The proper distribution of cellular organelles and protein complexes is important for maintaining cellular organization and function. Transport in eukaryotic cells requires three motor proteins, kinesin, dynein, and myosin, attached to specific cargoes mostly by adaptor proteins. This work focuses on the mechanism of microtubule-dependent organelle transport in vivo. This study examines three questions. Firstly, are motors of opposite polarity coordinated while transporting the same cargo? Intracellular organelles move along microtubules bidirectionally by kinesin and dynein, but how theses motors work together is not known. This study showed that the depletion of kinesin-1 or dynein inhibited transport of peroxisomes in both directions, suggesting that peroxisomes require activities of both motors for transport. Secondly, does dynactin, the dynein adaptor protein complex, function as a regulator between motors? Does it affect cargo transport through its microtubule binding activity? In vitro studies showed that dynactin increased motor processivity, but, its in vivo roles by microtubule binding are not known. This study used a p150glued mutant (ΔN-p150glued) that was truncated in the microtubule binding domain. Movement of organelles required dynactin, but ΔN- p150glued did not affect the processivity, run length and velocity of transport. Dynactin binding to microtubules is not required for cargo transport in vivo and although dynactin may influence transport, this effect is not mediated through microtubule binding. Lastly, is organelle transport affected by microtubule movements? Are microtubules motile? Where do the forces that move microtubules originate? There are not many studies done on microtubule transport. This study showed that microtubules display sliding, looping, and bending movement. These microtubule movements were significantly inhibited by depletion of kinesin-1. We suggest that kinesin-1 moves (or slides) microtubules by cross-linking microtubules through its C-terminal microtubule-binding domain. We also showed that microtubule movement/sliding did not affect organelle transport. The research presented here contributes to the understanding of microtubule-dependent transport in three ways: first, by providing evidence for interdependence between kinesin-1 and dynein; second, by exploring the role of the microtubule binding by dynactin on transport; and third, by providing a mechanism of microtubule movements mediated by kinesin-1.

Last modified

• 09/06/2018

Creator

• Hwajin Kim

DOI

• https://doi.org/10.21985/N24Q7N

Subject

• Chemical and Biological Engineering

Keyword

• Cell Biology

Date created

• 2008-03-14

Resource type

• Dissertation

Rights statement

• In Copyright