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Molecular Mechanisms of Motility Deduced from in Vitro Reconstituted Microtubule- and Actin-Based Motor Complexes

Award Number: R35GM136288
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Our overall approach is to focus on multi-component in vitro reconstitutions that will provide insight into complex biological processes such as cargo transport and cytokinesis. Expressed proteins used in the reconstitutions will be biochemically characterized, and single-molecule and biochemical/biophysical techniques will assess motor function. Cytoplasmic dynein-1 and kinesins drive long-distance motion on microtubules, which is required for cell polarity and function. Dynein moves to the minus-end of the polar MT and drives retrograde transport, while kinesins of class 1, 2 and 3 power motion to the opposite plus-end and drive anterograde transport. The biological cargoes of these motors include membrane-bound vesicles, organelles and mRNA. Defects in trafficking contribute to developmental and neurodegenerative diseases (e.g. Huntington’s and amyotrophic lateral sclerosis). Bidirectional motion of cellular cargoes as well as purified organelles are driven by motors of opposite directionality in many organisms and cell types. Dynein requires both dynactin and an activating adaptor for full motor activity, and these adaptors are emerging as scaffolds for coupling both dynein and kinesin motors. A major goal is to build on our in vitro reconstituted complex containing dynein-dynactin, the adaptor protein Bicaudal D, the mRNA-binding protein Egalitarian, and mRNA cargo by the addition of kinesin-1. Preliminary data show that this complex recapitulates the bidirectional motion seen in the cell. We will use biophysical and single molecule techniques (TIRF and iSCAT microscopy) to determine the stepping patterns and force dependence of these complexes to understand how the motors co-ordinate and/or compete to achieve this motion. We will determine if coupling dynein with different classes of transporting kinesins (kinesin-1, kinesin-2, or kinesin-3) affects the outcome, and how microtubule-associated proteins (MAPs) regulate these transport complexes. To generalize findings, we will reconstitute a dynein-kinesin-1 complex based on the scaffolding protein huntingtin, because it plays a causative role in Huntington’s disease. A second goal is to further our biochemical/biophysical characterization of fission yeast myosins involved in cytokinesis. A major driving force for cytokinesis is the interaction between myosin and actin that powers constriction of the contractile ring. The complexity of this process in animal cells has led to the use of fission yeast as a favored model system. To propose a more detailed molecular mechanism for cytokinesis in fission yeast it is essential to have an in depth characterization of the principal contractile components. Here we will use biochemical/biophysical techniques to characterize the two class II myosins involved in cytokinesis (Myo2 and Myp2), and determine how light chain phosphorylation regulates their speed and force output. Lastly, we will pursue via collaboration how track geometry influences transport of cargo (liposomes) with bound myoVa and kinesin-1 on suspended actin and microtubule tracks, which is relevant to both the initiation and termination of motility.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2024 ( Subtotal = $351,001 )
2024	2024	UNIVERSITY OF VERMONT & STATE AGRICULTURAL COLLEGE	85 S PROSPECT STREET	BURLINGTON	VT	05405	CHITTENDEN	USA	Biomedical Research and Research Training	000	5	3/6/2024	NON-COMPETING CONTINUATION	$351,001
														Subtotal = $351,001

Issue Date FY: 2023 ( Subtotal = $438,552 )
2023	2023	UNIVERSITY OF VERMONT & STATE AGRICULTURAL COLLEGE	85 S PROSPECT ST	BURLINGTON	VT	05405	CHITTENDEN	USA	Biomedical Research and Research Training	000	4	3/8/2023	NON-COMPETING CONTINUATION	$390,000
2023	2023	UNIVERSITY OF VERMONT & STATE AGRICULTURAL COLLEGE	85 S PROSPECT ST	BURLINGTON	VT	05405	CHITTENDEN	USA	Biomedical Research and Research Training	001	4	5/8/2023	SUPPLEMENT FOR EXPANSION	$48,552
														Subtotal = $438,552

Issue Date FY: 2022 ( Subtotal = $409,010 )
2022	2022	UNIVERSITY OF VERMONT & STATE AGRICULTURAL COLLEGE	85 S PROSPECT ST	BURLINGTON	VT	05405	CHITTENDEN	USA	Biomedical Research and Research Training	001	3	7/6/2022	NON-COMPETING CONTINUATION	$39,003
2022	2022	UNIVERSITY OF VERMONT & STATE AGRICULTURAL COLLEGE	85 S PROSPECT ST	BURLINGTON	VT	05405	CHITTENDEN	USA	Biomedical Research and Research Training	000	3	3/15/2022	NON-COMPETING CONTINUATION	$350,997
2022	2022	UNIVERSITY OF VERMONT & STATE AGRICULTURAL COLLEGE	85 S PROSPECT ST	BURLINGTON	VT	05405	CHITTENDEN	USA	Biomedical Research and Research Training	002	3	7/22/2022	SUPPLEMENT FOR EXPANSION	$19,010
														Subtotal = $409,010

Issue Date FY: 2021 ( Subtotal = $390,000 )
2021	2021	UNIVERSITY OF VERMONT & STATE AGRICULTURAL COLLEGE	85 S PROSPECT ST	BURLINGTON	VT	05405	CHITTENDEN	USA	Biomedical Research and Research Training	000	2	3/9/2021	NON-COMPETING CONTINUATION	$390,000
														Subtotal = $390,000

Issue Date FY: 2020 ( Subtotal = $390,000 )
2020	2020	UNIVERSITY OF VERMONT & STATE AGRICULTURAL COLLEGE	85 S PROSPECT ST	BURLINGTON	VT	05405	CHITTENDEN	USA	Biomedical Research and Research Training	000	1	3/25/2020	NEW	$390,000
														Subtotal = $390,000

Grand Total All Awards = $1,978,563

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Molecular Mechanisms of Motility Deduced from in Vitro Reconstituted Microtubule- and Actin-Based Motor Complexes

Award Number: R35GM136288

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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