Single-molecule studies of kinesin mechanochemistry
Abstract/Contents
- Abstract
- Kinesin is a class of ATP-powered cytoskeletal motor proteins that translocate along, or exert forces against, microtubules (MTs). Kinesin motors mediate key aspects of a wide range of cellular processes, including cell division as well as long-range transport in neurons and cilia. One of these motors, kinesin-1—the founding member of the kinesin superfamily—is a highly-processive, dimeric motor, capable of transporting cargo micron-scale distances along its MT track. My earliest work explored fundamental aspects of how the ATP hydrolysis cycle and mechanical transitions of kinesin-1 are coordinated to give rise to its remarkable processivity. A key structural element for coordinating the two motor domains, or "heads", of kinesin-1 is the neck linker (NL), which connects each head to a common stalk. By investigating the motility properties of kinesin-1 motors under load using single-molecule optical trapping approaches, we discovered that the structural change driving kinesin motility, most likely NL docking, is completed only upon ATP hydrolysis, rather than immediately after ATP binding, as commonly suggested. In related work, we found that although tension transmitted through the NL when both heads of kinesin-1 are bound to the MT enhances motor velocity, it is not crucial for inter-head coordination. Instead, it is the spatial orientation of the NL that facilitates coordination of biochemical states of the two catalytic heads of a kinesin dimer. My subsequent work entailed leveraging single-molecule kinesin motility assays to extend our mechanistic understanding of cellular processes that require multiple, different kinesins to coordinate their motility in vivo. Intraflagellar transport (IFT)—cargo transport inside cilia—is one such process and is mediated by two different kinesin-2 motors: the slower KIF3AB and the faster KIF17. Using single-molecule characterizations of the motility of both motors, we were able determine the mechanism by which ciliary cargos attain in vivo velocities that are well in excess of the velocity of KIF3AB. Due to their key roles in important cellular processes, kinesins are also central to disease states that arise when these processes go awry, including cancer. Eg5, a kinesin-5 motor, has long been a target of anti-cancer drug development efforts due to its key role in mitosis. Unfortunately, no Eg5 inhibitor has progressed beyond clinical trials, in part because a kinesin-12 motor, KIF15, can rescue cell division when Eg5 is inhibited. However, unlike Eg5, little was known about KIF15, and no studies investigating a KIF15 inhibitor were available in the peer-reviewed literature prior to our work. To address this knowledge gap, my colleagues and I characterized KIF15 motility in detail at the single-molecule level. We also characterized a small-molecule inhibitor of KIF15, and obtained experimental support for earlier proposals that a combination drug therapy employing inhibitors of both KIF15 and Eg5 may be a viable strategy for overcoming KIF15-mediated resistance to inhibitors of Eg5.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2019; ©2019 |
| Publication date | 2019; 2019 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Milic, Bojan |
|---|---|
| Degree supervisor | Block, Steven M |
| Thesis advisor | Block, Steven M |
| Thesis advisor | Fordyce, Polly |
| Thesis advisor | Greenleaf, William James |
| Degree committee member | Fordyce, Polly |
| Degree committee member | Greenleaf, William James |
| Associated with | Stanford University, Biophysics Program. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Bojan Milic. |
|---|---|
| Note | Submitted to the Biophysics Program. |
| Thesis | Thesis Ph.D. Stanford University 2019. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2019 by Bojan Milic
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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