Calcium-Triggered Exocytosis
Blank, Stastna, Amrisha Verma, Bezrukov, Coorssen, Humphrey, Rahamimoff, Whalley, Yin, Zimmerberg
Calcium-triggered exocytosis is the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents. Although immunoblotting, or Western blotting, is widely used for detection of specific proteins, it is generally thought to be poor as a quantitative tool for measuring the concentration of specific proteins. However, for testing hypotheses at the level of quantitative science, such a tool is essential. Analysis and understanding of the molecular mechanism of exocytosis requires the unambiguous identification and quantitative assessment of the surface density of specific molecules. Here, using newly refined immunoblotting and analysis paradigms, we provide a fully quantitative analysis of the SNARE protein complement of functional secretory vesicles. Our findings demonstrate the routine quantitation of femtomole to attomole amounts of known proteins and indicate that native secretory vesicles of the sea urchin egg, like other regulated secretory vesicles, undergo Ca2+-triggered fusion despite an endogenous SNARE complement that is about 50-fold lower than that required for relatively inefficient fusion in model vesicle systems.

Membrane Fusion
Biswas, Kumar, Anil Verma, Chizmadzhev, Fan, Kuzmin, Ratinov, Bradlow, Zimmerberg
At the heart of exocytosis is membrane fusion. We considered the theoretical energetics of a fusion pathway starting from the contact site where two apposed membranes each locally protrude (as "nipples") toward each other. The equilibrium distance between the tips of the two nipples is determined by a balance of physical forces: repulsion caused by hydration and attraction generated by fusion proteins. The energy required to create the initial stalk, caused by bending of cis monolayer leaflets, is much less when the stalk forms between nipples rather than between parallel flat membranes. The stalk cannot, however, expand by bending deformations alone, as such expansion would necessitate the creation of a hydrophobic void of prohibitively high energy. But small movements of the lipids out of the plane of their monolayers allow transformation of the stalk into a modified stalk. This intermediate stalk, not previously considered, is a low-energy structure that can reconfigure into a fusion pore via an additional intermediate, the prepore. The lipids of the latter structure are oriented as in a fusion pore, but the bilayer is locally compressed. All membrane rearrangements occur in a discrete local region without creation of an extended hemifusion diaphragm. Importantly, all steps of the proposed pathway are energetically feasible.

Remodeling of Biological Membranes in Fusion, Fission, and Poration
Basanez, Biswas, Glushakova, Kumar, Li, Anil Verma, Chanturiya, Chizmadzhev, Cruz, Fan, Frolov, Hollenberg, Kuzmin, Ratinov, Yin, Bradlow, Zimmerberg
The project is aimed at understanding the physicochemical mechanisms of membrane remodeling during physiological and pathogenic events. During apoptotic cell death, cells usually release apoptogenic proteins such as cytochrome c from the mitochondrial intermembrane space. If Bcl-2 family proteins induce such release by increasing outer mitochondrial membrane permeability, then the pro-apoptotic, but not anti-apoptotic, activity of these proteins should correlate with their permeabilization of membranes to cytochrome c. Over the past year, we tested our hypothesis by using prosurvival full-length Bcl-xL and prodeath Bcl-xL cleavage products (DN61Bcl-xL and DN76Bcl-xL). Unlike Bcl-xL, DN61Bcl-xL and DN76Bcl-xL caused the release of cytochrome c from mitochondria in vivo and in vitro. Recombinant DN61Bcl-xL and DN76Bcl-xL, as well as Bcl-xL, cleaved in situ by caspase 3, possessed intrinsic pore-forming activity, as demonstrated by their ability to permeabilize pure lipid vesicles efficiently. Furthermore, only DN61Bcl-xL and DN76Bcl-xL, but not Bcl-xL, formed pores large enough to release cytochrome c and to destabilize planar lipid bilayer membranes through reduction of pore line tension. Because Bcl-xL and its C-terminal cleavage products bound similarly to lipid membranes and formed oligomers of the same size, neither lipid affinity nor protein-protein interactions appear to be solely responsible for the increased membrane-perturbing activity elicited by Bcl-xL cleavage. Taken together, the data are consistent with the hypothesis that pro-apoptotic forms of Bcl-2 family proteins permeabilize the outer mitochondrial membrane through a multistep process, ultimately leading to liberation of intermembrane apoptogenic factors into the cytosol.

Since the molecular mechanism by which Bcl-2 prevents apoptosis still remains elusive, we have also studied recombinant human Bcl-2 with the deletion of 22 residues at the C-terminal membrane-anchoring region (rhBcl-2Delta22). Characterization of rhBcl-2Delta22 showed that the recombinant protein is homogeneous and monodisperse in nondenaturing solutions, stable at room temperature in the presence of a metal chelator, and an alpha-helical protein with unfolding of secondary structure at a T(m) of 62.8ºC. Optimal membrane pore formation by rhBcl-2Delta22 required negatively charged phospholipids. We demonstrated the existence of a hydrophobic groove in rhBcl-2Delta22 by the fluorescence enhancement of the hydrophobic ANS probe with which a pro-apoptotic Bak BH3 peptide competed. The respiratory inhibitor antimycin A also bound to the hydrophobic groove of rhBcl-2Delta22 with a K(d) of 0.82 microM. We predicted the optimal binding conformation of antimycin A from molecular docking of antimycin A with the hBcl-2 model created by homology modeling. Antimycin A selectively induces apoptosis in cells overexpressing Bcl-2, suggesting that hydrophobic groove-binding compounds may act as selective apoptotic triggers in tumor cells.

In the infection of cells by enveloped viruses, we found that the sialic acid analog 4-GU-DANA (zanamivir) (as well as DANA and 4-AM-DANA) inhibited the neuraminidase activity of human parainfluenza virus type 3 (HPF3). The viral neuraminidase activity is attributable to hemagglutinin-neuraminidase (HN), an envelope protein essential for viral attachment and for fusion mediated by the other envelope protein, F. While there is no evidence that HN's neuraminidase activity is essential for receptor binding and syncytium formation, we found that 4-GU-DANA prevented hemadsorption and fusion of persistently infected cells with uninfected cells. In plaque assays, 4-GU-DANA reduced both the number (but not the area) of plaques if plaques were present only during the adsorption period and the plaque area (but not number) if added after the 90-minute adsorption period. 4-GU-DANA also reduced the area of plaques formed by a neuraminidase-deficient variant, confirming that 4-GU-DANA’s interference with cell-cell fusion is unrelated to inhibition of neuraminidase activity. Given that 4-GU-DANA (and also DANA and 4-AM-DANA) inhibit plaque area formation and fusion by 50 percent at concentrations that are an order of magnitude lower than those required for reducing plaque number or blocking hemadsorption, these sialic acid analogs appear to be particularly efficient in interfering with cell-cell fusion. In cell lines expressing influenza virus hemagglutinin (HA) as the only viral protein, we found that 4-GU-DANA had no effect on hemadsorption but did inhibit HA2b-red blood cell fusion, as judged by both lipid mixing and content mixing. Thus, 4-GU-DANA can interfere with both influenza virus- and HPF3-mediated fusion. The results indicate that in HPF3, 4-GU-DANA and its analogs have an affinity not only for the neuraminidase active site of HN but also for sites important for receptor binding and cell fusion and that sialic acid-based inhibitors of influenza virus neuraminidase can also exert a direct, negative effect on the fusogenic function of the other envelope protein, HA.