Graduate Thesis Or Dissertation
 

Mechanisms and Regulation of the Mitotic Kinesin-14 KlpA

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  • Faithful segregation of genetic material during cell division is essential to all life on earth. In eukaryotes, the mitotic spindle – a bipolar array consisting of cytoskeletal filaments called microtubules – is the cellular machinery responsible for this function. The mitotic spindle requires both exquisite spatiotemporal organization and the generation of force to physically separate replicated chromosomes. Protein-based nanomachines called molecular motors are responsible for much of this activity within the mitotic spindle. Molecular motors interact directly with microtubules and convert the chemical energy in ATP into mechanical force and movement. One class of these molecular motors, termed kinesins, perform several essential processes within the mitotic spindle. Despite extensive study, the mechanisms that guide kinesin functions in the mitotic spindle remain an open question. Within this dissertation, the mechanisms and regulation of one such mitotic kinesin motor protein are investigated and discussed. These investigations include the development of a new technique that expands our ability to gain insight into kinesin mechanisms. Original work is presented in three chapters in the form of primary research reports. Chapter 2 describes the discovery and characterization of a novel mitotic kinesin-14, KlpA, from the filamentous fungus, Aspergillus nidulans. KlpA is the first processive homodimeric kinesin-14 to be discovered. KlpA is also the first kinesin-14 to exhibit plus end-directed directionality on microtubules. Moreover, KlpA is the first kinesin-14 observed to be bidirectional, relying on its tail domain to maintain a context-dependent directionality. Both contexts that were explored are present within mitotic spindles, suggesting this functional regulation may be conserved in other mitotic kinesins. In chapter 3, the function and regulation of KlpA within the mitotic spindle is further explored. By using a conserved pair of mitotic proteins, TinA and AnWdr8, a regulatory pathway of KlpA was discovered. We found that TinA can cause KlpA to change its direction on microtubules and TinA and AnWdr8 can form a ternary complex with KlpA to anchor it on microtubules. This discovery enabled the proposal of a more complete model for KlpA function within the mitotic spindle. Furthermore, this work revealed that TinA is a microtubule binding protein whose affinity for microtubules is enhanced by AnWdr8. This information enabled the proposal of a potential mechanism for kinesin-14-dependent spindle microtubule anchoring at spindle pole bodies (SPBs) via TinA and AnWdr8. More importantly, this is the first proposed mechanism for kinesin-14-dependent anchoring, which is required for proper function of the mitotic spindle during chromosome segregation. In Chapter 4, an entirely new and powerful method for the generation of kinesin heterodimers is presented. This work was undertaken to expand our ability to probe the fundamental mechanisms of kinesin function and regulation. In order to overcome the shortcomings of existing techniques for kinesin heterodimer formation, genetic code expansion was used to introduce biorthogonal chemistries via noncanonical amino acid incorporation. These chemistries were then used to click together distinct monomeric kinesin motors via a small-molecule linker. Importantly, this artificial tethering did not compromise kinesin motility and enabled formation of a novel heterodimer that was used to probe the mechanics of processive motility of the kinesin-8 protein, Kip3. Lastly, in chapter 5, I revisit the main points and themes of previous chapters in order to discuss the impacts and future directions of my work.
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  • Ongoing Research
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  • 2018-06-21 to 2020-07-22

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