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Structural basis of dynamin-mediated membrane fission actin bundling and interaction with binding partners.

Award Number: R00GM140220
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Project Summary/Abstract Dynamin GTPases have critical roles in mediating endocytosis by wrapping around the neck of budding vesicles to catalyze membrane fission necessary for the release of nascent vesicles from the plasma membrane. Recently, we discovered a novel role for dynamin in bundling numerous actin filaments, which has implications for actin-mediated processes, such as cell-cell fusion and migration. While structural and biophysical studies have elucidated the mechanism of dynamin assembly and constriction of membranes, several unanswered questions remain including how dynamin is actually organized and mediates fission within cells, how dynamin forms the final pre-fission state where it wraps around lipid tubules in a superconstricted state, how dynamin binds substrates via the proline rich domain (PRD), and how substrate binding regulates dynamin activity. The long-term objective of this application is to elucidate the mechanism of dynamin-mediated membrane fission from in vitro and in vivo studies, and to define the mechanism of dynamin interaction with actin and SH3 domain- containing proteins that recruit dynamin to sites of endocytosis. These objectives will be addressed by the following specific aims: (1) determine the atomic model of dynamin in the superconstricted prefission state and define the structure of the PRD; (2) investigate the assembly of the dynamin helical polymer on membranes within cells; and (3) Elucidate the mechanism of PRD interaction with actin filaments and SH3 domain-containing binding partners. The rationale for these aims is that: (1) there is no atomic model describing the structural basis by which dynamin constricts membranes to 3.4 nm in the superconstricted state where spontaneous hemifission and membrane fission occurs; (2) how dynamin is actually organized in cells has not been reported; and (3) the structure of the critical PRD and how it binds dynamin substrates including actin, amphiphysin and intersectin is unknown. The research design and methods are as follows : Aim 1, Apply cryo-electron microscopy (cryo-EM) to determine the structure of full-length dynamin (containing the PRD) organized around lipid tubules in the superconstricted state; Aim 2, obtain cryo-electron tomograms and subtomogram averages of dynamin within cells transfected with a GTPase-deficient dynamin mutant which delays membrane fission and extends the lifetime of dynamin helices on cellular membranes; Aim 3, obtain the cryo-EM structure of actin filaments decorated with PRD from dynamin, as well as complexes of dynamin/amphiphysin and dynamin/intersectin. This work is of


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 = $224,100 )
2024	2024	THE TRUSTEES OF PRINCETON UNIVERSITY	1 NASSAU HALL	PRINCETON	NJ	08544	MERCER	USA	Biomedical Research and Research Training	000	4	2/1/2024	NON-COMPETING CONTINUATION	$224,100
														Subtotal = $224,100

Issue Date FY: 2023 ( Subtotal = $249,000 )
2023	2023	THE TRUSTEES OF PRINCETON UNIVERSITY	1 NASSAU HALL	PRINCETON	NJ	08544	MERCER	USA	Biomedical Research and Research Training	000	3	12/30/2022	NON-COMPETING CONTINUATION	$224,101
2023	2023	THE TRUSTEES OF PRINCETON UNIVERSITY	1 NASSAU HALL	PRINCETON	NJ	08544	MERCER	USA	Biomedical Research and Research Training	001	3	2/22/2023	NON-COMPETING CONTINUATION	$24,899
														Subtotal = $249,000

Issue Date FY: 2022 ( Subtotal = $249,000 )
2022	2022	TRUSTEES OF PRINCETON UNIVERSITY, THE	1 NASSAU HALL	PRINCETON	NJ	08544	MERCER	USA	Biomedical Research and Research Training	000	2	12/13/2021	EXTENSION WITH OR WITHOUT FUNDS	$249,000
														Subtotal = $249,000

Grand Total All Awards = $722,100

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Structural basis of dynamin-mediated membrane fission actin bundling and interaction with binding partners.

Award Number: R00GM140220

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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