In this work the functions of the microtubule motors kinesin-1, -2 and -3 have been analysed in various settings. The reconstitution of microtubule-dependent motor activity in vitro has been primarily used to dissect the contributions of individual motors to cargo motility in two specific scenarios. Initially the regulation of kinesin-1 in a cell cycle-dependent manner has been examined by studying the ability of rat liver endoplasmic reticulum (ER) tubules to move in cytosols prepared from Xenopus laevis egg extracts arrested in interphase, meiosis or mitosis. It was found that kinesin-1-driven ER motility is significantly disrupted during metaphase in vitro. This is likely due to the recruitment or loss of binding partners which has a concomitant influence upon kinesin-1 activity. This work presents the first evidence that kinesin-1-driven ER movement, and not simply network morphology, varies during cell division. Furthermore, it is postulated that the replication of such regulation of kinesin-1 activity in vivo may contribute to the well documented changes in organelle positioning and cargo transit through membrane trafficking pathways which occur during cell division.The fungal metabolite brefeldin A (BFA) induces tubulation of several compartments located within the secretory and endocytic pathways in a microtubule-dependent fashion. The identity of the motor(s) responsible for this motility remains unconfirmed and controversial since several reports with conflicting data have been published. The contributions of kinesin-1, -2 and -3 to these processes have been investigated using in vitro motility assays in which rat liver Golgi membranes were combined with Xenopus laevis egg extract cytosol in the presence of BFA. Function blocking antibodies and dominant negative proteins were used to perturb the activities of various kinesin motors. This data indicates a particular isoform of kinesin-3, KIF1C, is solely responsible for the movement of BFA-induced tubules in vitro. This work was complemented by in vivo immunofluorescence studies using the HeLaM cultured cell line. Transient transfections of dominant negative proteins, or siRNA-mediated depletion, were used to disrupt the activities of various kinesin motors, either in isolation or in combination with each other. This approach revealed a contribution of KIF1C and kinesin-1 to the movement of early endosomal BFA-induced tubules in vivo.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:529203 |
| Date | January 2010 |
| Creators | Brownhill, Kim |
| Contributors | Allan, Victoria |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/function-and-regulation-of-kinesin1-2-and-3(572d409a-6afb-446a-8f84-832383201666).html |
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