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D E Chandler

Publications and source records attributed to D E Chandler.

At least 37 records · Page 2Linked to original sources
PubMed1990

Stepwise transformation of the vitelline envelope of Xenopus eggs at activation: a quick-freeze, deep-etch analysis.

The extracellular matrix of Xenopus laevis eggs was analyzed at fixed intervals after prick-activation using quick-freeze, deep-etch, rotary-shadow electron microscopy. This technique revealed that the modifications of the matrix seen at fertilization do not occur simultaneously, but that instead there is an orderly progression of alterations at activation. The first modification, conversion of the vitelline envelope (VE) to the altered vitelline envelope (VE), occurs within 2 to 3 min after activation. Intermediate stages of the VE to VE transformation can be visualized traveling around the egg in a wave-like fashion. Upon completion of the wave, the loosely woven outer surface of the VE, believed to be the prefertilization layer, remains unaltered. Subsequent formation of the fertilization (F) layer at this VE-jelly interface occurs between 4 and 8 min postactivation. Finally, between 10 and 15 min postactivation, the smooth (S) layer forms on the tips of the microvilli and surrounds the entire egg.

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PubMed1990

The fluorescent probe BCECF has a heterogeneous distribution in sea urchin eggs.

Sea urchin eggs were loaded with the pH-sensitive fluorescent probe BCECF. Homogenization of these eggs, followed by centrifugation, resulted in 25% of the total homogenate fluorescence remaining with the pellet. Fluorescence microscopy revealed brightly fluorescing punctate organelles whose fluorescence was not quenched by acidification. The excitation spectrum of intracellular BCECF was markedly red-shifted compared to probe calibrated in buffer. Model excitation spectra based on a two compartment model mimicked the intracellular BCECF spectrum, therefore, supporting the possibility that organelles in sea urchin eggs accumulate large amounts of BCECF in a relatively pH-insensitive form.

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PubMed1989

Quick-freeze, deep-etch, rotary-shadow views of the extracellular matrix and cortical cytoskeleton of Xenopus laevis eggs.

The quick-freeze, deep-etch, rotary-shadow technique provides a powerful tool to study the structural dynamics of extracellular matrices. Using this technique, we show that the extracellular investments of the Xenopus laevis egg are multilayered and securely anchored to the egg surface. The cortical cytoskeleton within the egg contains embedded cortical granules with surrounding endoplasmic reticulum and is capped by a thin reticular sheet that contacts the inner surface of the plasma membrane. The extracellular matrix undergoes three distinct changes at fertilization: a) formation of a "smooth" layer below the vitelline envelope (VE), b) transformation of the VE itself to an altered VE composed of concentric fibrous sheets, and c) formation of a dense, "briar-patch"-like fertilization layer at the upper surface of the VE.

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PubMed1989

Hyperosmolality inhibits exocytosis in sea urchin eggs by formation of a granule-free zone and arrest of pore widening.

Hyperosmolality is known to inhibit membrane fusion during exocytosis. In this study cortical granule exocytosis in sea urchin eggs is used as a model system to determine at what step this inhibition occurs. Strongylocentrotus purpuratus eggs were incubated in hyperosmotic seawater (Na2SO4, sucrose or sodium HEPES used as osmoticants), the eggs activated with 20 microM A23187 to trigger exocytosis, and then quick frozen or chemically fixed for electron microscopy. Thin sections and freeze-fracture replicas show that at high osmolality (2.31 osmol/kg), there is a decrease in cortical granule size, a 90% reduction in granule-plasma membrane fusion, and formation of a granule-free zone between the plasma membrane and cortical granules. This zone averages 0.64 micron in thickness and prevents the majority of granules from docking at the plasma membrane. The remaining granules (approximately 10%) exhibit early stages of fusion which appear to have been stabilized; the matrix of these granules remains intact. We conclude that exocytosis is blocked by two separate mechanisms. First, the granule-free zone prevents granule-plasma membrane contact required for fusion. Second, in cases where fusion does occur, opening of the pocket and dispersal of the granule contents are slowed in hyperosmotic media.

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PubMed1989

The coelomic envelope of Xenopus laevis eggs: a quick-freeze, deep-etch analysis.

The extracellular matrix of Xenopus laevis oocytes was analyzed before and after meiotic maturation using quick-freeze, deep-etch, rotary-shadow electron microscopy. The perivitelline space (PS) of the meiotically immature oocyte contains a filamentous network which connects microvilli (MV) and follicle cell macrovilli to the folded oocyte surface below. The envelope overlying the PS is composed of bundles of large fibers which course between the tips of the MV. Spaces between these bundles contain smaller fibrils which secure the egg envelope to the microvillar tips. Meiotic maturation is accompanied by flattening of the oocyte plasma membrane, formation of an orderly array of MV, and elevation of the egg envelope. In the coelomic eggs, the reorganized envelope is composed of loosely bundled large fibers which course above the microvillar tips rather than between them. The spaces between these bundles contain small fibers similar to those seen in the meiotically immature oocyte. This reorganized envelope, however, will not bind sperm; further modifications must transpire during passage through the oviduct to render it sperm receptive.

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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.

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PubMed1988

Freeze-fracture analysis of structural reorganization during meiotic maturation in oocytes of Xenopus laevis.

During meiotic maturation, the cortex of oocytes of Xenopus laevis undergoes structural reorganization, visualized in this study by freeze-fracture electron microscopy. In the full-grown but immature oocyte, annulate lamellae are dispersed throughout the subcortex of the egg, 5 to 20 micron from the plasma membrane. The annulate lamellae consist of well-organized stacks of membrane with visible pores. Stimulation of meiotic maturation by progesterone leads to disruption of the annulate lamellae and formation of an elaborate cortical endoplasmic reticulum which surrounds the cortical granules and intertwines throughout the cortex of the mature egg. Pore-like structures similar to those previously observed in the subcortical annulate lamellae are observed in the mature cortical endoplasmic reticulum. The cortical endoplasmic reticulum is often in close apposition with the plasma membrane and with membranes of cortical granules, but no junctions are visualized. This study provides further evidence that the cortical endoplasmic reticulum develops during progesterone-stimulated meiotic maturation in vitro, and that the annulate lamellae are precursors to the cortical endoplasmic reticulum.

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PubMed1988

In vitro formation of the "S" layer, a unique component of the fertilization envelope in Xenopus laevis eggs.

The extracellular matrix (ECM) of unfertilized Xenopus laevis eggs consists of an elaborate filamentous network in the perivitelline space (PS) and a thick fibrillar vitelline envelope (VE), with a thin layer of horizontal filaments (HF) separating the two. At fertilization this ECM is converted into the fertilization envelope (comprised of the fertilization (F) layer and altered VE), and a third layer, the smooth (S) layer, is formed at the upper boundary of the PS (Larabell and Chandler, 1988). In this report, we use quick-freeze, deep-etch, rotary-shadow electron microscopy to show that an intact S layer can be formed in vitro by incubation of unfertilized eggs in an exudate obtained from cortical granules. Within 5 min numerous 36-nm-diameter particles assemble in a highly ordered array at the microvillar tips. These particles appear to "melt" and to form patches of smooth material and within 10 min one continuous sheet has formed. The presence of the VE is required for formation of the S layer, and we suggest that the HF layer is the site of assembly.

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PubMed1988

The extracellular matrix of Xenopus laevis eggs: a quick-freeze, deep-etch analysis of its modification at fertilization.

Eggs of the amphibian, Xenopus laevis, were quick-frozen, deep-etched, and rotary-shadowed. The structure of the extracellular matrix surrounding these eggs, including the perivitelline space and the vitelline envelope (VE), was visualized in platinum replicas by electron microscopy. The perivitelline space contains an elaborate filamentous glycocalyx which connects microvillar tips to the plasma membrane, to adjacent microvilli, and to the overlying VE. The VE is comprised of two layers, the innermost of which is a thin network of horizontal fibrils lying on the tips of the microvilli. The outermost is a thicker layer of large, cable-like fibers which twist and turn throughout the envelope. Upon fertilization, three dramatic modifications of the matrix occur. A thin sheet of smooth material, termed the smooth layer, is deposited on the tips of the microvilli and separates the egg from the overlying envelopes. The VE above is transformed from a thick band of cable-like fibers to concentric fibrous sheets, the altered VE. Finally, an ornate band of particles, corresponding to the fertilization layer in previous studies, is deposited at the altered VE/jelly interface. The altered VE and the fertilization layer comprise the fertilization envelope, which effects the structural block to polyspermy.

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PubMed1988

Hyperosmotic inhibition of calcium signals and exocytosis in rabbit neutrophils.

Previous studies have shown that hyperosmotic media inhibit secretion in many cells and this has been interpreted to be a direct effect on membrane fusion during exocytosis. This conclusion is complicated, however, by our recent study in neutrophils (Biochim. Biophys. Acta 931: 175-179, 1987) showing that both calcium signals and enzyme release are inhibited by hyperosmolality. In this report, we extend these observations by demonstrating that chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP)-induced enzyme release, secretory granule-plasma membrane fusion, and cytosolic calcium signals are all inhibited by similar increases in medium osmolality. The extent of beta-glucuronidase release is decreased in hyperosmotic media and its dose dependence is shifted to higher FMLP concentrations. Inhibition is rapid, reversible, and independent of osmoticant. Freeze-fracture replicas of quick-frozen neutrophils show that granules of cells stimulated in hyperosmotic media do not undergo fusion nor do they have specialized interactions with the plasma membrane or with membranes of adjacent granules. Calcium signals monitored and quantitated by indo-1 fluorescence during secretion confirmed the presence of three phases the calcium dependence of which we have described previously: 1) an initial peak of calcium that is independent of extracellular calcium and is inhibited 70% at high osmolality; 2) a broad shoulder of elevated calcium levels 30-90 s after stimulation that is dependent on extracellular calcium and is totally blocked at high osmolality; and 3) a plateau of lower but above basal calcium 2-5 min after stimulation that is dependent on extracellular calcium but is relatively unaffected by high osmolality. These results suggest that the FMLP-elicited calcium signal is a composite of multiple signaling events and that hyperosmotic inhibition of secretion, at least in neutrophils, may result from an impaired calcium signal in addition to the direct effect it has on exocytosis.

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PubMed1987

Calcium signals in neutrophils can be divided into three distinct phases.

Rabbit neutrophils were loaded with the fluorescence probe indo-1 and cytosolic free calcium levels were monitored during chemotactic peptide (fMet-Leu-Phe) stimulation. The fMet-Leu-Phe-induced calcium signal consisted of three consecutive phases: (1) an initial peak that was independent of extracellular calcium, (2) a secondary shoulder that required extracellular calcium but was totally blocked by hyperosmolality and (3) a final plateau of elevated calcium that was dependent on extracellular calcium but insensitive to hyperosmolality.

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PubMed1986

Extracellular coats on the surface of Strongylocentrotus purpuratus eggs: stereo electron microscopy of quick-frozen and deep-etched specimens.

Sea urchin (Strongylocentrotus purpuratus) eggs were fixed, quick-frozen, deep-etched, and rotary-replicated, and the three-dimensional structure of the external surface of the egg visualized using stereo electron microscopy. The cell surface is coated with three layers of filaments: the sheetlike vitelline layer adhering closely to the plasma membrane, a second layer of oblique fibrils extending from microvillar tips to the vitelline layer below, and a third, outermost layer of horizontal filaments coursing in bundles over the microvillar tips. After fertilization, the newly elevated vitelline envelope is transformed into a three-layered structure, the central layer being a tightly knit network of fine filaments decorated on each side with a loose network of thicker fibrils. Subsequently, the envelope becomes coated with 'paracrystalline' protein released from the cortical granules, and microvillar casts are reshaped into angular, jagged peaks having two to five sides. The final structure of the fertilization envelope consists of a thick central layer of compact fibrillar material that is coated on each side with thin plates of paracrystalline protein.

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PubMed1986

Chemotactic peptide-induced exocytosis in neutrophils: granule fusion patterns depend on the source of messenger calcium.

Rabbit neutrophils, exposed either to partially purified human C5a or to formylmethionyl-leucyl-phenylalanine in the presence of 5 micrograms ml-1 cytochalasin B underwent a rapid, compound exocytosis. The pattern of granule fusion, as visualized in freeze-fracture replicas, differed depending on the source of messenger calcium. In the presence of extracellular calcium, a linearly directed pattern, consisting of finger-like invaginations converging at the cell centre, was prominent at 10-20 s after stimulation. After stimulation for 20-40 s, further fusion of granule membranes created extremely convoluted surfaces, some consisting of up to a dozen granule membranes connected by narrow pores or flat ribbons of membrane. In many cells the peripheral cytoplasm was constricted to form lobes or finger-like protrusions. Neutrophils stimulated in the absence of extracellular calcium exhibited granule fusion in the directed pattern, with only occasional involvement of the convoluted patterns seen when calcium is present. In contrast, neutrophils depleted of intracellular calcium before stimulation (and thereby forced to use extracellular calcium for triggering secretion) exhibited the convoluted pattern of granule fusion almost exclusively. These results suggest that the directed pattern of fusion is initiated by release of intracellular calcium or a calcium-independent pathway and that the non-directed, convoluted pattern of fusion is initiated by entry of extracellular calcium.

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PubMed1985

Cortical granule exocytosis in sea urchin eggs is inhibited by drugs that alter intracellular calcium stores.

In sea urchin eggs fertilization is accompanied by cortical granule exocytosis, a secretory event thought to be initiated by release of intracellularly sequestered calcium. We have examined the effect of two drugs on this process: chlortetracycline (CTC), a known chelator of intracellular calcium, and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an antagonist of intracellular calcium release in both skeletal and smooth muscle. Preincubation of eggs for 10 min with either CTC or TMB-8 blocked sperm entry, inhibited the burst of 45Ca2+ efflux normally seen postinsemination, and prevented fertilization envelope elevation. Half-maximal inhibition occurred with 200 microM CTC and 60 microM TMB-8. Electron microscopy confirmed that cortical granule exocytosis had been blocked, although inhibition was not due to a direct effect on exocytosis. CTC and TMB-8 had no effect on Ca2+-stimulated granule fusion in isolated egg cortices. Rather, these drugs block the early events in egg activation: sperm incorporation and triggering of exocytosis. These two effects appear to be independent since addition of either drug just before insemination permits sperm entry but inhibits calcium release and cortical granule exocytosis.

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PubMed1984

Exocytosis in vitro: ultrastructure of the isolated sea urchin egg cortex as seen in platinum replicas.

Sea urchin egg cortices, isolated on polylysine-coated cover glasses by the method of V.D. Vacquier (1975, Dev. Biol. 43, 62-74), have been fixed with 2% glutaraldehyde, critical point-dried, and rotary shadowed with platinum-carbon. The replicas produced show that the isolated cortex consists of a plasma membrane having a regular array of microvillar evaginations each containing a filamentous core. Attached to the plasma membrane is a densely packed layer of cortical granules; each granule is linked to the plasma membrane and to four to six adjacent granules by fine, 6-nm-diameter filaments. Between the plasma membrane and cortical granule layer runs a tubular endoplasmic reticulum consisting of bulb-like varicosities joined in chains that are arranged in a network of polygons. Interspersed among the cortical granules are occasional spherical organelles which correspond in size to osmiophilic granules seen in the intact egg cortex. Addition of 1000 microM or 50 microM Ca2+ to the cortex initiates fusion of individual granules with the plasma membrane, as well as extensive fusion between granules, producing extended, branched exocytic pockets. Frequently, fusion patterns are observed that suggest a propagation of granule fusion outward from one or more foci within a single cortex.

Actin Cytoskeleton↗
PubMed1984

Comparison of quick-frozen and chemically fixed sea-urchin eggs: structural evidence that cortical granule exocytosis is preceded by a local increase in membrane mobility.

Eggs of the purple sea-urchin, Strongylocentrotus purpuratus, were fertilized and fixed with 2% glutaraldehyde at various stages during cortical granule exocytosis. Fixation resulted in membrane blebs being formed precisely at the point of incipient granule fusion. These blebs pinched off to form the membranous vesicles frequently seen in exocytic pockets and in the perivitelline space. In contrast, eggs that were fixed with osmium tetroxide or were quick-frozen without chemical fixation, showed no signs of bleb or vesicle formation. Rather, fusion of each granule appeared to begin at a single minute pore, 30-50 nm in diameter, which then enlarged. We suggest that formation of blebs during glutaraldehyde fixation is an artifact that is caused by a highly localized and transient increase in membrane mobility. Normally, this increased mobility facilitates fusion of granule and plasma membranes, but in the presence of glutaraldehyde it leads to large-scale distortions of these fusing membranes.

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PubMed1983

Intracellular or extracellular calcium can be used to trigger C5a-stimulated enzyme secretion from rabbit neutrophils.

The ability of C5a to stimulate lysosomal enzyme release and 45Ca2+ efflux from rabbit neutrophils was studied. C5a stimulated beta-glucuronidase release from cytochalasin B-treated neutrophils either in the presence or absence of extracellular calcium. Depletion of cell calcium by pretreatment with the calcium ionophore A23187 blocked both the ability of C5a to elicit enzyme release in the absence of extracellular calcium and its ability to stimulate 45Ca2+ efflux. Both actions were dose-dependent over the same concentration range (10(-8)-10(-6) M ionophore A23187). In contrast, ionophore pretreatment had no effect on C5a-stimulated enzyme release in the presence of extracellular calcium. These results suggest that (a) release of cell calcium is required for enzyme secretion in the absence of extracellular calcium, and (b) C5a can trigger near-maximal enzyme release by using calcium from either of two sources: the extracellular space or an intracellular site.

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