Mechanistic Studies of the Microtubule-Based Motors Dynein and Kinesin-8

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Mechanistic Studies of the Microtubule-Based Motors Dynein and Kinesin-8

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Title: Mechanistic Studies of the Microtubule-Based Motors Dynein and Kinesin-8
Author: Hernandez-Lopez, Rogelio Antonio
Citation: Hernandez-Lopez, Rogelio Antonio. 2015. Mechanistic Studies of the Microtubule-Based Motors Dynein and Kinesin-8. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
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Abstract: The precise delivery and organization of intracellular factors in space and time relies on a set of molecules that move along and regulate the dynamics of cytoskeletal filaments. The two families of microtubule-based motors-- dyneins and kinesins-- power vital biological processes such as intracellular transport, chromosome segregation and more broadly cell-cell communication and cell polarization. Despite their role in such diverse activities, their molecular mechanisms remain poorly understood. Combining biochemistry, cryo-electron microscopy, molecular dynamics simulations and single molecule biophysics, we provide novel insights into the mechanistic basis of how dynein and kinesin-8 interact with microtubules (MTs) to regulate their function.
Cytoplasmic dynein is a homodimer that moves for long distances along MTs without dissociating, a property known as processivity. Its movement requires coupling cycles of ATP binding and hydrolysis to changes in affinity for its track. Intriguingly, the main site of ATP hydrolysis in the motor is separated from the microtubule binding domain (MTBD) by 25 nm. How do these sites communicate with each other? What are the changes responsible for modulating the affinity between the motor and its track during dynein’s mechanochemical cycle? Furthermore, it has been shown that dynein’s stepping behavior is highly variable. Dynein walks by taking a broad distribution of step sizes; some of its steps are sideways and some are backwards. Is dynein’s stepping behavior dictated by the motor’s ATPase activity or dynein’s affinity for MTs? To address these important questions, first, we solved a cryo-EM reconstruction of dynein’s MTBD bound to the MT. We found that upon MT binding, dynein’s MTBD undergoes a large conformational change underlying changes in its affinity for MTs. Our structural model suggested specific negatively charged residues within the MTBD that tune dynein’s affinity for MTs. We mutated these residues to alanine and show a dramatic increase in dynein’s MT binding affinity resulting in enhanced (~5-6 fold) motor processivity. These mutants provide us with a tool to explore the role of MT-binding affinity in dynein’s stepping behavior. We characterized, using single molecule experiments, the stepping pattern of the high MT binding affinity dyneins. We found that an increased MT-binding affinity reduces dynein’s stepping rate and impairs the coupling between ATPase activity and stepping. Together, our results provide a model for how dynein has evolved a finely tuned mechanism that allows its MTBD to communicate MT-binding to its motor domain. This mechanism also regulates dyneins’s affinity for the MT and motor’s processivity.
We then sought to understand the unique functional properties of kinesin-8. Unlike other kinesins that have the ability to either move along microtubules or regulate the dynamics of MT-ends, kinesin-8s can do both. Kip3, the budding yeast kinesin-8, is a highly processive motor capable of dwelling at the MT plus-end and it is a MT depolymerase. Given the highly conserved sequence and structure of kinesin’s motor domain, how is that Kip3 can perform these two distinct functions? Does Kip3 interact with the MT-lattice in the same manner than that at the MT-end? We characterized, structurally, how Kip3 binds to microtubules that mimic the MT-lattice and the MT-end. We have identified and tested specific residues within Kip3 that are responsible for Kip3’s processivity, MT-end dwelling and depolymerization activity.
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Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467496
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