The long-term goal of this research project is to understand how the microtubule (MT)-based motor cytoplasmic dynein (""""""""dynein"""""""") is regulated. Dynein is the largest and most complex of all cytoskeletal motors; this >1 MDa dimeric complex contains numerous mechanical elements whose movements must be coordinated over strikingly long molecular distances to achieve processive motility along MTs. In addition to its enormous size, dynein is also the most versatile of the molecular motors;in sharp contrast to the 45 kinesins and 39 myosins present in humans, a single dynein gene product is responsible for transporting macromolecules within neurons, constructing the mitotic spindle, polarizing cells, and anchoring mRNAs during development. To give dynein the functional plasticity necessary for carrying out its many roles, several ubiquitous co-factors interact with dynein, including Lis1 and the dynactin complex. This project will apply our combined expertise in biochemistry, single-molecule biophysics and cryo-electron microscopy to address the structural and mechanistic bases of dynein's interaction with MTs and its regulation by Lis1 and dynactin. We recently showed that binding of dynein to MTs is accompanied by conformational changes in its MT- binding domain and that Lis1 acts as a """"""""clutch"""""""" to uncouple MT binding and release from ATP hydrolysis, promoting a strongly MT-attached state. Dynactin, a 1.2 MDa complex, enhances dynein's processivity and is required for nearly all dynein functions in cells, but its mechanism of action is poorly understood. This grant will address major mechanistic questions about dynein and its regulation. What are the structural and mechanistic bases of MT binding (Aim 1), and of regulation by Lis1 (Aim 2) and dynactin (Aim 3)?

Public Health Relevance

Dynein is a large and complex molecular motor that transports a wide variety of cellular cargos. This is crucial in neurons where defects in dynein-mediated transport lead to neurodevelopmental and neurodegenerative diseases. This research will provide insight into these diseases at a molecular level by investigating how dynein's multiple mechanical elements are coordinated to allow movement, and how this coordination is regulated by other proteins to give dynein the functional plasticity it needs to carry out its many cellular roles.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Gindhart, Joseph G
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Harvard University
Anatomy/Cell Biology
Schools of Medicine
United States
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Cianfrocco, Michael A; Lahiri, Indrajit; DiMaio, Frank et al. (2018) cryoem-cloud-tools: A software platform to deploy and manage cryo-EM jobs in the cloud. J Struct Biol 203:230-235
Salogiannis, John; Reck-Peterson, Samara L (2017) Hitchhiking: A Non-Canonical Mode of Microtubule-Based Transport. Trends Cell Biol 27:141-150
DeSantis, Morgan E; Cianfrocco, Michael A; Htet, Zaw Min et al. (2017) Lis1 Has Two Opposing Modes of Regulating Cytoplasmic Dynein. Cell 170:1197-1208.e12
Redwine, William B; DeSantis, Morgan E; Hollyer, Ian et al. (2017) The human cytoplasmic dynein interactome reveals novel activators of motility. Elife 6:
Salogiannis, John; Egan, Martin J; Reck-Peterson, Samara L (2016) Peroxisomes move by hitchhiking on early endosomes using the novel linker protein PxdA. J Cell Biol 212:289-96
Cianfrocco, Michael A; Leschziner, Andres E (2015) Low cost, high performance processing of single particle cryo-electron microscopy data in the cloud. Elife 4:
Reck-Peterson, Samara L (2015) Dynactin revealed. Nat Struct Mol Biol 22:359-60
Cianfrocco, Michael A; DeSantis, Morgan E; Leschziner, Andres E et al. (2015) Mechanism and regulation of cytoplasmic dynein. Annu Rev Cell Dev Biol 31:83-108
Cianfrocco, Michael A; Leschziner, Andres E (2014) Traffic control: adaptor proteins guide dynein-cargo takeoff. EMBO J 33:1845-6
Toropova, Katerina; Zou, Sirui; Roberts, Anthony J et al. (2014) Lis1 regulates dynein by sterically blocking its mechanochemical cycle. Elife 3:

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