Bandar Fahad Alharbi1, Dhurgham Al-Fahad2, Philip Richard Dash3. 1. Department of clinical laboratory, College of Applied Medical Science, University of Hail, Hail, 81411, Saudi Arabia. 2. Department of Pathological Analysis, College of Science, University of Thi-Qar, Thi-Qar, 64001 Iraq. 3. Department of Biomedical, School of Biological Sciences, University of Reading, Reading, RG6 6UB, United Kingdom.
Abstract
BACKGROUND: Focal adhesion (FA) play a critical role in many biological processes which include cell survival and cell migration. They serve as cellular anchor, allowing cells to stay attached to the extracellular matrix (ECM), and can also regulate cellular transduction. Previously, it has been suggested that vesicles such as endosomes could interact directly with FA or be implicated in their turnover. In this study, we investigated whether there is a relationship between FA and the early endocytic machinery in MDA-MB-231 cells. METHODS: In this study, cell culture, transfection, time laps confocal microscopies, immunocytochemistry, western blotting, Cell fractionation and immunoprecipitation techniques were performed. RESULTS: Cells acutely treated with Dynasore, an inhibitor of dynamin, or with Pitstop 2, an inhibitor of clathryn-dependent endocytosis showed a reduction in the expression of early endosome biomarkers such as Rab5 and EEA1. Additionally, cells treated with these endocytic inhibitors exhibited an increase number and size of FA, as well as an increase FA turnover duration. This data was consistent with the reduction of the speed of cell migration. We demonstrated that Rab5- and EEA1-positive early endosomes were found to be colocalized with internalized FA. CONCLUSION: The present study suggests that there is a link between FA and early endosome markers, which indicates that the early endosomes may be involved in FA dynamics.
BACKGROUND: Focal adhesion (FA) play a critical role in many biological processes which include cell survival and cell migration. They serve as cellular anchor, allowing cells to stay attached to the extracellular matrix (ECM), and can also regulate cellular transduction. Previously, it has been suggested that vesicles such as endosomes could interact directly with FA or be implicated in their turnover. In this study, we investigated whether there is a relationship between FA and the early endocytic machinery in MDA-MB-231 cells. METHODS: In this study, cell culture, transfection, time laps confocal microscopies, immunocytochemistry, western blotting, Cell fractionation and immunoprecipitation techniques were performed. RESULTS: Cells acutely treated with Dynasore, an inhibitor of dynamin, or with Pitstop 2, an inhibitor of clathryn-dependent endocytosis showed a reduction in the expression of early endosome biomarkers such as Rab5 and EEA1. Additionally, cells treated with these endocytic inhibitors exhibited an increase number and size of FA, as well as an increase FA turnover duration. This data was consistent with the reduction of the speed of cell migration. We demonstrated that Rab5- and EEA1-positive early endosomes were found to be colocalized with internalized FA. CONCLUSION: The present study suggests that there is a link between FA and early endosome markers, which indicates that the early endosomes may be involved in FA dynamics.
Entities:
Keywords:
Cell migration; Early endosome; Endocytosis; Focal Adhesion
Authors: Marina N Sharifi; Erin E Mowers; Lauren E Drake; Chris Collier; Hong Chen; Marta Zamora; Stephanie Mui; Kay F Macleod Journal: Cell Rep Date: 2016-05-12 Impact factor: 9.423
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