Membrane fusion during secretion: cortical granule exocytosis in sex urchin eggs as studied by quick-freezing and freeze-fracture.

Chandler, Douglas E.; Heuser, John E.

doi:10.1083/jcb.83.1.91

Cited by 194 publications

(78 citation statements)

References 33 publications

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“…pores in eggs, however, was problematic because in that cell the glycerol used for cryoprotection caused artifactual membrane fusion (6). In the present situation, however, multiple pores are found in mast cells that have been exposed to neither fixatives or éryoprotectants ; thus it is more likely that such multiplicity of fusion actually does occur.…”

Section: Discussionmentioning

confidence: 48%

“…Formation of multiple openings into a single granule has been described in sea urchin eggs (6) and zoospores (27). Indeed, in sea urchin eggs these openings appeared to be preceded by numerous small contacts between the granule and plasma membrane (6) . Interpretation of multiple contacts and FIGURE 17 An IMP-cleared area on the P face of the plasma membrane of a glutaraldehyde-fixed mast cell .…”

Section: Discussionmentioning

confidence: 98%

“…All specimens were stimulated for 17 s with 4 pg/ml 48/80, then quick-frozen . tion (6) . This fusion involved formation ofan IMP-free bilayer continuous with both "fixed" membranes .…”

Section: Discussionmentioning

confidence: 99%

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Arrest of membrane fusion events in mast cells by quick-freezing.

Chandler¹,

Heuser

1980

The Journal of Cell Biology

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238

122

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We have used quick-freezing and freeze-fracture to study early stages of exocytosis in rat peritoneal mast cells. Mast cells briefly stimulated with 48/80 (a synthetic polycation and well-known histamine-releasing agent) at 22°C displayed single, narrow-necked pores (some as small as 0.05 pm in diameter) joining single granules with the plasma membrane . Pores that had become as large as 0.1 lm in diameter were clearly etchable and thus represented aqueous channels connecting the granule interior with the extracellular space. Granules exhibiting pores usually did not have wide areas of contact with the plasma membrane, and clearings of intramembrane particles, seen in chemically fixed mast cells undergoing exocytosis, were not present on either plasma or granule membranes. Fusion of interior granules later in the secretory process also appeared to involve pores; granules were often joined by one pore or a group of 2-4 pores . Also found were groups of extremely small, etchable pores on granule membranes that may represent the earliest aqueous communication between fusing granules .Ultrastructural studies have shown that histamine secretion by mast cells, in response to either antigens or synthetic polycations, is mediated by a rapid, compound exocytosis (I, 3, 15, 16, 18, 19, 28). Early in the secretory process, single granules fuse with the plasma membrane, whereas at later stages membranes of adjacent granules in the cell interior fuse in succession to produce deep invaginations in the cell surface (1,19,28). These morphological observations correlate well with chemical measurements of release showing that 40-80% of the total cell histamine is secreted within 2 min (3, 28). There is good evidence that secretion is initiated by a rise in intracellular calcium activity (9,10,16,17,22), and for this reason the mast cell represents an important model of calcium-mediated stimulus-secretion coupling (8,12).This rapid exocytotic response of the mast cell has proven extremely useful for studying membrane fusion because numerous fusion events occur within a short period ; electron microscopy studies have already demonstrated what appear to be preliminary stages in this process. In thin sections of stimulated mast cells, granule membranes often contact the plasma membrane at sites where the intervening cytoplasm has been expressed (7,21), forming what Palade and Bruns (25) termed a "pentalaminar figure ." The occurrence of these structures in many types of secretory cells during exocytosis has suggested that they are a preliminary stage in membrane fusion (2,24,29,30) . In mast cells these contacts can be extensive and are often seen where the granule bulges against the plasma membrane. In freeze-fracture, these bulges frequently have plasma membrane domes that have been cleared of intramembrane particles (IMP) (7, 21). Furthermore, Lawson et al. (21) have shown in thin sections that a variety of ferritin-conjugated surface ligands will not bind to the plasma membrane where it contacts an underlying secretory...

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Section: Discussionmentioning

confidence: 48%

Section: Discussionmentioning

confidence: 98%

See 1 more Smart Citation

Arrest of membrane fusion events in mast cells by quick-freezing.

Chandler¹,

Heuser

1980

The Journal of Cell Biology

Self Cite

238

122

View full text Add to dashboard Cite

show abstract

“…Because only a portion of the membrane lipids contain free primary amino groups, many of the lipids will not be fixed immediately, and thus remain in a mobile state allowing vesicles to either pinch off or fuse with the plasma membrane. The work of Buckley (1973) and Chandler and Heuser (1979) suggests that fixation with glutaraldehyde occurs more rapidly at the cell surface, and that the penetration of glutaraldehyde into the cell and fixation of the cytoplasm are much slower. This may indicate that, following initial exposure to glutaraldehyde, many cytoplasmic vesicles are still capable of directed morion and may move to the surface of the cell and fuse with the plasma membrane, thereby giving an unrealistic view of the relative number of vesicles and caveoiae.…”

Section: Discussionmentioning

confidence: 99%

Morphology of rapidly frozen aortic endothelial cells. Glutaraldehyde fixation increases the number of caveolae.

McGuire¹,

Twietmeyer²

1983

Circulation Research

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SUMMARY. Using a Polaron E7200 quick freeze unit, we investigated the distribution of vesicles (caveolae) bound to the plasma membrane of aortic endothelial cells from adult Sprague-Dawley rats. Counts of caveolae from replicas of rapidly frozen, unfixed samples were made and compared with counts from both aldehyde-fixed, conventionally frozen, and rapidly frozen aortic samples. Aldehyde-fixed samples prepared for freeze fracture by either of the freezing methods revealed a significantly greater number of caveolae than did unfixed, rapidly frozen samples. These findings suggest that the relative number of caveolae is artifactually increased by exposure to aldehyde fixatives. We conclude that previous estimates of the rates of pinocytotic activity in vascular endothelial cells may contain substantial errors based on overestimates of caveolae and underestimates of subplasmalemmal 'free ' vesicles. (Circ Res 53: 424-429, 1983)

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“…This was interpreted as evidence against protein involvement in exocytotic membrane fusion. Recent studies indicate that the particle clearing has originated artifactually during processing for electron microscopy [70,71].…”

Section: Lnvolvement Of Different Membrane Componentsmentioning

confidence: 99%

Membrane fusion of secretory vesicles and liposomes Two different types of fusion

Ekerdt

Dahl²,

Gratzl³

1981

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Secretory vesieles isolated from adrenal medulla were found to fuse in vitro in response to incubation with Ca 2 +. Intervesicular fusion was detected by electron microscopy and was indicated by the appearance of twinned vesieies in freeze-fractured suspensions of vesieles and in thin-sectioned pellet. Two types of fusion could be distinguished: Type I, occurring between 10-7 M and 10-4 M Ca 2 +, was specific for ea 2 +, was inhibited by other divalent cations and was abolished by pretreatment of vesieles with glutaraldehyde, neuraminidase or trypsin. Fusion type I was linear with temperature. A second type of intervesicular fusion was elicited by Ca 2 + in concentrations higher than 2.5 mM and was morphologically characterized by multiple fusions of secretory vesicles. This type of fusion was found to be similar to fusion of liposomes prepared from the membrane lipids of adrenal medullary secretory vesieles: ea 2 + could be replaced by other divalent cations, the effect of different divalent cations was additive and pretreatments attacking membrane proteins were ineffective. Fusion type 11 of intact secretory vesieies as weil as liposome fusion was discontinuous with temperature. Liposome fusion could be detected within 35 ms and persisted for 180 min. Using liposomes containing defined Ca 2 + concentrations we have not found a major influence of Ca 2 + asymmetry on fusion. Incorporation of the ganglioside GM 3 , which is present in the membranes of intact adrenal medullary secretory vesicles did not change the properties of liposomes fusion. Using a Ca 2 +-selective electrode we have identified in secretory vesiele membranes both high affinity binding sites for Ca 2 + (/(} = 1.6 . 10-6 M) and low affinity sites (/(} = 1.2· 10-4 M).

show abstract

Membrane fusion during secretion: cortical granule exocytosis in sex urchin eggs as studied by quick-freezing and freeze-fracture.

Cited by 194 publications

References 33 publications

Arrest of membrane fusion events in mast cells by quick-freezing.

Arrest of membrane fusion events in mast cells by quick-freezing.

Morphology of rapidly frozen aortic endothelial cells. Glutaraldehyde fixation increases the number of caveolae.

Membrane fusion of secretory vesicles and liposomes Two different types of fusion

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