Molecular motors drive key biological processes such as intracellular cargo transport and cell division. Two dimeric motors, kinesin and cytoplasmic dynein, can take many consecutive steps along microtubules to transport cargos over long distances. This continuous movement, termed processivity, requires coordination between the two motor domains to prevent premature release from the microtubule. Detailed structural and mechanistic models exist for kinesin, but the mechanism and coordination of dynein motility remains largely unknown. Dynein's unconventional structure and distinct origin suggest that it has different mechanistic features than other cytoskeletal motors. Dynein forms a large multisubunit complex, the core of which consists of a ring of AAA ATPase domains. Conformational changes driven by ATP hydrolysis within the ring underlie dynein force generation and motion. Recent structural and biochemical studies have identified the major conformational states of monomeric dynein constructs. However, studies of active dynein dimers are lacking. As a result, the molecular basis by which ATP driven structural changes lead to unidirectional motion of a dimer as a whole is unknown. In our preliminary work, we have used S. cerevisiae to express recombinant dynein motors and characterized dynein stepping behavior in vitro. In this proposal, using single-molecule imaging methods, we propose to dissect the coordination between the nucleotide and conformational states of the motor domains in native and engineered dynein constructs. We have three specific aims. First, using multicolor tracking methods, we will directly observe how the AAA ring domains coordinate their nucleotide cycles and move relative to each other. The specific roles of distinct AAA domains will be studied by selectively mutating out the ATPase sites in one ring. Second, we will investigate how ATP-driven conformational states of the motor domain drive the dynein powerstroke and alter microtubule-binding affinity. The ability to perform these measurements as dynein walks will allow us to demonstrate whether the mechanical cycle of one head is gated until the other head completes its forward step. Third, we will establish the structural basis of dynein's minus-end directionality. Together, our proposed research represents a focused investigation of the conformational and chemical states of dynein at a single-molecule level, as active dynein dimers move along surface-immobilized MTs. We hope to significantly advance understanding of dynein's fundamental mechanochemistry and learn how it achieves retrograde transport of intracellular cargos.

Public Health Relevance

Consistent with its fundamental roles in neurobiology and cell development, complete knockouts of dynein stop the entire microtubule transport machinery and inhibit mitosis. Mutations that alter the processivity or velocity of dynein movement lead to pathogenesis of motor neuron degeneration, including the Alzheimer's disease and ALS. Detailed studies of dynein-related diseases require replacement of engineered dynein mutants whose motility properties have been altered in predictable ways. Dissecting the mechanism of dynein motility is a prerequisite of understanding the molecular basis of these diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM094522-03
Application #
8450779
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Gindhart, Joseph G
Project Start
2011-04-01
Project End
2016-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
3
Fiscal Year
2013
Total Cost
$274,680
Indirect Cost
$91,330
Name
University of California Berkeley
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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Cleary, Frank B; Dewitt, Mark A; Bilyard, Thomas et al. (2014) Tension on the linker gates the ATP-dependent release of dynein from microtubules. Nat Commun 5:4587
Belyy, Vladislav; Yildiz, Ahmet (2014) Processive cytoskeletal motors studied with single-molecule fluorescence techniques. FEBS Lett 588:3520-5
Can, Sinan; Dewitt, Mark A; Yildiz, Ahmet (2014) Bidirectional helical motility of cytoplasmic dynein around microtubules. Elife 3:e03205
Nan, Beiyan; Bandaria, Jigar N; Moghtaderi, Amirpasha et al. (2013) Flagella stator homologs function as motors for myxobacterial gliding motility by moving in helical trajectories. Proc Natl Acad Sci U S A 110:E1508-13
DeWitt, Mark A; Chang, Amy Y; Combs, Peter A et al. (2012) Cytoplasmic dynein moves through uncoordinated stepping of the AAA+ ring domains. Science 335:221-5