Membrane fusion is central to many areas of endocrine and exocrine physiology, and imbalances in these processes give rise to important diseases, such as diabetes. As a result of the work supported by this grant during the current cycle of funding, the core principle of cellular membrane fusion is now well established, consisting of the assembly of cognate SNARE proteins initially residing in apposing membranes to yield a stable, bridging complex that triggers the bilayers to merge. How does the assembly of SNARE proteins between membranes drive membrane fusion? A mechanistic understanding of the fusion event, in which as many as four separate proteins fold together to fuse apposing bilayers, will require the combined power of a variety of biophysical approaches. Such an undertaking has become possible only recently, thanks to continuing advances in both SNARE fusion biochemistry and biophysical membrane technologies. We will use different complementary technologies (cellular and molecular biology, electrophysiology, surface force/adhesion, and optical imaging), to follow SNARE dynamics and function in real time. SNARE proteins will be reconstituted in both synthetic and biological membranes, thus allowing measurements in fully reconstituted membrane environments as well as in less flexible but more physiological cellular membranes. Within these systems, we will determine the energetics of SNARE-assembly, the consequences of membrane composition and membrane tension on fusion, and the dynamics of the fusion pore itself. Insights gained in the different approaches can be linked by comparing the effects of critical mutations and other perturbations. By following this program, whose value has been proven with viral fusion proteins, we expect new and important information concerning cellular membrane fusion to emerge during the next five years.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
3R37DK027044-34S1
Application #
8032992
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Haft, Carol R
Project Start
2010-03-23
Project End
2011-08-31
Budget Start
2010-03-23
Budget End
2011-08-31
Support Year
34
Fiscal Year
2010
Total Cost
$165,500
Indirect Cost
Name
Yale University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Bello, Oscar D; Jouannot, Ouardane; Chaudhuri, Arunima et al. (2018) Synaptotagmin oligomerization is essential for calcium control of regulated exocytosis. Proc Natl Acad Sci U S A 115:E7624-E7631
Rothman, James E; Krishnakumar, Shyam S; Grushin, Kirill et al. (2017) Hypothesis - buttressed rings assemble, clamp, and release SNAREpins for synaptic transmission. FEBS Lett 591:3459-3480
Wang, Jing; Li, Feng; Bello, Oscar D et al. (2017) Circular oligomerization is an intrinsic property of synaptotagmin. Elife 6:
Li, Feng; Tiwari, Neeraj; Rothman, James E et al. (2016) Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion. Proc Natl Acad Sci U S A 113:10536-41
Xu, Weiming; Nathwani, Bhavik; Lin, Chenxiang et al. (2016) A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion. J Am Chem Soc 138:4439-47
Bello, Oscar D; Auclair, Sarah M; Rothman, James E et al. (2016) Using ApoE Nanolipoprotein Particles To Analyze SNARE-Induced Fusion Pores. Langmuir 32:3015-23
Xu, Weiming; Wang, Jing; Rothman, James E et al. (2015) Accelerating SNARE-Mediated Membrane Fusion by DNA-Lipid Tethers. Angew Chem Int Ed Engl 54:14388-92
Zorman, Sylvain; Rebane, Aleksander A; Ma, Lu et al. (2014) Common intermediates and kinetics, but different energetics, in the assembly of SNARE proteins. Elife 3:e03348
Li, Feng; K├╝mmel, Daniel; Coleman, Jeff et al. (2014) A half-zippered SNARE complex represents a functional intermediate in membrane fusion. J Am Chem Soc 136:3456-64
Shi, Lei; Howan, Kevin; Shen, Qing-Tao et al. (2013) Preparation and characterization of SNARE-containing nanodiscs and direct study of cargo release through fusion pores. Nat Protoc 8:935-48

Showing the most recent 10 out of 27 publications