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 #
5R01GM094522-02
Application #
8242076
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
2012-04-01
Budget End
2013-03-31
Support Year
2
Fiscal Year
2012
Total Cost
$285,279
Indirect Cost
$95,279
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
Bandaria, Jigar N; Qin, Peiwu; Berk, Veysel et al. (2016) Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin. Cell 164:735-46
Belyy, Vladislav; Schlager, Max A; Foster, Helen et al. (2016) The mammalian dynein-dynactin complex is a strong opponent to kinesin in a tug-of-war competition. Nat Cell Biol 18:1018-24
Wu, Robert Alexander; Dagdas, Yavuz S; Yilmaz, S Tunc et al. (2015) Single-molecule imaging of telomerase reverse transcriptase in human telomerase holoenzyme and minimal RNP complexes. Elife 4:
Nan, Beiyan; Bandaria, Jigar N; Guo, Kathy Y et al. (2015) The polarity of myxobacterial gliding is regulated by direct interactions between the gliding motors and the Ras homolog MglA. Proc Natl Acad Sci U S A 112:E186-93
DeWitt, Mark A; Cypranowska, Caroline A; Cleary, Frank B et al. (2015) The AAA3 domain of cytoplasmic dynein acts as a switch to facilitate microtubule release. Nat Struct Mol Biol 22:73-80
Dogan, Merve Yusra; Can, Sinan; Cleary, Frank B et al. (2015) Kinesin's front head is gated by the backward orientation of its neck linker. Cell Rep 10:1967-73
Belyy, Vladislav; Yildiz, Ahmet (2014) Processive cytoskeletal motors studied with single-molecule fluorescence techniques. FEBS Lett 588:3520-5
Ray, Sujay; Bandaria, Jigar N; Qureshi, Mohammad H et al. (2014) G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding. Proc Natl Acad Sci U S A 111:2990-5
Belyy, Vladislav; Hendel, Nathan L; Chien, Alexander et al. (2014) Cytoplasmic dynein transports cargos via load-sharing between the heads. Nat Commun 5:5544
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

Showing the most recent 10 out of 14 publications