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 range 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 distinct structural features compared to the other two cytoskeletal motor protein families, kinesin and myosin. Dynein is also exceptionally large (~1.4 MDa) and structure function studies on dynein have been limited until recently 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 the dynein heavy chain and dynactin's largest subunit, p150glued, which contains dynactin?s putative microtubule-binding domain, cause devastating neurological diseases. However, mechanistic knowledge of dynein?s function?and therefore its dysfunction? is limited compared to kinesin and myosin, which poses a major barrier for the development of targeted therapies. In this grant, we seek to overcome these limitations by combining ultrasensitive single-molecule assays with mutagenesis and structure-function studies. We will employ S. cerevisiae, insect and human cell-based expression systems 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 decipher the molecular mechanism underlying the processive motion of the dynein-dynactin complex and determine how dynactin regulates dynein force generation. This information will provide insights into cellular physiology and identify targets within the dynein-dynactin complex for therapeutic interventions.

Public Health Relevance

Cytoplasmic dynein is vital to various eukaryotic activities, and mutations in dynein and 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098469-10
Application #
9900796
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Ainsztein, Alexandra M
Project Start
2012-08-01
Project End
2022-03-31
Budget Start
2020-04-01
Budget End
2021-03-31
Support Year
10
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
DUNS #
081266487
City
Bronx
State
NY
Country
United States
Zip Code
10461
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) 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
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
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