In vitro reconstitution of exocytosis from sea urchin egg plasma membrane and isolated cortical vesicles

1987 ◽  
Vol 7 (5) ◽  
pp. 399-409 ◽  
Author(s):  
Joseph H. Crabb ◽  
Paul A. Modern ◽  
Robert C. Jackson
Keyword(s):  
Plasma Membrane ◽  
Human Red Blood Cells ◽  
Sea Urchin Egg ◽  
Cytoplasmic Face ◽  
In Vitro Reconstitution ◽  
Isotonic Buffer ◽  
Cortical Vesicles ◽  
Coated Glass Slide ◽  
Egg Cortex

We have succeeded in reconstituting an exocytotically active egg cortex fraction by recombining purified cortical vesicles (CVs) with egg plasma membrane (PM). CVs were dislodged from a suspension of egg cortex by gentle homogenization in a dissociative buffer with a pH of 9.1, and purified by two rounds of differential centrifugation. Egg PM was prepared by shearing the cortical vesicles from a cortical lawn preparation with a jet of isotonic buffer. PM lawns produced by this procedure consist of an array of CV-free PM fragments attached via their extracellular surface to a polylysine coated glass slide. When a neutralized suspension of CVs was recombined with a PM lawn, CVs reassociated with the cytoplasmic face of the plasma membrane to form a reconstituted lawn (RL). RLs undergo a morphological change in response to Ca2+-containing buffers that is similar to the exocytotic release of CV contents from cortical lawns. In both reactions CV contents are vectorially transferred from the cytoplasmic to the extracytoplasmic face of the egg PM. A quantitative binding assay was developed and used to show that adherence of CVs to a heterologous PM lawn prepared from human red blood cells is minimal.

scholarly journals In vitro reconstitution of exocytosis from plasma membrane and isolated secretory vesicles.

1985 ◽  
Vol 101 (6) ◽  
pp. 2263-2273 ◽  
Author(s):  
J H Crabb ◽  
R C Jackson
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Secretory Vesicles ◽  
Cytoplasmic Face ◽  
In Vitro Reconstitution ◽  
Transport Assay ◽  
Complex Preparation ◽  
A Cell ◽  
Vectorial Transport

We describe the reconstitution of exocytotic function through recombination of purified cortical secretory vesicles (CVs) and plasma membrane from sea urchin eggs. CVs were dislodged from a cell surface complex preparation by gentle homogenization in an isotonic dissociation buffer, and purified by differential centrifugation. CV-free plasma membrane fragments were obtained by mechanically dislodging CVs from cortical lawn (CL) preparations with a jet of CL isolation buffer. This procedure produced a "plasma membrane lawn" preparation, consisting of plasma membrane fragments attached via their vitelline layer (an extracellular glycocalyx) to a polylysine-coated microscope slide. When freshly prepared CVs were incubated with plasma membrane lawns, CVs reassociated with the cytoplasmic face of the plasma membrane, forming an exocytotically competent, reconstituted cortical lawn (RL). Exocytosis in RLs was monitored by phase-contrast microscopy, and quantitated with a sensitive microphotometric assay. Half-maximal exocytosis in RLs occurred at 18.5 microM free Ca2+; half-maximal exocytosis in control lawns occurred at 5.7 microM free Ca2+. Greater than 90% of the purified CVs that were not attached to a plasma membrane lawn remained intact when bathed in a buffer containing millimolar Ca2+. This result excluded the possibility that Ca2+-triggered CV lysis was responsible for our observations, and confirmed that the association of CVs with the plasma membrane was required for exocytosis in RLs. Evidence that the Ca2+-stimulated release of CV contents in CLs and RLs is the in vitro equivalent of exocytosis was obtained with an immunofluorescence-based vectorial transport assay, using an antiserum directed against a CV content protein: stimulation of RLs or partially CV-depleted CLs with Ca2+ resulted in fusion of the CV and plasma membranes, and the vectorial transport of CV contents from the cytoplasmic to the extracytoplasmic face of the egg plasma membrane.

scholarly journals Isolation and partial characterization of the plasma membrane of the sea urchin egg.

1980 ◽  
Vol 87 (1) ◽  
pp. 248-254 ◽  
Author(s):  
W H Kinsey ◽  
G L Decker ◽  
W J Lennarz
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Sea Urchin ◽  
Plasma Membranes ◽  
Binding Property ◽  
Marker Enzyme ◽  
Surface Complex ◽  
Sea Urchin Egg ◽  
Peripheral Proteins ◽  
Cortical Vesicles

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.

scholarly journals Control of neurotransmitter release by two distinctmembrane-binding faces of the Munc13-1 C­1C2B region

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Marcial Camacho ◽  
Bradley Quade ◽  
Thorsten Trimbuch ◽  
Junjie Xu ◽  
Levent Sari ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Point Mutations ◽  
Short Term ◽  
In Vitro Reconstitution ◽  
Presynaptic Plasticity ◽  
Hippocampal Cultures

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane through distinct interactions of the C­1C2B region with the plasma membrane: i) one involving a polybasic face that is expected to yield a perpendicular orientation of Munc13-1 and hinder release; and ii) another involving the DAG-Ca2+-PIP2-binding face that is predicted to result in a slanted orientation and facilitate release. Here we have tested this model and investigated the role of the C­1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays, and synaptic vesicle priming in primary murine hippocampal cultures. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that two distinct faces of this region control neurotransmitter release and short-term presynaptic plasticity.

scholarly journals Control of neurotransmitter release and presynaptic plasticity by re-orientation of membrane-bound Munc13-1

2021 ◽  
Author(s):  
Josep Rizo ◽  
Marcial Camacho ◽  
Bradley Quade ◽  
Thorsten Trimbuch ◽  
Junjie Xu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Neurotransmitter Release ◽  
Point Mutations ◽  
Short Term ◽  
In Vitro Reconstitution ◽  
C1 Domain ◽  
Presynaptic Plasticity ◽  
Membrane Bound

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane in two different orientations mediated by distinct interactions of the C1C2B region with the plasma membrane: i) one involving a polybasic face that yields a perpendicular orientation of Munc13-1 and hinders release; and ii) another involving the DAG-Ca2+-PIP2-binding face that induces a slanted orientation and facilitates release. Here we have tested this model and investigated the role of the C1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair synaptic vesicle priming in primary murine hippocampal cultures, and Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that re-orientation of Munc13-1 controls neurotransmitter release and short-term presynaptic plasticity.

scholarly journals Regulation of extracellular matrix assembly: in vitro reconstitution of a partial fertilization envelope from isolated components.

1987 ◽  
Vol 105 (1) ◽  
pp. 561-567 ◽  
Author(s):  
P J Weidman ◽  
B M Shapiro
Keyword(s):  
Binding Sites ◽  
Divalent Cations ◽  
Matrix Assembly ◽  
Sea Urchin Egg ◽  
In Vitro Reconstitution ◽  
Cross Links ◽  
Necessary And Sufficient ◽  
Secreted Peptides

At fertilization, the glycocalyx (vitelline layer) of the sea urchin egg is transformed into an elevated fertilization envelope by the association of secreted peptides and the formation of intermolecular dityrosine bonds. Dityrosine cross-links are formed by a secreted ovoperoxidase that exists in a Ca2+-stabilized complex with proteoliaisin in the fertilization envelope. By using purified proteins, we now show that proteoliaisin is necessary and sufficient to link ovoperoxidase to the egg glycocalyx. Specifically, we have found that ovoperoxidase can associate with the vitelline layer only when complexed with proteoliaisin; proteoliaisin binds to the vitelline layer independently of its association with ovoperoxidase; proteolytic modification of the vitelline layer is not required for this interaction to occur; the binding of proteoliaisin to the vitelline layer is mediated by the synergistic action of the two major seawater divalent cations, Ca2+ and Mg2+; the number of proteoliaisin-binding sites on the vitelline layer of unfertilized eggs is equivalent to the amount of proteoliaisin secreted at fertilization; and the binding of ovoperoxidase to the vitelline layer, via proteoliaisin, permits the in vitro cross-linking of these two in vivo substrates. The association of purified ovoperoxidase and proteoliaisin with the vitelline layer of unfertilized eggs reconstitutes part of the morphogenesis of the fertilization envelope.

N-ethylmaleimide-sensitive protein(s) involved in cortical exocytosis in the sea urchin egg: localization to both cortical vesicles and plasma membrane

1990 ◽  
Vol 96 (2) ◽  
pp. 313-321
Author(s):  
R.C. Jackson ◽  
P.A. Modern
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Cellular Localization ◽  
Plasma Membranes ◽  
Protein S ◽  
Covalent Modification ◽  
Sea Urchin Egg ◽  
Contrast Microscopy ◽  
Cortical Vesicles ◽  
Secretory Products

The exocytotic release of secretory products from fragments of sea urchin egg cortex has been shown to be inhibited by covalent modification of membrane sulfhydryl groups with N-ethylmaleimide (NEM). Exocytotically competent preparations of reconstituted cortex, formed by recombination of purified cortical vesicles (CVs) with fragments of egg plasma membrane (PM) were also inhibited by treatment with NEM. The cellular localization of sulfhydryl-containing constituent(s) responsible for inhibition was investigated by treating CVs and/or PM with NEM prior to reconstitution. Both native cortex and cortex reconstituted with NEM-treated components were challenged with calcium-containing buffers. Exocytosis was monitored by phase-contrast microscopy, and quantitated by light scattering. Evidence for CV-PM fusion was obtained with an immunofluorescence-based assay that permits visualization of the transport of CV content proteins across the PM. Cortex reconstituted by recombination of NEM-treated CVs with untreated PM or by recombination of untreated CVs with NEM-treated PM was exocytotically competent, whereas cortex formed by recombination of NEM-treated CVs with NEM-treated PM was inactive. These results: (1) support the hypothesis that the mechanism of exocytosis in native and reconstituted cortex is the same; (2) provide evidence that both CV and plasma membranes participate in the release of CV contents from reconstituted cortex; and (3) suggest that sulfhydryl-containing protein(s) present on the surface of purified CVs and plasma membrane are involved in exocytosis.

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

1985 ◽  
Vol 101 (6) ◽  
pp. 2398-2410 ◽  
Author(s):  
J Zimmerberg ◽  
C Sardet ◽  
D Epel
Keyword(s):  
Light Scattering ◽  
Sea Urchin ◽  
Freeze Fracture ◽  
Calcium Dependent ◽  
Sea Urchin Egg ◽  
Attachment Sites ◽  
En Face ◽  
Cortical Vesicles ◽  
Kinetics Of

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.

In vitro exocytosis in sea urchin eggs requires a synaptobrevin-related protein

1997 ◽  
Vol 110 (14) ◽  
pp. 1555-1561 ◽  
Author(s):  
J. Avery ◽  
A. Hodel ◽  
M. Whitaker
Keyword(s):  
Sea Urchin ◽  
Protein Sequence ◽  
In Vitro Model ◽  
Molecular Mechanisms ◽  
Related Protein ◽  
Sea Urchin Eggs ◽  
Sea Urchin Egg ◽  
Vitro Model ◽  
Egg Cortex

Sea urchin eggs provide an efficient in vitro model of exocytosis. We have identified proteins in sea urchin eggs that cross-react with antibodies to mammalian synaptobrevin, synaptotagmin, SNAP-25, syntaxin and rab3a. We show that these proteins are localized to the sea urchin egg cortex, using western blotting and immunocytochemistry. Tetanus toxin light chain cleaves the synaptobrevin-related protein in vitro and inhibits calcium-induced exocytosis. These data demonstrate a conservation between phyla of protein sequence and molecular mechanisms thought to facilitate exocytosis and show that the sea urchin egg provides a unique in vitro exocytotic model with which to study the conserved protein machinery of membrane fusion during secretion.

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