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J Zimmerberg

Publications and source records attributed to J Zimmerberg.

At least 91 records · Page 5Linked to original sources
PubMed1989

Large aqueous channels in membrane vesicles derived from the rough endoplasmic reticulum of canine pancreas or the plasma membrane of Escherichia coli.

Voltage clamp conditions were used to study the membrane permeability properties of rough microsomes (RM) derived from the rough endoplasmic reticulum of canine pancreas and inverted vesicles (InV) derived from the plasma membrane of Escherichia coli. Membrane vesicles of RM or InV were fused to a planar lipid bilayer that was formed in a hole of a partition separating two chambers. Fusion of a single RM vesicle yielded a single-step conductance increase. Some preparations yielded unitary conductances of 20, 55, 80, and 115 pS in 45 mM potassium glutamate. These channels were largely open at negative membrane potential on the cytoplasmic side of the RM membrane, mostly closed at positive voltages, permeable to amino acids, and slightly more selective for anions than cations. There was a dramatic increase in the number of open channels when 100 microM GTP was added to the cytoplasmic side of the fused RM, whereas 100 microM guanosine 5'-[gamma-thio]triphosphate caused closing of channels. ATP had no effect. A large channel of 115 pS at 45 mM potassium glutamate was also detected after the fusion of InV. As both RM and InV share the ability to translocate secretory proteins, it is possible that the 115-pS channel in both membranes represents a protein-conducting channel.

Animals↗
PubMed1989

Initial stages of influenza hemagglutinin-induced cell fusion monitored simultaneously by two fluorescent events: cytoplasmic continuity and lipid mixing.

We have monitored the mixing of both aqueous intracellular and membrane-bound fluorescent dyes during the fusion of human red blood cells to influenza hemagglutinin-expressing fibroblasts using fluorescence spectroscopy and low light, image-enhanced video microscopy. The water-soluble fluorescent dye, N-(7-nitrobenzofurazan-4-yl)taurine, was incorporated into intact human red blood cells. The fluorescence of the dye in the intact red blood cell was partially quenched by hemoglobin. The lipid fluorophore, octadecylrhodamine, was incorporated into the membrane of the same red blood cell at self-quenching concentrations (Morris, S. J., D. P. Sarkar, J. M. White, and R. Blumenthal. 1989. J. Biol. Chem. 264: 3972-3978). Fusion, which allowed movement of the water-soluble dye from the cytoplasm of the red blood cell into the hemagglutinin-expressing fibroblasts, and movement of octadecylrhodamine from membranes of red blood cell to the plasma membrane of the fibroblasts, was observed by fluorescence microscopy as a spatial relocation of dyes, and monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, fluorescence increased after a delay of about 30 s at 37 degrees C, reaching a maximum within 3 min. The kinetics, pH profile, and temperature dependence were similar for both fluorescent events measured simultaneously, indicating that influenza hemagglutinin-induced fusion rapidly establishes bilayer continuity and exchange of cytoplasmic contents.

Animals↗
PubMed1989

High molecular weight polymers block cortical granule exocytosis in sea urchin eggs at the level of granule matrix disassembly.

Recently, we have shown that high molecular weight polymers inhibit cortical granule exocytosis at total osmolalities only slightly higher than that of sea water (Whitaker, M., and J. Zimmerberg. 1987. J. Physiol. 389:527-539). In this study, we visualize the step at which this inhibition occurs. Lytechinus pictus and Strongylocentrotus purpuratus eggs were exposed to 0.8 M stachyose or 40% (wt/vol) dextran (average molecular mass of 10 kD) in artificial sea water, activated with 60 microM of the calcium ionophore A23187, and then either fixed with glutaraldehyde and embedded or quick-frozen and freeze-fractured. Stachyose (2.6 osmol/kg) appears to inhibit cortical granule exocytosis by eliciting formation of a granule-free zone (GFZ) in the egg cortex which pushes granules away from the plasma membrane thus preventing their fusion. In contrast, 40% dextran (1.58 osmol/kg) does not result in a GFZ and cortical granules undergo fusion. In some specimens, the pores joining granule and plasma membranes are relatively small; in other cases, the exocytotic pocket has been stabilized in an omega configuration and the granule matrix remains intact. These observations suggest that high molecular weight polymers block exocytosis because of their inability to enter the granule matrix: they retard the water entry that is needed for matrix dispersal.

Animals↗
PubMed1988

Ionic and permeability requirements for exocytosis in vitro in sea urchin eggs.

We study exocytosis in the planar isolated cortex of the egg of the sea urchin Lytechinus pictus. Solutions bathing the exocytotic apparatus need not contain appreciable amounts of ions: fusion follows addition of submicromolar calcium to solutions containing only nonelectrolyte. We examine the effects of altering the granule membrane permeability to small molecules with ionophores and digitonin. Introducing holes in the secretory granule membrane to the extent of allowing free passage of small molecules does not cause secretion in vitro. We add the amphipathic compound digitonin at 12 to 15 microM concentrations and demonstrate that the granule membrane can become permeable to lucifer yellow, yet that granules remain intact. Granules still undergo exocytosis after digitonin treatment at such concentrations upon subsequent addition of calcium. Higher concentrations of digitonin lead to granule content swelling and vesicle bursting. We conclude that cortical granule hydration during exocytosis is not mediated by small ionic channels.

Animals↗
PubMed1987

Water movement during channel opening and closing.

By applying the osmotic stress of a nonpenetrating polymer, we have measured the change delta nu in polymer-inaccessible internal water volume of a voltage-gated ionic channel. The voltage-dependent anion channel (VDAC) from mitochondrial outer membranes shows a delta nu comparable in magnitude to the full channel volume estimated from solute penetrability, single-channel conductance, or image reconstruction. It thus appears that channel "gating" involves significant structure reorganization and water movement rather than the minimal changes caused by a local constriction or blockade. Hydration of the inner channel surface may be an important factor in channel gating as is the hydration of molecular surfaces in controlling macromolecular interaction in solution.

Body Water↗
PubMed1987

Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells.

Mast cells show dramatic morphological changes when undergoing exocytosis. We have investigated whether the first of those morphological changes, swelling of the secretory granule, precedes--and therefore possibly initiates--secretion or whether it occurs after fusion of the granule and plasma membranes. We used cell membrane capacitance to detect the moment when granule and plasma membrane become continuous. We measured large capacitance increases, often preceded by transients in capacitance. The rise-times of the capacitance increases were half-maximal at 2-59 msec. We observed cells with high-resolution video microscopy while these measurements were done. The capacitance increase always preceded the granular swelling that leads to exocytosis. To rule out the possibility that fusion was induced by a mechanical stress imparted by the internal pressure of a taut granule, we performed control experiments using cells in which vesicles were shrunken with hyperosmotic solutions. With these flaccid granules, again, the capacitance rise always preceded the swelling of the granules. We conclude that swelling cannot be the driving force for membrane fusion in this system.

Animals↗
PubMed1987

Inhibition of secretory granule discharge during exocytosis in sea urchin eggs by polymer solutions.

1. We have investigated the effects of the osmotic pressure exerted by polymer solutions on exocytosis in sea urchin eggs. 2. Exocytosis is prevented by including a variety of polymers of different chemical composition and molecular weight in the sea water surrounding the eggs. Inhibition is complete at a polymer osmolality of 250 mosmol/kg. 3. The increase in membrane capacitance which occurs during exocytosis and which corresponds to the fusion of secretory granules with the plasma membrane is not substantially altered by inhibitory concentrations of polymer. 4. In the absence of the ions present in sea water, these polymers inhibit exocytosis at an osmolality of 950 mosmol/kg. 5. Calcium, magnesium and sodium ions reduce the polymer osmolality required to prevent exocytosis. 6. A comparison of the effects of monovalent and divalent ions indicates that the divalent ions bind to and stabilize the secretory granule contents. 7. These results demonstrate that polymers prevent exocytosis by preventing dispersal of the granule contents once fusion of the secretory granule with the plasma-lemma has occurred. The ions present in sea water do not promote dispersal, rather, they hinder it. The contribution of ionic fluxes to granule swelling during exocytosis is discussed.

Animals↗
PubMed1986

Osmotic swelling of vesicles: its role in the fusion of vesicles with planar phospholipid bilayer membranes and its possible role in exocytosis.

In order for either lipid bilayer membranes or biological membranes to undergo fusion, stresses must somehow be generated in the region of membrane contact. In the fusion of phospholipid vesicles with planar bilayer membranes, the stress can be produced by osmotic swelling of vesicles contacting the planar membrane. On the other hand, fully swollen vesicles may be sufficiently stretched that the additional stress experienced from their adhesion to the planar membrane may in itself suffice to produce fusion (see also Rand & Parsegian, this volume). There is considerable circumstantial evidence that osmotic swelling of vesicles may also be a driving force in exocytosis. This evidence centers both on experiments demonstrating inhibition of exocytosis when vesicles are in a hyperosmotic medium, and on observations of vesicle swelling during the secretory process. This article has not reviewed all of the examples in the literature supporting an osmotic mechanism for fusion, but has attempted to suggest the diversity of cell types from which the examples are drawn and to indicate that the evidence is not conclusive. The unambiguous establishment of vesicle swelling prior to fusion would go far in establishing an osmotic mechanism of exocytosis. We must also be prepared to find that osmotic swelling may not be the only biological mechanism of stressing vesicle membranes contacting plasma membranes. The viral membrane fusion proteins provide the precedent for agents that can apparently sufficiently perturb membranes to cause fusion without any additionally imposed stresses, and even direct membrane mechanical stretching may act biologically as a fusogenic stress.

Animals↗
PubMed1985

Exocytosis of sea urchin egg cortical vesicles in vitro is retarded by hyperosmotic sucrose: kinetics of fusion monitored by quantitative light-scattering microscopy.

We have used the isolated planar cortex of sea urchin eggs to examine the role of osmotic forces in exocytosis by morphological and physiological methods. Electron micrographs of rotary-shadowed replicas show an en face view of exocytosis and demonstrate fusion of cortical vesicles to the underlying oolemma upon addition of calcium. Freeze-fracture replicas of rapidly frozen cortices reveal specialized attachment sites between cortical vesicles and the oolemma, and between the cortical vesicles themselves. We describe a novel light scattering assay for the kinetics of fusion which allows rapid changes of solutions and monitors exocytosis in real time. The rate and extent of fusion are found to be calcium dependent. The removal of calcium halts exocytosis. The validation of exocytosis in this system and development of tools for kinetic analysis allowed us to test predictions of the osmotic hypothesis of exocytosis: hyperosmotic media should inhibit exocytosis; calcium should cause vesicular swelling. Cortical vesicles were found to be permeant to sucrose, glucose, and urea. In media made hyperosmotic with 1.7 M sucrose, cortical vesicles were seen to shrink. Addition of calcium in hyperosmotic media led to a 10-fold decrease in the rate of exocytosis compared with the isotonic rate. The rate, while triggered by calcium, was no longer calcium-dependent. This slowing of exocytosis allowed us to photograph the swelling of cortical vesicles caused by calcium. Removal of calcium had no effect on subsequent exocytosis. Return of cortices to isotonic medium without calcium led to immediate exocytosis. These results are consistent with the idea that swelling of cortical vesicles is required for fusion of biological membranes.

Animals↗
PubMed1984

Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes.

It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion, it does affect the rate of fusion and causes the fusion process to be dependent on stirring in the cis compartment.

Hydrocarbons↗
PubMed1980

Micromolar Ca2+ stimulates fusion of lipid vesicles with planar bilayers containing a calcium-binding protein.

Fusion of phospholipid vesicles with a planar phospholipid bilayer membrane that contains a calcium-binding protein appears to mimic the essential aspects of cytoplasmic-vesicle fusion with plasma membranes (exocytosis) in that (i) there is a low basal rate of fusion in the absence of Ca2+, (ii) this basal rate is enormously increased by micromolar (approximately 10 microM) amounts of Ca2+, and (iii) this rate is not increased by millimolar Mg2+. Essential to this process is an osmotic gradient across the planar membrane, with the side containing the vesicles hyperosmotic to the opposite side. Similar osmotic gradients or their equivalent may be crucial for biological fusion events.

Calcium↗
PubMed1980

Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. I. Discharge of vesicular contents across the planar membrane.

Multilamellar phospholipid vesicles are introduced into the cis compartment on one side of a planar phospholipid bilayer membrane. The vesicles contain a water-soluble fluorescent dye trapped in the aqueous phases between the lamellae. If a vesicle containing n lamellae fuses with a planar membrane, an n-1 lamellar vesicle should be discharged into the opposite trans compartment, where it would appear as a discernible fluorescent particle. Thus, fusion events can be assayed by counting the number of fluorescent particles appearing in the trans compartment. In the absence of divalent cation, fusion does not occur, even after vesicles have been in the cis compartment for 40 min. When CaCl2 is introduced into the cis compartment to a concentration of greater than or equal to 20 mM, fusion occurs within the next 20 min; it generally ceases thereafter because of vesicle aggregation in the cis compartment. With approximately 3 x 10(8) vesicles/cm3 in the cis compartment, about 25-50 fusion events occur following CaCl2 addition. The discharge of vesicular contents across the planar membrane is the most convincing evidence of vesicle-membrane fusion and serves as a model for that ubiquitous biological phenomenon--exocytosis.

Exocytosis↗
PubMed1980

Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane.

Fusion of multilamellar phospholipid vesicles with planar phospholipid bilayer membranes was monitored by the rate of appearance in the planar membrane of an intrinsic membrane protein present in the vesicle membranes. An essential requirement for fusion is an osmotic gradient across the planar membrane, with the cis side (the side containing the vesicles) hyperosmotic to the opposite (trans) side; for substantial fusion rates, divalent cation must also be present on the cis side. Thus, the low fusion rates obtained with 100 mM excess glucose in the cis compartment are enhanced orders of magnitude by the addition of 5-10 mM CaCl2 to the cis compartment. Conversely, the rapid fusion rates induced by 40 mM CaCl2 in the cis compartment are completely suppressed when the osmotic gradient (created by the 40 mM CaCl2) is abolished by addition of an equivalent amount of either CaCl2, NaCl, urea, or glucose to the trans compartment. We propose that fusion occurs by the osmotic swelling of vesicles in contact with the planar membrane, with subsequent rupture of the vesicular and planar membranes in the region of contact. Divalent cations catalyze this process by increasing the frequency and duration of vesicle-planar membrane contact. We argue that essentially this same osmotic mechanism drives biological fusion processes, such as exocytosis. Our fusion procedure provides a general method for incorporating and reconstituting transport proteins into planar phospholipid bilayer membranes.

Calcium Chloride↗
PubMed1975

Peptidyl proline hydroxylase in adult, developing, and neoplastic rat tissues.

A sensitive assay system, optimally supplemented with tritiated protocollagen substrate and cofactors, is described which is suitable for determining the peptidyl proline hydroxylase (PPH) content of a wide spectrum of rat tissues. In most tissues, less than 50 percent of the total activity was soluble; the particulate portion of the activity (concentrated in the mitochondrial and microsomal fractions) was doubled by pretreatment with Triton X-100. Among normal adult tissues, lung had the highest total PPH activity (2.4 times that of liver) and small intestine had the lowest (25 percent that of liver). In brain and lactating mammary gland, the activity was similar to that in skin (60 percent of that in liver). Fetal tissues contained 3 to 8 times more PPH than the corresponding adult tissues, and a much lower portion of the total activity was soluble. In four tissues studied in detail (lung, liver, kidney, and brain), the total PPH declined rapidly during the last few days of gestation; brain attained its low adult value before term, whereas the other three tissues continued to decrease in the course of postnatal development. An injection of cortisol to fetal rats enhanced the decline of PPH in lung, liver, and skull. These experiments suggest that during normal differentiation the decline in collagen synthesis is initiated by fetal glucocorticoid secretion which is maximal on the 19th gestational day. PPH activity appears to be a sensitive indicator of neoplastic growth. In renal, mammary, muscle, and hepatic tumors, the PPH activities were 4 to 10 times higher than in the cognate adult tissue. Even in well-differentiated, slowgrowing tumors, the activity was considerably higher than in any normal, mature, or immature tissue, with the exception of the skull and lung of the 19-day-old fetus.

Animals↗
PubMedDate not supplied

Irreversible swelling of secretory granules during exocytosis caused by calcium.

The fusion of the limiting membrane of a secretory granule with the plasmalemma during exocytosis is equivalent to the fusion and release of contents that occurs when phospholipid vesicles fuse with planar bilayers. Experiments with bilayers demonstrate that phospholipid vesicles must swell if they are to fuse. Also, inhibition of exocytosis in solutions of high osmolarity occurs in several types of secretory cell. We report here experiments on the cortical granule exocytosis of sea-urchin eggs. Exocytosis is prevented when the osmolality of the medium surrounding the eggs is raised from 1 to 2 osmol kg-1. High osmolality also prevents calcium-dependent exocytosis in vitro. Prior treatment with calcium at high osmolality triggers fusion when normal osmolality is restored, even if calcium is removed before dilution. Addition of calcium causes the cortical granules to swell. The large increase in membrane capacitance which normally accompanies fusion is absent in eggs activated in solutions of high osmolarity. Our data are consistent with the idea that a secretory granule must swell to fuse with the plasma membrane and support the hypothesis of an osmotically driven fusion step during exocytosis.

Animals↗