The cellular contents of eukaryotic organisms are highly dynamic, yet organized spatially and temporally. Microtubules and their motors play central roles in these processes and defects in this machinery cause neurological diseases. We focus on cytoplasmic dynein-1 (?dynein?), the motor responsible for nearly all minus- end-directed (typically towards the cell interior) transport along microtubules. The basic dynein machine consists of a dimer of motor subunits and 5 additional subunits that are each present in two copies. Mammalian dynein exists in a closed ?Phi? conformation that converts to an ?Open? conformation. Binding to dynactin, a large regulatory complex, and a coiled coil-containing activating adaptor stabilizes the Open conformation. This DDX (Dynein, Dynactin, X = an activating adaptor) complex moves processively on microtubules. There are about a dozen activating adaptors, which also link dynein to its cargo and some activating adaptors recruit two dynein dimers (D2DX). S. cerevisiae dynein does not form a stable Phi particle, and as a result is processive on its own, making it an ideal model system for studying basic questions about dynein regulation. In this proposal we focus on how two dynein regulators, Lis1 and Nudel, which are conserved from yeast to human alter the activity of yeast and human dynein. Building on our finding in the previous funding cycle that Lis1 regulates yeast dynein in opposing ways depending on the stoichiometry of its interaction with dynein, we will determine the mechanism of this unique form of regulation. We will also determine how Lis1 affects dynein?s response to load using wild-type dynein and mutants that can?t bind Lis1 at one of its two binding sites. We will then turn our focus to regulation of human dynein by Lis1, NDE1 and NDEL1, the two human Nudel genes. Based on our preliminary findings, we will test the hypothesis that Lis1 and Nudel regulate the Phi to Open transition of dynein. Next we will determine how Lis1 and Nudel regulate active DDX or D2DX complexes, measuring parameters such as stabilization of the active complex, its motile properties, and its response to load. For all of these experiments we will use a combination of cryo-electron microscopy to solve structures, single-molecule motility assays, optical trapping, and biochemical reconstitutions and live-cell imaging to test our hypotheses. Finally, we have identified the human Lis1 and Nudel protein interactomes and we will determine how novel protein interactions we identified affect Lis1 and Nudel regulation of human dynein.
Dynein is a large and complex molecular motor that transports many 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 motility is regulated by other proteins, giving dynein the functional plasticity it needs to carry out its many cellular roles.
|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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