Funded by this grant, my lab has combined biochemistry and cell biology to define the role of the GTPase dynamin in endocytic clathrin-coated vesicle (CCV) formation. Our data, and results of others, now suggest that dynamin plays a dual role in clathrin-mediated endocytosis (CME). During early stages of CME, unassembled dynamin is localized to coated pits and functions as a regulatory GTPase to monitor coat assembly, cargo capture and membrane curvature. At late stages, dynamin self-assembles around the neck of deeply invaginated coated pits and rapid, assembly-stimulated GTP hydrolysis drives a concerted conformational change necessary for membrane fission. Thus, a consensus has emerged that dynamin is a mechanochemical enzyme, but little is known about the molecular nature of dynamin's mechanochemical conformational changes that govern dynamin-mediated membrane fission. Over the next 4 years we will rigorously apply biochemical, biophysical and structural approaches to define the structural and enzymological basis for dynamin's mechanochemical properties and to identify how the enzymatic and mechanochemical activities of dynamin function together with coat proteins and other endocytic accessory factors to mediate CCV formation.
In Aim 1 we will characterize new classes of dynamin mutants that differentially affect basal and assembly- stimulated GTPase activity and test the functions of these distinct dynamin activities in CCV formation in vivo by reconstitution of conditional dynamin-2 knock-out cells.
In Aim 2, we will use X-ray crystallography and cryo-EM, together with helical reconstruction techniques to define the structural bases for basal and assembly-stimulated GTPase activities.
In Aim 3 we will use site- specific labeling of dynamin and multiple independent fluorescence spectroscopy techniques to define the nucleotide-dependent, and effector-regulated conformational changes in dynamin that account for its mechanochemical properties towards lipid bilayers. Finally, in Aim 4 we will develop and utilize new fluorescence-based assays to directly measure dynamin-dependent steps in CCV formation from supported proteolipid bilayers. Together these studies should provide a detailed mechanistic understanding of dynamin function in membrane fission. As dynamin is a prototypical member of a diverse class a GTPases involved in membrane fission, the results from our studies will have broad impact on many areas of cell biology.

Public Health Relevance

Clathrin-mediated endocytosis is the primary mechanism by which cells communicate with and adapt to changes in their environment, through nutrient uptake, regulation of signaling receptors and remodeling of plasma membrane composition. The GTPase dynamin is the master regulator of clathrin-mediated endocytosis and drives membrane fission. We will use biochemical, biophysical, structural and cell biological approaches to elucidate its mechanism of action.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042455-24
Application #
8436194
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
1989-07-01
Project End
2016-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
24
Fiscal Year
2013
Total Cost
$368,872
Indirect Cost
$136,877
Name
University of Texas Sw Medical Center Dallas
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Srinivasan, Saipraveen; Dharmarajan, Venkatasubramanian; Reed, Dana Kim et al. (2016) Identification and function of conformational dynamics in the multidomain GTPase dynamin. EMBO J 35:443-57
Bendris, Nawal; Stearns, Carrie J S; Reis, Carlos R et al. (2016) Sorting nexin 9 negatively regulates invadopodia formation and function in cancer cells. J Cell Sci 129:2804-16
Bendris, Nawal; Williams, Karla C; Reis, Carlos R et al. (2016) SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases. Mol Biol Cell :
Mattila, Juha-Pekka; Shnyrova, Anna V; Sundborger, Anna C et al. (2015) A hemi-fission intermediate links two mechanistically distinct stages of membrane fission. Nature 524:109-13
Reis, Carlos R; Chen, Ping-Hung; Srinivasan, Saipraveen et al. (2015) Crosstalk between Akt/GSK3β signaling and dynamin-1 regulates clathrin-mediated endocytosis. EMBO J 34:2132-46
Elkin, Sarah R; Bendris, Nawal; Reis, Carlos R et al. (2015) A systematic analysis reveals heterogeneous changes in the endocytic activities of cancer cells. Cancer Res 75:4640-50
Srinivasan, Saipraveen; Mattila, Juha-Pekka; Schmid, Sandra L (2014) Intrapolypeptide interactions between the GTPase effector domain (GED) and the GTPase domain form the bundle signaling element in dynamin dimers. Biochemistry 53:5724-6
Neumann, Sylvia; Pucadyil, Thomas J; Schmid, Sandra L (2013) Analyzing membrane remodeling and fission using supported bilayers with excess membrane reservoir. Nat Protoc 8:213-22
Liu, Ya-Wen; Mattila, Juha-Pekka; Schmid, Sandra L (2013) Dynamin-catalyzed membrane fission requires coordinated GTP hydrolysis. PLoS One 8:e55691
Shnyrova, Anna V; Bashkirov, Pavel V; Akimov, Sergey A et al. (2013) Geometric catalysis of membrane fission driven by flexible dynamin rings. Science 339:1433-6

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