Kinesin-2 motors carry out anterograde transport in cilia and flagella, as well as other bidirectional transport processes in cells. Although many aspects of kinesin mechanochemistry are understood, the specific motor activities and regulation that underlie multi-motor and bidirectional cargo transport are not well understood. Deficiencies in Kinesin-2 transport lead to abnormal development, photoreceptor degradation and polycystic kidney disease. The goals of this project are to understand the mechanism by which Kinesin-2 motors walk along microtubules, and the degree to which Kinesin-2 motor properties are tuned for its specific transport tasks. A notable difference from the canonical Kinesin-1 motor family is that neck linker domain of Kinesin-2, which connects the core motor head to the coiled-coil domain, is 17 amino acids compared to only 14 in Kinesin-1. Because the neck linker serves as a mechanical element connecting each head to their shared coil-coil, it is expected that extending the neck linker will diminish mechanochemical coupling between the head domains. Consistent with this, we found that extending the Kinesin-1 neck linker diminishes its processivity and shortening the Kinesin-2 neck linker enhances processivity. In addition, we found that the force dependence of Kinesin-2 velocity and run length differ from Kinesin-1, suggesting the motor may be optimally tuned for bidirectional cargo transport rather than long distance unidirectional transport. Using single-molecule and multi-motor experiments in conjunction with computational modeling of the kinesin chemomechanical cycle, we will uncover the structural basis of mechanistic differences between Kinesin-1 and Kinesin-2 motors, with the goal of understanding bidirectional transport in vivo. This work involves structural studies to determine the role played by the neck linker and neck coil domains in processive movement along microtubules. We will also investigate specific steps in the Kinesin-2 chemomechanical cycle to determine how the motor biochemistry is controlled by intramolecular tension between the two head domains, as well as by external loads applied by optical tweezers. These investigations into the mechanism of Kinesin-2 motility will provide a framework in which to understand Kinesin-2 transport in cells. By benchmarking against Kinesin-1, these measurements will establish universal themes underlying kinesin mechanochemistry that will help to better understand the molecular basis of neurodegenerative diseases and aid the effort to develop anti-tumor therapies targeting mitotic kinesins.

Public Health Relevance

Kinesin-2 motors transport intracellular cargo along microtubule filaments in the cell and in cilia and flagella, and deficiencies in Kinesin-2 transport lead to abnormal development, photoreceptor degradation and polycystic kidney disease. This study will investigate the molecular mechanism of Kinesin-2 motility to understand the cellular role of this motor and to develop general paradigms for understanding kinesin-driven transport. Experimental approaches include single-molecule studies and measuring biochemical kinetics, and results will be interpreted in the context of mathematical models of this mechanoenzyme.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM076476-06
Application #
8287008
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Gindhart, Joseph G
Project Start
2006-05-01
Project End
2015-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
6
Fiscal Year
2012
Total Cost
$296,877
Indirect Cost
$87,877
Name
Pennsylvania State University
Department
Biomedical Engineering
Type
Schools of Engineering
DUNS #
003403953
City
University Park
State
PA
Country
United States
Zip Code
16802
Milic, Bojan; Andreasson, Johan O L; Hancock, William O et al. (2014) Kinesin processivity is gated by phosphate release. Proc Natl Acad Sci U S A 111:14136-40
Chen, Yalei; Rolls, Melissa M; Hancock, William O (2014) An EB1-kinesin complex is sufficient to steer microtubule growth in vitro. Curr Biol 24:316-21
Hancock, William O (2014) Bidirectional cargo transport: moving beyond tug of war. Nat Rev Mol Cell Biol 15:615-28
Hoeprich, Gregory J; Thompson, Andrew R; McVicker, Derrick P et al. (2014) Kinesin's neck-linker determines its ability to navigate obstacles on the microtubule surface. Biophys J 106:1691-700
Hughes, John; Shastry, Shankar; Hancock, William O et al. (2013) Estimating Velocity for Processive Motor Proteins with Random Detachment. J Agric Biol Environ Stat 18:204-217
Hughes, John; Hancock, William O; Fricks, John (2012) Kinesins with extended neck linkers: a chemomechanical model for variable-length stepping. Bull Math Biol 74:1066-97
Shastry, Shankar; Hancock, William O (2011) Interhead tension determines processivity across diverse N-terminal kinesins. Proc Natl Acad Sci U S A 108:16253-8
Cai, Dawen; Kaul, Neha; Lionberger, Troy A et al. (2010) Recording single motor proteins in the cytoplasm of mammalian cells. Methods Enzymol 475:81-107
Kutys, Matthew L; Fricks, John; Hancock, William O (2010) Monte Carlo analysis of neck linker extension in kinesin molecular motors. PLoS Comput Biol 6:e1000980
Shastry, Shankar; Hancock, William O (2010) Neck linker length determines the degree of processivity in kinesin-1 and kinesin-2 motors. Curr Biol 20:939-43

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