The active transport of organelles, membrane vesicles, and proteins across great distances along the microtubule (MT) cytoskeleton within axons is critical to the function of our nervous system. This ATP-fueled shuttling of vital cytoplasmic components is accomplished by two types of motors - kinesins, which transport cargo towards the axon tips, and dyneins, which move in the retrograde direction. While the molecular basis of kinesin-based transport is well- characterized, our understanding of the more sophisticated dynein motor is severely lacking. Cargo-binding to dynein is mediated by a multiprotein complex named dynactin, which interacts with the dynein tail as well as MTs, and significantly increases dynein processivity. Dynein movement along MTs is also heavily influenced by a wide array of MT-associated proteins (MAPs), which also play a critical role in stabilizing the MT highways in neurons. The list of neurological diseases linked to deficiencies in dynein/dynactin-dependent transport include Huntington's, Parkinson's, Alzheimer's, microcephaly, lissencephaly, Perry syndrome, and spino-cerebellar ataxia to name a few, underscoring the biomedical relevance of this system. Using cryoEM, I plan to elucidate the structure-function relationships that give rise to the loading and transport of intracellular cargo by the dynein-dynactin complex along MAP- stabilized MTs. We have already obtained a low-resolution 3D structure of dynactin, and developed algorithms for analyzing the flexible components of dynein. Over the next few years, we will resolve the molecular details of dynactin recruitment to dynein with near-atomic precision, outlining the specific interactions that influenc cargo tethering and promote dynein processivity along MTs. Concurrently, we will explore the structural motifs involved in MAP-induced MT stabilization. The resulting mechanistic framework for this cargo transport system will provide a structural context for the known mutations leading to the aforementioned diseases, highlighting key interactions or conformational switches that are required for proper neurological function. These specific dynein and dynactin components will then be further probed by cryoEM using recombinant expression systems in order to identify the precise causes for abrogation of function, providing the basis for future work aimed at restoring native behavior to mutated subunits. Through the course of these studies, we will additionally develop and optimize a novel molecular labeling technique for protein localization by EM, which will have far-reaching implications in the field.

Public Health Relevance

Modern science's ability to combat neurological disorders is significantly hampered by our limited understanding of the motors that transport nutrients within neuronal circuits. Using high- resolution 3D imaging, I will solve the structure of the critical moor system that is involved in clearing dangerous protein tangles away from neurons, bringing us substantially closer to developing cures for neurodegenerative diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
NIH Director’s New Innovator Awards (DP2)
Project #
1DP2EB020402-01
Application #
8756639
Study Section
Special Emphasis Panel (ZRG1-MOSS-C (56))
Program Officer
Conroy, Richard
Project Start
2014-09-30
Project End
2019-06-30
Budget Start
2014-09-30
Budget End
2019-06-30
Support Year
1
Fiscal Year
2014
Total Cost
$2,842,500
Indirect Cost
$1,342,500
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037