Our long-term goal is to elucidate the molecular mechanism of the cytoplasmic dynein-dynactin motor complex, and to define the molecular bases of dynein-related diseases in humans. Dynein is the primary vehicle for microtubule minus-end-directed transport in eukaryotic cells. The function and dysfunction of this vital motor and its regulatory proteins contribute to a broad set of cellular functions and human diseases. Despite increasing efforts to define dynein's functional properties, the molecular mechanisms that govern dynein's mechanochemistry remain poorly understood. This deficiency largely stems from dynein's structural complexity. Dynein belongs to the AAA+ class of ATP-hydrolyzing mechanoenzymes that assemble into ring- shaped structures, and therefore, possesses characteristically distinct structural features compared to the other two cytoskeletal motor protein families, kinesin and myosin. Dynein is also exceptionally large (~1.2 MDa) and structure-function studies on dynein have been limited by the availability of functional recombinant dynein. Adding to dynein's complexity, dynein associates with multiple accessory chains and the dynactin complex, all of which are essential for nearly every cellular function of dynein. Mutations in dynactin's largest subunit, p150glued, which contains dynactin's putative microtubule-binding domain, cause Perry syndrome and motor neuron degeneration in humans. Yet, the role of p150glued in dynein function remains unknown. In this grant, we seek to overcome these limitations by combining ultrasensitive single-molecule assays with protein engineering. We will use S. cerevisiae, the only source for recombinant full-length dynein and dynactin, to produce stable wildtype and mutant versions of both multiprotein complexes. Using these biochemical tools and multicolor single-molecule fluorescence and optical tweezers methods, we will resolve 1) how dynein's AAA+ motor domains are coordinated within dynein's mechanochemical cycle, 2) how dynactin modulates and regulates dynein function, and 3) how human p150glued mutations disrupt the function of the dynein-dynactin complex. This information will provide insight into cellular physiology and pathophysiology, and potentially identify targets within the dynein-dynactin complex for therapeutic interventions.

Public Health Relevance

Cytoplasmic dynein is vital to various eukaryotic activities, and mutations in its largest regulatory complex dynactin, cause human neurological disease. We are studying the molecular mechanisms that underlie the function and dysfunction of the dynein-dynactin complex.

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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Albert Einstein College of Medicine
Anatomy/Cell Biology
Schools of Medicine
United States
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Rao, Lu; Hülsemann, Maren; Gennerich, Arne (2018) Combining Structure-Function and Single-Molecule Studies on Cytoplasmic Dynein. Methods Mol Biol 1665:53-89
Nicholas, Matthew P; Berger, Florian; Rao, Lu et al. (2015) Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains. Proc Natl Acad Sci U S A 112:6371-6
Nicholas, Matthew P; Höök, Peter; Brenner, Sibylle et al. (2015) Control of cytoplasmic dynein force production and processivity by its C-terminal domain. Nat Commun 6:6206
Nicholas, Matthew P; Rao, Lu; Gennerich, Arne (2014) Covalent immobilization of microtubules on glass surfaces for molecular motor force measurements and other single-molecule assays. Methods Mol Biol 1136:137-69
Nicholas, Matthew P; Rao, Lu; Gennerich, Arne (2014) An improved optical tweezers assay for measuring the force generation of single kinesin molecules. Methods Mol Biol 1136:171-246
Gennerich, Arne (2014) Molecular motors: DNA takes control. Nat Nanotechnol 9:11-2
Rao, Lu; Romes, Erin M; Nicholas, Matthew P et al. (2013) The yeast dynein Dyn2-Pac11 complex is a dynein dimerization/processivity factor: structural and single-molecule characterization. Mol Biol Cell 24:2362-77