1 Peking University, Beijing, China, 2 Institut de Neurobiologie Alfred Fessard, Gif sur Yvette, France, 3 Singapore Bioimaging Consortium, Agency for Science, Technology, and Research, Singapore, Singapore. Exocytosis of transmitter releasing vesicles is elicited by an elevation of intra- cellular Ca 2þ concentration ([Ca 2þ ] i ). Given the existing Ca 2þ sensor receptor (CaSR), although [Ca 2þ ] i -induced exocytosis is fully established, however, whether extracellular Ca 2þ (concentration [Ca 2þ ] o ) directly regulates exocyto- sis is not rigorously examined yet. Here we report that extracellular Ca 2þ in- hibited exocytosis following moderate [Ca 2þ ] i rises (2-3 mM), which were triggered by either photolysis of caged Ca 2þ or caffeine. IC 50 of extracellular Ca 2þ inhibition on exocytosis (ECIE) was 1.38 mM, so that a physiological re- duction (~30%) of [Ca 2þ ] o significantly increased the evoked exocytosis. On single vesicle level, the quantal size and release frequency were significantly regulated by physiological [Ca 2þ ] o . Involvement of CaSR in ECIE was ex- cluded by experiments of pharmacology and molecular biology. Thus, as an ex- tension of the classic Ca 2þ hypothesis of synaptic release, physiological extracellular Ca 2þ plays dual roles in evoked exocytosis by providing source of Ca 2þ influx, and by directly regulating quantal size and release probability in neuronal cells. 1021-Plat Strong, Positive Cooperativity of SNARES For Fusion Pore Opening Stud- ied At the Single-Molecule Level Woori Bae 1 , Mal-Gi Choi 2 , Yeon-Kyun Shin 2,3 , Tae-Young Yoon 1 . 1 KAIST, Daejeon, Korea, Republic of, 2 POSTECH, Pohang, Korea, Republic of, 3 Iowa State University, Ames, IA, USA. Single vesicle fluorescence assay detects the fluorescence signals from surface- immobilized nano-scale vesicles (typically diameter of 50 nm). There are three principal labeling positions: vesicle membranes, luminal contents and mem- brane proteins, each of which allows for study of different aspects of mem- brane-related biological processes. During the past five years, we have reported two realizations of these possibilities: Measuring the kinetics of single vesicle-vesicle fusion by labeling vesicle membranes [1-3] and detecting fusion pore opening in such single vesicle fusion by encapsulating fluorescently-la- beled DNA hairpins inside vesicles [4]. In this work, by using fluorescently la- beled SNARE proteins, we report on the kinetics of SNARE complex formation observed at the single-molecule level. For this purpose, we have developed an advanced single-molecule FRET technique, in which we track not one, but up to 10 proteins at the same time while keeping the precision at the single-mol- ecule level. The measured kinetics of SNARE complex formation shows strong, positive cooperativity. We finally discuss whether we can make two sin- gle-molecule measurements in one experimental setting, for example, detecting the moment of fusion pore opening while tracking formation of multiple SNARE complexes. Such experiment would reveal quantitative correlation be- tween multimeric structure of SNARE proteins and its functional effect on fu- sion pore opening. [1] Tae-Young Yoon, Burak Okumus, Fan Zhang, Yeon-Kyun Shin and Taek- jip Ha. PNAS 103, 19731 (2006). [2] Tae-Young Yoon, Xiaobing Lu, Jiajie Diao, Taekjip Ha and Yeon-Kyun Shin. Nat. Struct. Mol. Biol. 15, 707 (2008). [3] Han-Ki Lee, Yoosoo Yang, Changbong Hyeon, Tae-Sun Lee, Hong-Won Lee, Dae-Hyuk Kweon, Yeon-Kyun Shin and Tae-Young Yoon. Science 328, 760 (2010). [4] Jiajie Diao, Zengliu Su, Yuji Ishitsuka, Bin Lu, Kyung Suk Lee, Ying Lai, Yeon-Kyun Shin and Taekjip Ha. Nature Communications 1, 54 (2010). 1022-Plat HIV Fusion Peptide Penetrates, Disorders and Softens T-Cell Membrane Mimics Stephanie Tristram-Nagle 1 , Rob Chan 1 , Edgar E. Kooijman 2 , Wei Qiang 3 , David P. Weliky 4 , John F. Nagle 1 . 1 Carnegie Mellon University, Pittsburgh, PA, USA, 2 Kent State University, Kent, OH, USA, 3 Michigan State University, East Lansin, MI, USA, 4 Michigan State University, East Lansing, MI, USA. This work investigates the interaction of N-terminal gp41 fusion peptide (FP) of HIV-1 virus with model membranes in order to elucidate how FP leads to fusion of HIV and T-cell membranes. FP constructs were (i) wildtype FP23 (23 N-terminal amino acids of gp41), (ii) water soluble monomeric FP that adds six lysines on the C-terminus of FP23 (FPwsm) and (iii) the C-terminus covalently linked trimeric version (FPtri) of FPwsm. Model membranes were (i) LM3 (a T-cell mimic), (ii) DOPC, (iii) DOPC/30 mole% cholesterol, (iv) diC22:1PC and (v) diC22:1PC/ 30 mole% cholesterol. Diffuse synchrotron low- angle x-ray scattering (LAXS) from fully hydrated samples, supplemented by volumetric data, showed that FP23 and FPtri penetrate into the hydrocarbon re- gion and cause membranes to thin. Depth of penetration appears to depend upon a complex combination of factors including bilayer thickness, presence of cholesterol and electrostatics. X-ray data showed an increase in curvature in hexagonal phase DOPE which further indicates that FP23 penetrates into the hydrocarbon region rather than residing in the interfacial headgroup region. LAXS data also yielded bending moduli K C , a measure of membrane stiffness, and wide-angle x-ray scattering (WAXS) yielded the S xray orientational order parameter. Both FP23 and FPtri decreased K C and S xray considerably, while the weak effect of FPwsm suggests that it did not partition strongly into LM3 model membranes. Our results are consistent with the HIV fusion peptide disordering and softening the T-cell membrane, thereby lowering the activation energy for viral membrane fusion. This research was supported by NIH Grant GM 44976 (STN,RC,JFN) and NIH AI 47153(WQ,DPW). 1023-Plat PE Lipids in Single SNARE Vesicle Fusion Assay on Supported Mem- branes Promote Docking and Reduce the Number of SNARE Complexes Required for Fast Fusion Marta K. Domanska, Volker Kiessling, Lukas K. Tamm. University of Virginia, Charlottesville, VA, USA. SNARE proteins are the core of the membrane fusion machinery in mammalian cells. Both in vitro and in vivo studies focus mostly on neuronal SNAREs (syn- taxin1A, SNAP25, synaptobrevin2), which are involved in Ca2þ regulated exocytosis of synaptic vesicles. A recently developed single vesicle SNARE- mediated fusion assay in planar supported bilayers allows us to study individual docking and fusion events with a millisecond time resolution in a well-con- trolled lipid environment. Although, the fusion time in this assay is differently defined than in cellular settings, various parameters describing SNARE-medi- ated membrane fusion can be obtained, including the efficiency of vesicle dock- ing and the probability of fusion after docking. Additionally, the number of particles in the fusion site and their activation rates can be determined from modeling of the fusion kinetics data. To better mimic the composition of the plasma membrane, we added various concentrations of PE and PS to our standard PC/Chol mixture. We observed a 1.5- to 3-fold increase in docking efficiency when the PE content was raised from 0 to 30%. In contrast, the fusion probability decreased 3- to 6-fold com- pared to PC/Chol membranes. Interestingly, including PE only in the vesicle membrane was sufficient to cause these effects. A detailed analysis of the fu- sion kinetics revealed that the fusion rate did not change significantly while the number of SNARE complexes that drive fast membrane fusion decreased with 3 complexes constituting the minimal fusion site. These results are ratio- nalized with a model, in which fewer SNAREs are required to overcome the lower activation energy barrier in the presence of negative curvature-promoting PE than the higher activation energy that is likely present in the absence of PE. 1024-Plat The Pathway to Fusion in Synthetic Membrane Systems Jason M. Warner, Ben O’Shaughnessy. Columbia University, New York, NY, USA. Considerable evidence indicates the pathway to fusion of synthetic and biolog- ical membranes consists of two stages. The first is a transition to a hemifused intermediate where only contacting leaflets are fused and distal leaflets engaged in a hemifusion diaphragm (HD). The second consists in rupture of the growing or fully developed HD to give fusion. Here we present a quantitative model for this pathway in protein-free systems. The first stage is described by our model of hemifusion (presented elsewhere) which outputs time-evolutions of HD ten- sion and area. We find that complete fusion results only if the HD tension, ini- tially twice that outside the HD, excedes the lysis threshold for sufficiently long. Fusion does not occur instantaneously after hemifusion nucleation since membranes transiently tolerate tensions above lysis as previously demonstrated by micropipet aspiration of vesicles [Evans et al, Biophys J, 2003]. Tension re- laxes rapidly with HD size such that a growing HD surviving the initial high tension episode matures into a dead-end hemifused state. Thus fusion and dead-end hemifusion are alternative outcomes; the favored outcome depends critically on cation concentration, lipid composition and other physical factors. We find higher salt levels create higher tensions which favor complete fusion. Predicted fusion times are in quantitative agreement with a study of giant ves- icles where HD rupture yielded fusion after ~1-2 s in 6 mM Ca 2þ [Nikolaus et al, Biophys J, 2010]. We predict dead-end hemifusion at lower salt, consistent with stable HDs reported in 2 mM Mg 2þ . In suspended bilayer-vesicle systems we find cation-induced vesicle tensions are dissipated upon hemifusion and thus Ca 2þ drives dead-end hemifusion only. This is in agreement with the con- sistently reported experimental finding that calcium alone cannot fuse vesicles to bilayers, and fusion requires additional osmotic swelling. 186a Monday, March 7, 2011