab27a, regulates transport of lysosome-related organelles in specialized cells, such as melanosomes and lytic granules. Very little is known about the related isoform, Rab27b. Here we used genetically modified mice to study the involvement of the Rab27 proteins in mast cells, which play key roles in allergic responses. Both Rab27a and Rab27b isoforms are expressed in bone marrow-derived mast cells (BMMC) and localize to secretory granules. Nevertheless, secretory defects as measured by beta-hexosaminidase release in vitro and passive cutaneous anaphylaxis in vivo were found only in Rab27b and double Rab27 knockout (KO) mice. Immunofluorescence studies suggest that a subset of Rab27b and double Rab27-deficient BMMCs exhibit mild clustering of granules. Quantitative analysis of live-cell time-lapse imaging revealed that BMMCs derived from double Rab27 KO mice showed almost 10-fold increase in granules exhibiting fast movement (>1.5 microm/s), which could be disrupted by nocodazole. These results suggest that Rab27 proteins, particularly Rab27b, play a crucial role in mast cell degranulation and that their action regulates the transition from microtubule to actin-based motility.
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Affiliation(s)
- Kouichi Mizuno
- Molecular and Cellular Medicine, National Heart and Lung Institute, Imperial College London SW7 2AZUK
| | - Tanya Tolmachova
- Molecular and Cellular Medicine, National Heart and Lung Institute, Imperial College London SW7 2AZUK
| | - Dmitry S Ushakov
- Biological Nanoscience, National Heart and Lung Institute, Imperial College London SW7 2AZUK
| | - Maryse Romao
- Institut Curie, CNRS UMR144, Structure and Membrane Compartments, 75248 ParisFrance
| | - Magnus Åbrink
- Department of Medical Biochemistry and Microbiology, Uppsala University, 751 05 UppsalaSweden
| | - Michael A Ferenczi
- Biological Nanoscience, National Heart and Lung Institute, Imperial College London SW7 2AZUK
| | - Graça Raposo
- Institut Curie, CNRS UMR144, Structure and Membrane Compartments, 75248 ParisFrance
| | - Miguel C Seabra
- Molecular and Cellular Medicine, National Heart and Lung Institute, Imperial College London SW7 2AZUK
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Lopes VS, Ramalho JS, Owen DM, Karl MO, Strauss O, Futter CE, Seabra MC. The ternary Rab27a-Myrip-Myosin VIIa complex regulates melanosome motility in the retinal pigment epithelium. Traffic 2007; 8:486-99. [PMID: 17451552 PMCID: PMC1920545 DOI: 10.1111/j.1600-0854.2007.00548.x] [Citation(s) in RCA: 75] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2006] [Revised: 01/30/2007] [Accepted: 01/30/2007] [Indexed: 12/11/2022]
Abstract
The retinal pigment epithelium (RPE) contains melanosomes similar to those found in the skin melanocytes, which undergo dramatic light-dependent movements in fish and amphibians. In mammals, those movements are more subtle and appear to be regulated by the Rab27a GTPase and the unconventional myosin, Myosin VIIa (MyoVIIa). Here we address the hypothesis that a recently identified Rab27a- and MyoVIIa-interacting protein, Myrip, promotes the formation of a functional tripartite complex. In heterologous cultured cells, all three proteins co-immunoprecipitated following overexpression. Rab27a and Myrip localize to the peripheral membrane of RPE melanosomes as observed by immunofluorescence and immunoelectron microscopy. Melanosome dynamics were studied using live-cell imaging of mouse RPE primary cultures. Wild-type RPE melanosomes exhibited either stationary or slow movement interrupted by bursts of fast movement, with a peripheral directionality trend. Nocodazole treatment led to melanosome paralysis, suggesting that movement requires microtubule motors. Significant and similar alterations in melanosome dynamics were observed when any one of the three components of the complex was missing, as studied in ashen- (Rab27a defective) and shaker-1 (MyoVIIa mutant)-derived RPE cells, and in wild-type RPE cells transduced with adenovirus carrying specific sequences to knockdown Myrip expression. We observed a significant increase in the number of motile melanosomes, exhibiting more frequent and prolonged bursts of fast movement, and inversion of directionality. Similar alterations were observed upon cytochalasin D treatment, suggesting that the Rab27a-Myrip-MyoVIIa complex regulates tethering of melanosomes onto actin filaments, a process that ensures melanosome movement towards the cell periphery.
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Affiliation(s)
- Vanda S Lopes
- Molecular and Cellular Medicine, Faculty of Medicine, Imperial College LondonLondon SW7 2AZ, UK
| | - José S Ramalho
- Centre of Ophthalmology, Biomedical Institute for Research in Light and Image, University of Coimbra3000-354 Coimbra, Portugal
| | - Dylan M Owen
- Chemical Biology Centre, Imperial College LondonLondon, SW7 2AZ, UK
| | - Mike O Karl
- Bereich Experimentelle Ophthalmologie, Klinik und Poliklinik fuer Augenheilkunde, Universitaetsklinikum Hamburg-Eppendorf20246, Hamburg, Germany
| | - Olaf Strauss
- Bereich Experimentelle Ophthalmologie, Klinik und Poliklinik fuer Augenheilkunde, Universitaetsklinikum Hamburg-Eppendorf20246, Hamburg, Germany
| | - Clare E Futter
- Division of Cell Biology, Institute of Ophthalmology, University College LondonLondon EC1V 9EL, UK
| | - Miguel C Seabra
- Molecular and Cellular Medicine, Faculty of Medicine, Imperial College LondonLondon SW7 2AZ, UK
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Tolmachova T, Åbrink M, Futter CE, Authi KS, Seabra MC. Rab27b regulates number and secretion of platelet dense granules. Proc Natl Acad Sci U S A 2007; 104:5872-7. [PMID: 17384153 PMCID: PMC1831675 DOI: 10.1073/pnas.0609879104] [Citation(s) in RCA: 148] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2006] [Indexed: 12/16/2022] Open
Abstract
The Rab27 GTPase subfamily consists of two closely related homologs, Rab27a and Rab27b. Rab27a has been shown previously to regulate organelle movement and regulated exocytosis in a wide variety of secretory cells. However, the role of the more restrictedly expressed Rab27b remains unclear. Here we describe the creation of Rab27b knockout (KO) strain that was subsequently crossed with the naturally occurring Rab27a KO line, ashen, to produce double KO (Rab27a(ash/ash) Rab27b(-/-)) mice. Rab27b KO (and double KO) exhibit significant hemorrhagic disease in contrast to ashen mice. In vitro assays demonstrated impaired aggregation with collagen and U46619 and reduced secretion of dense granules in both Rab27b and double KO strains. Additionally, we detected a 50% reduction in the number of dense granules per platelet and diminished platelet serotonin content, possibly due to a dense granule packaging defect into proplatelets during megakaryocyte maturation. The presence of Rab27a partially compensated for the secretory defect but not the reduced granule number. The morphology and function of platelet alpha-granules were unaffected. Our data suggest that Rab27b is a key regulator of dense granule secretion in platelets and thus a candidate gene for delta-storage pool deficiency in humans.
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Affiliation(s)
- Tanya Tolmachova
- *Molecular and Cellular Medicine, National Heart and Lung Institute, Imperial College London, London SW7 2AZ, United Kingdom
| | - Magnus Åbrink
- Department of Medical Biochemistry and Microbiology, Uppsala University, 751 05 Uppsala, Sweden
| | - Clare E. Futter
- Institute of Ophthalmology, University College London, London EC1V 9EL, United Kingdom; and
| | - Kalwant S. Authi
- Cardiovascular Division, King's College London, London SE1 9NH, United Kingdom
| | - Miguel C. Seabra
- *Molecular and Cellular Medicine, National Heart and Lung Institute, Imperial College London, London SW7 2AZ, United Kingdom
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48
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Hu ZZ, Valencia JC, Huang H, Chi A, Shabanowitz J, Hearing VJ, Appella E, Wu C. Comparative Bioinformatics Analyses and Profiling of Lysosome-Related Organelle Proteomes. INTERNATIONAL JOURNAL OF MASS SPECTROMETRY 2007; 259:147-160. [PMID: 17375895 PMCID: PMC1828028 DOI: 10.1016/j.ijms.2006.09.024] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 05/14/2023]
Abstract
Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for 7 lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles.
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Affiliation(s)
- Zhang-Zhi Hu
- Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC
| | - Julio C. Valencia
- Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD
| | - Hongzhan Huang
- Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC
| | - An Chi
- Department of Chemistry, University of Virginia, Charlottesville, VA
| | | | - Vincent J. Hearing
- Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD
| | - Ettore Appella
- Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD
| | - Cathy Wu
- Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC
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Chintala S, Tan J, Gautam R, Rusiniak ME, Guo X, Li W, Gahl WA, Huizing M, Spritz RA, Hutton S, Novak EK, Swank RT. The Slc35d3 gene, encoding an orphan nucleotide sugar transporter, regulates platelet-dense granules. Blood 2006; 109:1533-40. [PMID: 17062724 PMCID: PMC1794067 DOI: 10.1182/blood-2006-08-040196] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Platelet dense granules are lysosome-related organelles which contain high concentrations of several biologically important low-molecular-weight molecules. These include calcium, serotonin, adenine nucleotides, pyrophosphate, and polyphosphate, which are necessary for normal blood hemostasis. The synthesis of dense granules and other lysosome-related organelles is defective in inherited diseases such as Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS). HPS and CHS mutations in 8 human and at least 16 murine genes have been identified. Previous studies produced contradictory findings for the function of the murine ashen (Rab27a) gene in platelet-dense granules. We have used a positional cloning approach with one line of ashen mutants to establish that a new mutation in a second gene, Slc35d3, on mouse chromosome 10 is the basis of this discrepancy. The platelet-dense granule defect is rescued in BAC transgenic mice containing the normal Slc35d3 gene. Thus, Slc35d3, an orphan member of a nucleotide sugar transporter family, specifically regulates the contents of platelet-dense granules. Unlike HPS or CHS genes, it has no apparent effect on other lysosome-related organelles such as melanosomes or lysosomes. The ash-Roswell mouse mutant is an appropriate model for human congenital-isolated delta-storage pool deficiency.
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Affiliation(s)
- Sreenivasulu Chintala
- Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
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50
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Mahoney TR, Liu Q, Itoh T, Luo S, Hadwiger G, Vincent R, Wang ZW, Fukuda M, Nonet ML. Regulation of synaptic transmission by RAB-3 and RAB-27 in Caenorhabditis elegans. Mol Biol Cell 2006; 17:2617-25. [PMID: 16571673 PMCID: PMC1474797 DOI: 10.1091/mbc.e05-12-1170] [Citation(s) in RCA: 133] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Rab small GTPases are involved in the transport of vesicles between different membranous organelles. RAB-3 is an exocytic Rab that plays a modulatory role in synaptic transmission. Unexpectedly, mutations in the Caenorhabditis elegans RAB-3 exchange factor homologue, aex-3, cause a more severe synaptic transmission defect as well as a defecation defect not seen in rab-3 mutants. We hypothesized that AEX-3 may regulate a second Rab that regulates these processes with RAB-3. We found that AEX-3 regulates another exocytic Rab, RAB-27. Here, we show that C. elegans RAB-27 is localized to synapse-rich regions pan-neuronally and is also expressed in intestinal cells. We identify aex-6 alleles as containing mutations in rab-27. Interestingly, aex-6 mutants exhibit the same defecation defect as aex-3 mutants. aex-6; rab-3 double mutants have behavioral and pharmacological defects similar to aex-3 mutants. In addition, we demonstrate that RBF-1 (rabphilin) is an effector of RAB-27. Therefore, our work demonstrates that AEX-3 regulates both RAB-3 and RAB-27, that both RAB-3 and RAB-27 regulate synaptic transmission, and that RAB-27 potentially acts through its effector RBF-1 to promote soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function.
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Affiliation(s)
- Timothy R. Mahoney
- *Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110
| | - Qiang Liu
- Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030; and
| | - Takashi Itoh
- Fukuda Initiative Research Unit, RIKEN, Wako, Saitama 351-0198, Japan
| | - Shuo Luo
- *Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110
| | - Gayla Hadwiger
- *Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110
| | - Rose Vincent
- *Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110
| | - Zhao-Wen Wang
- Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030; and
| | - Mitsunori Fukuda
- Fukuda Initiative Research Unit, RIKEN, Wako, Saitama 351-0198, Japan
| | - Michael L. Nonet
- *Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110
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