The primary objective of this research project is to develop a powerful new molecular-genetic methodology that will allow us and other researchers to connect the well characterized in vitro mechanochemistry of the molecular motor protein kinesin to its relatively poorly understood in vivo transport functions and to the pathology of motor neuron diseases. Kinesin heavy chain (Khc) is the force producing subunit of kinesin-1, which is an abundant microtubule motor that drives many cytoplasmic motility processes, including the transport of mitochondria, vesicles and RNA complexes. Its functionality is particularly important in neurons for delivering newly synthesized components into and along axons toward their synaptic terminals. Defective kinesin-1 function can have serious health consequences, as evidenced by the fact that 20 different mutant amino acid changes in KIF5A, a human Khc homolog, have been identified as causing hereditary spastic paraplegia (HSP) and Charcot-Marie-Tooth (CMT2) neurodegenerative diseases. To allow tests of the effects of specific changes in Khc structure and biophysics on its functions in an intact model organism, we will create a homologous gene replacement tool set in Drosophila that will allow rapid insertion of in vitro engineered versions of the Khc gene at the native locus. The strategy entails a genetically complicated and time consuming homologous recombination to replace the native Khc coding sequence with an enzyme-actuated attP "landing pad" (?Khc-attP). A "delivery plasmid" bearing the missing Khc sequence and a cognate attB integration site (Khc-attB) will be constructed that will allow fast straightforward insertion of modified Khc genes. The homologous recombination step, while somewhat arduous, will need to be accomplished just once. Subsequent insertion of modified Khc genes will be simple and fast. The landing pad/delivery plasmid approach will first be carefully tested with a normal Khc gene replacement to ensure that they recapitulate native expression patterns, normal axonal transport of mitochondria, and a normal developmental life-cycle of the organism. The system will then be used in a pilot project to address questions about how an intensely clustered set of mutant changes in Loop 12 of KIF5A cause HSP/CMT2 neurodegeneration. The new disease model lines will be used for detailed analysis of the effects of those mutations on the axonal transport behavior of mitochondria and neurosecretory vesicles in live animals. The results should allow us to start filling the gaps between structural information on the Loop 12-microtubule binding relationship, the in vivo transport function of kinesin-1, and the mechanisms of HSP/CMT2-like axon degeneration. Validation of the Khc gene replacement tool set by this pilot project will allow us and others to expand its use into many other questions about how kinesin accomplishes its functions in vivo.

Public Health Relevance

There is a wealth of biophysical and structural knowledge of how the microtubule motor protein kinesin generates force and processive movement in vitro, however the specifics of how those in vitro properties influence kinesin's in vivo transport functions are not well understood. Bridging that gap is particularly important, because mutations in kinesin cause debilitating neurodegenerative diseases. We will create, use, and share a powerful new molecular genetic tool set for Drosophila that will allow fast homologous gene replacement of kinesin heavy chain to initiate incisive studies of kinesin structure/function relationships in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Small Research Grants (R03)
Project #
1R03NS078554-01A1
Application #
8512459
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Gubitz, Amelie
Project Start
2013-03-01
Project End
2015-02-28
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
1
Fiscal Year
2013
Total Cost
$76,167
Indirect Cost
$26,167
Name
University of California Santa Cruz
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
125084723
City
Santa Cruz
State
CA
Country
United States
Zip Code
95064