Kinesin motors carry out bidirectional transport in neurons, organize the mitotic spindle during cell division, and are involved in a number of other vital cellular processes. Significant progress has been made in understanding the mechanochemical transitions that underlie kinesin stepping; however, identifying the key structural features and the rates of structural and biochemical transitions that underlie the functional diversity observed across the kinesin superfamily remains elusive. Our ongoing work suggests that: i) differences in unloaded processivity between kinesin families result from differences in the length of their neck linker domains, a 14-18 residue sequence connecting the catalytic core to the coiled-coil domain, and ii) differences in motors' response to load result from properties resident in the core motor domain. We hypothesize that the processivity of dimeric kinesins results from a race between attachment of the tethered head to the next tubulin binding site and dissociation of the bound motor from the microtubule. Until now, there has been no reliable way to the detachment/reattachment dynamics of one head in a processive kinesin dimer. We will attach gold nanoparticles to individual heads in a kinesin dimer and track the particles with nm and sub-msec resolution using Interferometric Scattering (iSCAT) and Total Internal Reflection Dark-Field (TIRDF) Microscopy. We recently showed that iSCAT can detect substeps at saturating ATP levels that have not been measured by optical tweezers, the benchmark in the field.
Aim 1 is to identify transitions in the mechanochemical cycles of kinesin-1, the intraflagellar transport motor kinesin-2, and the `superprocessive' kinesin-3 that govern their differing behaviors. To understand how kinesins transport cargo along the crowded microtubules found in cells, Aim 2 will identify features of the hydrolysis cycle that enable motors to step around roadblocks.
In Aim 3 these investigations will expand to the mitotic motor kinesin-5, which we recently found to be a microtubule polymerase. Experiments in Aim 3 will identify the specific features of the kinesin-5 chemomechanical cycle that lead to its plus-end-binding and microtubule polymerase activity. We will test the hypothesis that the contrasting influence of load on these motors results from what proportion of the hydrolysis cycle the motor resides in a vulnerable one-head-bound state. By applying new techniques to measure rapid mechanical transitions during kinesin stepping, this work will help to define a general model for kinesin mechanochemistry that can be applied to diverse motors in the superfamily. This understanding is necessary to extrapolate observations at the single-molecule level to the complex dynamics of multi-motor transport in cells in both normal and diseased states.

Public Health Relevance

Kinesin molecular motors transport cargo in neurons and organize the mitotic spindle during cell division, and defects in axonal transport are linked to neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Alzheimer's disease. It is now clear that motors from a number of kinesin families are involved in these processes, but how they interact and how their motor properties differ is not clear. Work in this project will use high resolution optical microscopy, biochemical measurements, and computational modeling to understand the inner workings of molecular motors involved in intracellular transport and cell division, which will help us to better understand the molecular basis of neurodegenerative diseases and aid the effort to develop anti-tumor therapies targeting mitotic kinesins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM076476-10
Application #
9336931
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Gindhart, Joseph G
Project Start
2006-05-01
Project End
2020-05-31
Budget Start
2017-06-01
Budget End
2018-05-31
Support Year
10
Fiscal Year
2017
Total Cost
$344,145
Indirect Cost
$122,605
Name
Pennsylvania State University
Department
Biomedical Engineering
Type
Schools of Engineering
DUNS #
003403953
City
University Park
State
PA
Country
United States
Zip Code
16802
Pisupati, Aditya; Mickolajczyk, Keith J; Horton, William et al. (2018) The S6 gate in regulatory Kv6 subunits restricts heteromeric K+ channel stoichiometry. J Gen Physiol 150:1702-1721
Gicking, Allison M; Qiu, Weihong; Hancock, William O (2018) Mitotic kinesins in action: diffusive searching, directional switching, and ensemble coordination. Mol Biol Cell 29:1153-1156
Feng, Qingzhou; Mickolajczyk, Keith J; Chen, Geng-Yuan et al. (2018) Motor Reattachment Kinetics Play a Dominant Role in Multimotor-Driven Cargo Transport. Biophys J 114:400-409
Guan, Ruifang; Zhang, Lei; Su, Qian Peter et al. (2017) Crystal structure of Zen4 in the apo state reveals a missing conformation of kinesin. Nat Commun 8:14951
Arellano-Santoyo, Hugo; Geyer, Elisabeth A; Stokasimov, Ema et al. (2017) A Tubulin Binding Switch Underlies Kip3/Kinesin-8 Depolymerase Activity. Dev Cell 42:37-51.e8
Chen, Geng-Yuan; Kang, You Jung; Gayek, A Sophia et al. (2017) Eg5 Inhibitors Have Contrasting Effects on Microtubule Stability and Metaphase Spindle Integrity. ACS Chem Biol 12:1038-1046
Mickolajczyk, Keith J; Hancock, William O (2017) Kinesin Processivity Is Determined by a Kinetic Race from a Vulnerable One-Head-Bound State. Biophys J 112:2615-2623
Hancock, William O (2016) The Kinesin-1 Chemomechanical Cycle: Stepping Toward a Consensus. Biophys J 110:1216-25
Chen, Geng-Yuan; Mickolajczyk, Keith J; Hancock, William O (2016) The Kinesin-5 Chemomechanical Cycle Is Dominated by a Two-heads-bound State. J Biol Chem 291:20283-20294
Andrecka, J; Takagi, Y; Mickolajczyk, K J et al. (2016) Interferometric Scattering Microscopy for the Study of Molecular Motors. Methods Enzymol 581:517-539

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