Zwetsloot, Alexander J. (2021) Bidirectional transport on microtubules : cooperation of Kif1C and dynein. PhD thesis, University of Warwick.

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Abstract

Cells utilise their microtubule cytoskeletal network for transport of cargoes ranging in size from small vesicles to large organelles, and even the nucleus. Kinesin motor proteins transport cargoes towards the plus end of microtubules, while dynein is responsible for minus end directed transport. Inside cells, cargoes can be seen to frequently change direction on microtubules, indicating that cargoes have both types of molecular motor bound, and that there exists a mechanism that allows switching between directionality. Bidirectionality likely allows cargoes to avoid obstacles and to rapidly search the cytoplasm for their destination, and kinesin and dynein’s functions are interlinked such that depletion of a kinesin is sufficient to disrupt dynein driven transport and vice versa. A mechanism of bidirectional transport that explains this codependence of opposite polarity motors is yet to be elaborated.

A potential key player in bidirectional transport is the kinesin3 KIF1C, which has been shown to interact with multiple dynein adaptors, and in doing so may link itself to dynein. Using in vitro reconstitution, we investigated the mechanism of activation of KIF1C and found that the dynein adaptor Hook3 is able to relieve its autoinhibition. In single molecule microscopy assays, Hook3, but not BICD2 and BICDR1, frequently forms comotile complexes. Using a rapamycin induced cargo transport assay in cells, we found Hook3 driven intracellular transport is rapid, bidirectional, and sensitive to the concentration of active KIF1C. Using an acutely inhibitable KIF1C, we revealed that KIF1C inside the cell is physically interlinked with dynein on short timescales. In vitro reconstitution of a cocomplex of KIF1C and dynein in the presence of dynactin and Hook3 showed bidirectional motility in single molecule microscopy assays, and codependence of opposite polarity motors was reconstituted for the first time as dynein’s motility improves in the presence of KIF1C by acting as a processivity tether. We developed a method to measure the intensity of running motors and found that KIF1C promotes the formation of complexes containing two dynein dimers. We propose that dynein/kinesin cocomplexes may be better able to avoid microtubule obstacles, and predict that other dynein adaptor proteins may permit the formation of yet uncharacterised dynein/kinesin cocomplexes.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QH Natural history Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Microtubules, Cytoskeletal proteins, Kinesin, Dynein
Official Date:	March 2021
Dates:	Date Event March 2021 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	Warwick Medical School
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Straube, Anne ; Nédélec François
Format of File:	pdf
Extent:	xii, 1, 1-161 leaves : illustrations
Language:	eng

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