The kinesin-1 motor drives the transport of numerous cellular cargoes toward the plus ends of microtubules. Kinesin-1 can inhibit its own activity via a direct interaction between its C-terminal tail and enzymatic motor domains. In the cell, kinesin-1 can be further regulated by interactions with cargoes as well as with the microtubule track. We investigated the effect of a microtubule-associated protein, ensconsin, on kinesin-1 activity.
RNAi-mediated depletion and live cell imaging studies in Drosophila S2 cells revealed that ensconsin is required for the primary function of kinesin-1, organelle transport, as well as microtubule-microtubule sliding, a newly described function of the motor. We found that ensconsin is required for organelle transport in Drosophila neurons, and Drosophila homozygous for ensconsin gene deletion are unable to survive to adulthood, highlighting the importance of this MAP in the animal's development.
We performed rescue experiments using truncated ensconsin mutants, and determined that a C-terminal region of ensconsin, while unable to bind microtubules, is required for kinesin-1-dependent organelle transport. This finding suggests that microtubule binding, via a separate, dedicated domain, may act as a mechanism to localize ensconsin's motor-activating domain near the microtubule surface.
Through analysis of kinesin-1 mutants, we demonstrated that a "hingeless" mutant of kinesin-1, which mimics the active conformation of the motor, does not require ensconsin for cargo transport in vivo. This suggests that ensconsin plays a role in relieving autoinhibition of kinesin-1. Together, this work leads to a model in which ensconsin provides regulatory control over kinesin-1 in vivo near the microtubule surface, and adds to a growing body of evidence demonstrating that microtubules act not only as passive tracks for transport, but may also directly influence motor proteins and cargo delivery.
|Advisor:||Gelfand, Vladimir I.|
|Commitee:||Green, Kathleen J., Mitchell, Brian J., Topczewski, Jacek|
|School Location:||United States -- Illinois|
|Source:||DAI-B 75/01(E), Dissertation Abstracts International|
|Subjects:||Biology, Cellular biology|
|Keywords:||Ensconsin, Kinesin, Kinesin-1 motor activity, Microtubule, Microtubule-binding proteins, Mitochondria, Regulation, Transport|
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