Biomedical subjects

J Zimmerberg

Publications and source records attributed to J Zimmerberg.

At least 37 records · Page 2Linked to original sources

PubMed1999

Sea urchin egg preparations as systems for the study of calcium-triggered exocytosis.

This paper reviews recent work in our laboratory on the mechanism of calcium-triggered exocytosis. Upon echinoderm egg fertilization, cortical secretory vesicle exocytosis is massive and synchronous. By combining physiological and molecular analyses with a variety of purified membrane isolates containing secretory vesicles that fuse to the plasma membrane or each other, we have characterized the final steps of this calcium-triggered exocytosis. Our kinetic analysis led to a functional definition of the fusion complex whose activation by calcium follows Poisson statistics. The properties of this complex are compared with the properties of the heterotrimeric SNARE protein complex that is present in the cortical vesicle system. Our data do not support the hypothesis that this particular heterotrimeric complex is by itself the biological fusogen.

Animals↗

PubMed1999

Membrane fusion.

Although catalyzed by different proteins, the energy barriers for lipid bilayer fusion in exocytosis, viral fusion, and trafficking seem to be the same as those for the fusion of protein-free phospholipid membranes. To minimize this energy, fusion will proceed through a minimal number of lipid molecules, probably localized in bent non-bilayer intermediates. Experiments on phospholipid bilayer membrane fusion show the pathway of contact, hemifusion, flickering fusion pore formation, and fusion pore enlargement caused by swelling of the vesicle. Lipid curvature determines the barriers to hemifusion and fusion pore formation, while swelling-induced membrane tension drives fusion pore enlargement. Experiments on viral protein-induced cell-cell fusion and exocytosis show the same pathway with the same fundamental effects of lipid curvature and membrane tension. Thus while proteins control these reactions, lipid energetics determine the basic reaction scheme for membrane fusion.

Journal Article↗

PubMed1999

Hole-istic medicine.

Explore the source record for details and available documents.

Biological Transport↗

PubMed1999

Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations.

Release of proteins through the outer mitochondrial membrane can be a critical step in apoptosis, and the localization of apoptosis-regulating Bcl-2 family members there suggests they control this process. We used planar phospholipid membranes to test the effect of full-length Bax and Bcl-xL synthesized in vitro and native Bax purified from bovine thymocytes. Instead of forming pores with reproducible conductance levels expected for ionic channels, Bax, but not Bcl-xL, created arbitrary and continuously variable changes in membrane permeability and decreased the stability of the membrane, regardless of whether the source of the protein was synthetic or native. This breakdown of the membrane permeability barrier and destabilization of the bilayer was quantified by using membrane lifetime measurements. Bax decreased membrane lifetime in a voltage- and concentration-dependent manner. Bcl-xL did not protect against Bax-induced membrane destabilization, supporting the idea that these two proteins function independently. Corresponding to a physical theory for lipidic pore formation, Bax potently diminished the linear tension of the membrane (i.e., the energy required to form the edge of a new pore). We suggest that Bax acts directly by destabilizing the lipid bilayer structure of the outer mitochondrial membrane, promoting the formation of a pore-the apoptotic pore-large enough to allow mitochondrial proteins such as cytochrome c to be released into the cytosol. Bax could then enter and permeabilize the inner mitochondrial membrane through the same hole.

Animals↗

PubMed1999

An analysis of the role of the target membrane on the Gp64-induced fusion pore.

Influenza hemagglutinin (HA) and GP64 of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus induce strikingly different initial fusion pores when mediating fusion between host cells that express these fusion proteins and target cells (Plonsky and Zimmerberg, 1996; Spruce et al., 1989, 1991; Zimmerberg et al., 1994). However, in these experiments, variations in host and target membranes confounded the analysis of the role that major components of the fusion reaction play in determining initial pore characteristics. To determine the contribution of the target cell plasma membrane to the fusion pore phenotype, we studied GP64-induced fusion of stably transfected cells (Sf9(Op1D)) to either red blood cells (RBCs) or Sf9 cells. Initial fusion pores in Sf9(Op1D)/RBC and Sf9(Op1D)/Sf9 cell pairs exhibited the same conductance, and pore flickering was not observed in either combination of cells. This indicates that the target cell determines neither the size nor the reversibility of the initial pore. However, the target cell does influence the kinetics of pore formation. The waiting time between triggering and pore appearance was shorter for Sf9(Op1D)/RBC fusion than for Sf9(Op1D)/Sf9 pairs. No correlation between pore waiting time and conductance was found. This argues against a molecular model that assumes aggregation of the pore wall from a nonfixed number of components as the rate-limiting step in GP64 fusion pore formation.

Animals↗

PubMed1999

Short-chain alcohols promote an early stage of membrane hemifusion.

Hemifusion, the linkage of contacting lipid monolayers of two membranes before the opening of a fusion pore, is hypothesized to proceed through the formation of a stalk intermediate, a local and strongly bent connection between membranes. When the monolayers' propensity to bend does not support the stalk (e.g., as it is when lysophosphatidylcholine is added), hemifusion is inhibited. In contrast, short-chain alcohols, reported to affect monolayer bending in a manner similar to that of lysophosphatidylcholine, were here found to promote hemifusion between fluorescently labeled liposomes and planar lipid bilayers. Single hemifusion events were detected by fluorescence microscopy. Methanol or ethanol (1.2-1.6 w/w %) added to the same compartment of the planar bilayer chamber as liposomes caused a 5-50 times increase in the number of hemifusion events. Alcohol-induced hemifusion was inhibited by lysophosphatidylcholine. Promotion of membrane hemifusion by short-chain alcohol was also observed for cell-cell fusion mediated by influenza virus hemagglutinin (HA). Alcohol promoted a fusion stage subsequent to the low pH-dependent activation of HA. We propose that binding of short-chain alcohol to the surface of membranes promotes hemifusion by facilitating the transient breakage of the continuity of each of the contacting monolayers, which is required for their subsequent merger in the stalk intermediate.

1-Butanol↗

PubMed1999

Lipid flow through fusion pores connecting membranes of different tensions.

When two membranes fuse, their components mix; this is usually described as a purely diffusional process. However, if the membranes are under different tensions, the material will spread predominantly by convection. We use standard fluid mechanics to rigorously calculate the steady-state convective flux of lipids. A fusion pore is modeled as a toroid shape, connecting two planar membranes. Each of the membrane monolayers is considered separately as incompressible viscous media with the same shear viscosity, etas. The two monolayers interact by sliding past each other, described by an intermonolayer viscosity, etar. Combining a continuity equation with an equation that balances the work provided by the tension difference, Deltasigma, against the energy dissipated by flow in the viscous membrane, yields expressions for lipid velocity, upsilon, and area of lipid flux, Phi. These expressions for upsilon and Phi depend on Deltasigma, etas, etar, and geometrical aspects of a toroidal pore, but the general features of the theory hold for any fusion pore that has a roughly hourglass shape. These expressions are readily applicable to data from any experiments that monitor movement of lipid dye between fused membranes under different tensions. Lipid velocity increases nonlinearly from a small value for small pore radii, rp, to a saturating value at large rp. As a result of velocity saturation, the flux increases linearly with pore radius for large pores. The calculated lipid flux is in agreement with available experimental data for both large and transient fusion pores.

Biomechanical Phenomena↗

PubMed1999

A discrete stage of baculovirus GP64-mediated membrane fusion.

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH-induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.

Amino Acid Sequence↗

PubMed1998

Biochemical and functional studies of cortical vesicle fusion: the SNARE complex and Ca2+ sensitivity.

Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca2+ activity curve comparable to exocytosis (CV-PM fusion). Here we show that Sr2+ and Ba2+ also trigger CV-CV fusion, and agents affecting different steps of exocytotic fusion block Ca2+, Sr2+, and Ba2+-triggered CV-CV fusion. The maximal number of active fusion complexes per vesicle, Max, was quantified by NEM inhibition of fusion, showing that CV-CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both Max and the Ca2+ sensitivity of fusion complex activation were comparable to that determined for CV-PM fusion. Using Ca2+-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV-CV and CV-PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca2+, Sr2+, and Ba2+, the complex remains Ca2+- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca2+ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca2+-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca2+ sensitivity of fusion.

Animals↗

PubMed1998

Calcium can disrupt the SNARE protein complex on sea urchin egg secretory vesicles without irreversibly blocking fusion.

The homotypic fusion of sea urchin egg cortical vesicles (CV) is a system in which to correlate the biochemistry and physiology of membrane fusion. Homologues of vesicle-associated membrane protein (VAMP), syntaxin, and SNAP-25 were identified in CV membranes. A VAMP and syntaxin immunoreactive band at a higher apparent molecular mass (approximately 70 kDa) was detected; extraction and analysis confirmed that the band contained VAMP, SNAP-25, and syntaxin. This complex was also identified by immunoprecipitation and by sucrose gradient analysis. VAMP in the complex was insensitive to proteolysis by tetanus toxin. All criteria identify the SNARE complex as that described in other secretory systems. Complexes exist pre-formed on individual CV membranes and form between contacting CV. Most notably, CV SNARE complexes are disrupted in response to [Ca2+]free that trigger maximal fusion. N-Ethylmaleimide, which blocks fusion at or before the Ca2+-triggering step, blocks complex disruption by Ca2+. However, disruption is not blocked by lysophosphatidylcholine, which transiently arrests a late stage of fusion. Since removal of lysophosphatidylcholine from Ca2+-treated CV is known to allow fusion, complex disruption occurs independently from the membrane fusion step. As Ca2+ disrupts rather than stabilizes the complex, the presumably coiled-coil SNARE interactions are not needed at the time of fusion. These findings rule out models of fusion in which SNARE complex formation goes to completion ("zippers-up") after Ca2+ binding removes a "fusion-clamp."

Animals↗

PubMed1998

The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation.

The mechanism of bilayer unification in biological fusion is unclear. We reversibly arrested hemagglutinin (HA)-mediated cell-cell fusion right before fusion pore opening. A low-pH conformation of HA was required to form this intermediate and to ensure fusion beyond it. We present evidence indicating that outer monolayers of the fusing membranes were merged and continuous in this intermediate, but HA restricted lipid mixing. Depending on the surface density of HA and the membrane lipid composition, this restricted hemifusion intermediate either transformed into a fusion pore or expanded into an unrestricted hemifusion, without pores but with unrestricted lipid mixing. Our results suggest that restriction of lipid flux by a ring of activated HA is necessary for successful fusion, during which a lipidic fusion pore develops in a local and transient hemifusion diaphragm.

Cells, Cultured↗

PubMed1998

Fusion pore conductance: experimental approaches and theoretical algorithms.

Time-resolved admittance measurements provide the basis for studies showing that membrane fusion occurs through the formation and widening of an initially small pore, linking two previously separated aqueous compartments. Here we introduce modifications to this method that correct the cell-pipette (source) admittance for attenuation and phase shifts produced by electrophysiological equipment. Two new approaches for setting the right phase angle are discussed. The first uses the displacement of a patch-clamp amplifier C-slow potentiometer for the calculation of phase. This calculation is based on amplitudes of observed and expected (theoretical) changes in the source admittance. The second approach automates the original phase adjustment, the validity of which we prove analytically for certain conditions. The multiple sine wave approach is modified to allow the calculation of target cell membrane parameters and the conductance of the fusion pore. We also show how this technique can be extended for measurements of the resting potential of the first (voltage-clamped) membrane. We introduce an algorithm for calculation of fusion pore conductance despite a concurrent change in the resistance of the clamped membrane. The sensitivity of the capacitance restoration algorithm to phase shift errors is analyzed, and experimental data are used to demonstrate the results of this analysis. Finally, we show how the phase offset can be corrected "off-line" by restoring the shape of the capacitance increment.

Algorithms↗

PubMed1998

Evidence for the HIV-1 phenotype switch as a causal factor in acquired immunodeficiency.

Both cellular and humoral immunodeficiency develop in vivo after prolonged infection with HIV-1, but the mechanisms are unclear. Initial infection with HIV-1 is transmitted by macrophage (M)-tropic/non-syncytia-inducing (NSI) viruses, which hyperactivate the immune system, and, in one view, cause immunodeficiency by "exhaustion" of lymphoid tissue. An alternative hypothesis is that immunodeficiency is caused by the replacement of M-tropic viruses by T cell (T)-tropic/syncytia-inducing (SI) viruses, which are known to be highly cytopathic in vitro and emerge late in infected individuals around the time of transition to AIDS (refs. 1, 7-9). To test these two possibilities, we have developed an ex vivo model of humoral immunity to recall antigens using human lymphoid tissue. This tissue supports productive infection with both M- and T-tropic HIV-1 isolates when cultured ex vivo. We found that specific immune responses were enhanced by productive infection of the tissue with M-tropic/NSI HIV-1 isolates, but were blocked by T-tropic/SI HIV-1 isolates. The mechanism involves specific irreversible effect on B-cell activity. Our results support the hypothesis that the phenotype switch to T-tropic viruses is a key determinant of acquired humoral immunodeficiency in patients infected with HIV.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Submaximal responses in calcium-triggered exocytosis are explained by differences in the calcium sensitivity of individual secretory vesicles.

A graded response to calcium is the defining feature of calcium-regulated exocytosis. That is, there exist calcium concentrations that elicit submaximal exocytotic responses in which only a fraction of the available population of secretory vesicles fuse. The role of calcium-dependent inactivation in defining the calcium sensitivity of sea urchin egg secretory vesicle exocytosis in vitro was examined. The cessation of fusion in the continued presence of calcium was not due to calcium-dependent inactivation. Rather, the calcium sensitivity of individual vesicles within a population of exocytotic vesicles is heterogeneous. Any specific calcium concentration above threshold triggered subpopulations of vesicles to fuse and the size of the subpopulations was dependent upon the magnitude of the calcium stimulus. The existence of multiple, stable subpopulations of vesicles is consistent with a fusion process that requires the action of an even greater number of calcium ions than the numbers suggested by models based on the assumption of a homogeneous vesicle population.

Animals↗

PubMed1998

The calcium sensitivity of individual secretory vesicles is invariant with the rate of calcium delivery.

Differences in the calcium sensitivity of individual secretory vesicles can explain a defining feature of calcium-regulated exocytosis, a graded response to calcium. The role of the time dependence of calcium delivery in defining the observed differences in the calcium sensitivity of sea urchin egg secretory vesicles in vitro was examined. The calcium sensitivity of individual secretory vesicles (i.e., the distribution of calcium thresholds) is invariant over a range of calcium delivery rates from faster than micromolar per millisecond to slower than micromolar per second. Any specific calcium concentration above threshold triggers subpopulations of vesicles to fuse, and the size of these subpopulations is independent of the time course required to reach that calcium concentration. All evidence supports the hypothesis that the magnitude of the free calcium is the single controlling variable that determines the fraction of vesicles that fuse, and that this fraction is established before the application of calcium. Submaximal responses to calcium cannot be attributed to alterations in the calcium sensitivity of individual secretory vesicles arising from the temporal properties of the calcium delivery. Models that attempt to explain the cessation of fusion using changes in the distribution of calcium thresholds arising from the rate of calcium delivery and/or adaptation are not applicable to this system, and thus cannot be general.

Animals↗

PubMed1997

Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers.

For the act of membrane fusion, there are two competing, mutually exclusive molecular models that differ in the structure of the initial pore, the pathway for ionic continuity between formerly separated volumes. Because biological "fusion pores" can be as small as ionic channels or gap junctions, one model posits a proteinaceous initial fusion pore. Because biological fusion pore conductance varies widely, another model proposes a lipidic initial pore. We have found pore opening and flickering during the fusion of protein-free phospholipid vesicles with planar phospholipid bilayers. Fusion pore formation appears to follow the coalescence of contacting monolayers to create a zone of hemifusion where continuity between the two adherent membranes is lipidic, but not aqueous. Hypotonic stress, causing tension in the vesicle membrane, promotes complete fusion. Pores closed soon after opening (flickering), and the distribution of fusion pore conductance appears similar to the distribution of initial fusion pores in biological fusion. Because small flickering pores can form in the absence of protein, the existence of small pores in biological fusion cannot be an argument in support of models based on proteinaceous pores. Rather, these results support the model of a lipidic fusion pore developing within a hemifused contact site.

Exocytosis↗

PubMed1997

Lymphocyte trafficking and HIV infection of human lymphoid tissue in a rotating wall vessel bioreactor.

The pathogenesis of HIV infection involves a complex interplay between both the infected and noninfected cells of human lymphoid tissue, the release of free viral particles, the de novo infection of cells, and the recirculatory trafficking of peripheral blood lymphocytes. To develop an in vitro model for studying these various aspects of HIV pathogenesis we have utilized blocks of surgically excised human tonsils and a rotating wall vessel (RWV) cell culture system. Here we show that (1) fragments of the surgically excised human lymphoid tissue remain viable and retain their gross cytoarchitecture for at least 3 weeks when cultured in the RWV system; (2) such lymphoid tissue gradually shows a loss of both T and B cells to the surrounding growth medium; however, this cellular migration is reversible as demonstrated by repopulation of the tissue by labeled cells from the growth medium; (3) this cellular migration may be partially or completely inhibited by embedding the blocks of lymphoid tissue in either a collagen or agarose gel matrix; these embedded tissue blocks retain most of the basic elements of a normal lymphoid cytoarchitecture; and (4) both embedded and nonembedded RWV-cultured blocks of human lymphoid tissue are capable of productive infection by HIV-1 of at least three various strains of different tropism and phenotype, as shown by an increase in both p24 antigen levels and free virus in the culture medium, and by the demonstration of HIV-1 RNA-positive cells inside the tissue identified by in situ hybridization. It is therefore reasonable to suggest that gel-embedded and nonembedded blocks of human lymphoid tissue, cocultured with a suspension of tonsillar lymphocytes in an RWV culture system, constitute a useful model for simulating normal lymphocyte recirculatory traffic and provide a new tool for testing the various aspects of HIV pathogenesis.

Animals↗

PubMed1997

Experimental HIV infection of human lymphoid tissue: correlation of CD4+ T cell depletion and virus syncytium-inducing/non-syncytium-inducing phenotype in histocultures inoculated with laboratory strains and patient isolates of HIV type 1.

It is now apparent that HIV infection leads to a gradual collapse of a complex system of lymphoid tissue. This collapse tends to be associated with a change in virus tropism from macrophages to T lymphocytes, and a change in phenotype from nonsyncytium inducing (NSI) to syncytium inducing (SI). An experimental system is required to study the role of this change in HIV pathogenesis in lymphoid tissue. Here we describe such a system. Histocultures of human lymphoid tissue preserve their general cytoarchitecture, including a network of follicular dendritic cells (FDCs). Histocultures of tonsils, adenoids, or lymph nodes support productive infection with various laboratory and primary isolates of HIV-1 of different tropism and phenotype and exhibit isolate-dependent CD4+ T lymphocyte depletion. A strong correlation between the extent of CD4+ T cell depletion and the SI/NSI phenotype of the isolates is demonstrated. AZT was used as a model drug to inhibit viral replication and CD4+ T cell depletion in lymphoid histocultures. HIV pathogenesis and the effect of antivirals can now be studied in human lymphoid tissue under controlled conditions in vitro.

Adenoids↗