The long term research goal of this project is to understand how cytoskeletal motors power the transport of diverse macromolecules within eukaryotic cells, enabling them to effectively organize their contents, move, divide, and respond to signals. This proposal focuses on cytoplasmic dynein, the largest, most complex, and least understood of the cytoskeletal motors. The specific objectives are to determine how single dynein dimers move processively, how ensembles of motors efficiently move cargo, and the role of processive movement in cells. A significant obstacle to understanding these important features of motility is a lack of tools to precisely control motor-motor and motor-cargo interactions in vitro. Using DNA nanotechnology, we have developed methods to achieve this. First, we generate stable, functional dynein heterodimers through DNA base pairing. Second, using three-dimensional (3D) DNA nanotechnology, we build synthetic cargo to which DNA-linked dynein or kinesin motors can be attached with defined numbers and spacing. To determine how dynein takes consecutive steps along microtubules, single-molecule techniques, including high-precision, multi-color fluorescence microscopy and single-molecule Forster resonance energy transfer (smFRET), will be applied to track the behavior of individual moving dynein molecules. The results of these experiments will be used to construct a model for how dynein moves processively on microtubules. To determine how coordination among dynein motors or between dynein and kinesin motors affects cargo motility, varying numbers of dynein or dynein mixed with kinesin will be attached to a 3D, synthetic DNA cargo. By analyzing the behavior of both the cargo and individual, cargo-attached motors in single-molecule motility assays, the biophysical properties of multi-motor-based transport will be determined. Long distance transport is thought to require processive motility. However, we recently discovered that dynein is sub-maximally processive. Using in vivo and in vitro approaches, we will test the hypothesis that sub-maximal processivity is especially critical for cytoplasmic dynein. Because cytoplasmic dynein is encoded by only a single gene in all eukaryotes but carries out a wide range of tasks, sub-maximal processivity may allow it to be tuned to perform a variety of cellular functions. This research will provide fundamental, mechanistic insights into how the ubiquitous and essential dynein motor works. In addition, the DNA nanotechnology tools generated here will serve as general engineering principles for studying the oligomerization state of other proteins or for studying arrays of any molecular motor in a more physiologically relevant manner.

Public Health Relevance

The molecular motor cytoplasmic dynein powers the transport of many cellular cargos. Long cells such as neurons are especially sensitive to defects in transport;mutations in motors or motor-associated subunits lead to neurodevelopmental and neurodegenerative diseases. In the proposed research we develop novel DNA nanotechnology tools that will allow us to determine how the dynein motor works, providing insight into 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 #
5R01GM100947-02
Application #
8601111
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Gindhart, Joseph G
Project Start
2013-01-01
Project End
2016-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
2
Fiscal Year
2014
Total Cost
$283,057
Indirect Cost
$112,057
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Roberts, Anthony J; Goodman, Brian S; Reck-Peterson, Samara L (2014) Reconstitution of dynein transport to the microtubule plus end by kinesin. Elife 3:e02641
Goodman, Brian S; Reck-Peterson, Samara L (2014) Engineering defined motor ensembles with DNA origami. Methods Enzymol 540:169-88