Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.720493
Title: KIF1C control of podosome formation in vascular smooth muscle cells
Author: Bachmann, Alice
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2016
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Abstract:
Podosomes are adhesion structures formed at the ventral side of cells whose physiological functions require an efficient cell migration across tissues. In addition to their adhesive properties, podosomes display a protrusive activity responsible for the local degradation of matrix components that helps cells to migrate across basement membranes. Because of their ability to degrade matrix components, podosome formation, activity and turnover need to be tightly regulated to avoid an uncontrolled protrusive activity that could damage tissues. The kinesin-3 KIF1C was shown to contribute to the regulation of podosome dynamics in macrophages and here, we used depletion-rescue and dominant negative approaches to show that KIF1C activity is required for podosome formation in A7r5 cells. Little is known about the mechanism regulating KIF1C activity. To better understand the molecular mechanism underlying the KIF1Ccontrol of podosome formation in A7r5 cells, we generated different truncations of the KIF1C tail, tested the ability of each construct to mediate podosome formation in these cells and identified a ~150 amino acid long region between KIF1C third and fourth coiled-coil domains that is required for podosome formation in A7r5 cells, suggesting that this ~150 amino acid long region of KIF1C tail participates in the regulation of KIF1C activity during the podosome formation process. This region of KIF1C tail is known to interact with two proteins that were both described for their involvement in the regulation of cellular adhesion: the non-muscle Myosin IIA and the Protein Tyrosine Phosphatase PTPD1. We addressed the involvement of the non-muscle Myosin IIA in podosome formation in A7r5 cells using two Myosin inhibitor: Blebbistatin and the Y27632 ROCK inhibitor. The treatment of A7r5 cells with one or the other Myosin IIA inhibitor had no effect on the ability of these cells to form podosomes, suggesting that the non-muscle Myosin IIA is not required for the formation of podosomes in A7r5 cells. We therefore ruled out its involvement in the regulation of KIF1C activity during the podosome formation process to focus our work on PTPD1. PTPD1 is a scaffolding tyrosine phosphatase known to regulate focal adhesion and stress fibre stability. Using a depletion-rescue approach, we showed that PTPD1 expression is absolutely required for podosome formation in A7r5 cells. As PTPD1 displays both a scaffolding and a phosphatase activity, we used the catalytic inactive mutant of the phosphatase PTPD1-C1108S to discriminate between these two activities and show that PTPD1 ability to mediate podosome formation in A7r5 cells does not require its catalytic activity. Because PTPD1 and KIF1C depletions performed independently of each other both led to a decrease of the ability of A7r5 cells to form podosomes and because these two proteins interact with each other, we tested the ability of PTPD1 and KIF1C to cooperate with each other to mediate podosome formation and showed that PTPD1-C1108S over-expression in KIF1C-depleted cells restored the ability of A7r5 cells to efficiently form podosomes. Taken together, results obtained suggested that the interaction of PTPD1 with KIF1C could stimulate the activity of the remaining pool of KIF1C motors. To test this hypothesis, we used a pre-established α5-integrin transport assay, as KIF1C is a known α5-integrin transporter and its motor activity can therefore be assessed by following its ability to transport GFP-tagged α5-integrin cargoes to the tail of migratory RPE-1 cells. Using this assay in KIF1C-depleted RPE-1 cells stably expressing GFP-α5-integrin, we showed that PTPD1 overexpression restores the transport of α5-integrin to the tail of RPE-1 cells, therefore stimulating the activity of the remaining pool of KIF1C motors. Results obtained in this study and in vitro data generated during the course of this work allowed us to establish the following model for KIF1C activation: in the absence of PTPD1, KIF1C is in its auto-inhibitory state and can't ensure its cellular function. In presence of PTPD1, the phosphatase binds to KIF1C tail, inducing the activation of the motor that can thus mediate podosome formation and transport α5-integrins to the tail of RPE-1 cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.720493  DOI: Not available
Keywords: QP Physiology
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