scholarly journals Beginning of exocytosis captured by rapid-freezing of Limulus amebocytes.

1981 ◽  
Vol 90 (1) ◽  
pp. 40-54 ◽  
Author(s):  
R L Ornberg ◽  
T S Reese
Keyword(s):  
Structural Changes ◽  
Secretory Granules ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Secretory Cells ◽  
Rapid Freezing ◽  
Cytoplasmic Filaments ◽  
Granule Membrane ◽  
Plasmalemma Invaginations

Structural changes underlying exocytosis evoked by the application of endotoxin to Limulus amebocytes were studied at the level of detail afforded by freeze-fracture and freeze-substitution techniques combined with the time resolution of direct rapid-freezing. The results with amebocytes prepared in this manner differed from those with other secretory cells prepared by conventional means. Exocytosis begins within seconds of endotoxin treatment when the plasmalemma invaginates to form pedestallike appositions with peripheral secretory granules. The juxtaposed membranes at these pedestal appositions form several punctate pentalaminar contacts, but examination of freeze-fractured pedestals failed to reveal any corresponding changes in the intramembrane particle distribution. Small secretory granule openings or pores, which are very infrequent, appear within the first 5 s after endotoxin treatment. These pores rapidly widen and this widening is immediately followed by the sequential dissolution of the granule contents, which then move into the surrounding extracellular space. Cytoplasmic filaments connecting the plasmalemma with the granule membrane are suitably deployed to be responsible for the plasmalemma invaginations. How pores begin is not certain, but the appearance of clear spaces between the granule core and the granule membrane at this point in exocytosis supports the possibility of a role of osmotic forces.

scholarly journals Light-induced structural changes of cytoskeleton in squid photoreceptor microvilli detected by rapid-freeze method.

1988 ◽  
Vol 106 (4) ◽  
pp. 1151-1160 ◽  
Author(s):  
S Tsukita ◽  
S Tsukita ◽  
G Matsumoto
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Plasma Membranes ◽  
Structural Changes ◽  
Physiological Role ◽  
Freeze Substitution ◽  
Photoreceptor Cells ◽  
Rapid Freezing ◽  
Light Stimulation

The cytoskeleton in squid photoreceptor microvilli was studied by freeze-substitution electron microscopy combined with rapid freezing using liquid helium, under dark-adapted and light-illuminated conditions. In the dark-adapted microvilli, actin filaments were regularly associated with granular structures on their surface; these granular structures were cross-linked to the rhodopsin-bearing plasma membranes through slender strands. Upon exposure to light, the granular components detached from the actin filaments, which then appeared to be fragmented and/or depolymerized. These observations have led us to conclude that light stimulation triggers the breakdown of the microvillar actin filament complex in squid photoreceptor cells. The results are discussed with special reference to the physiological role of actin filaments in photoreception.

Ultrastructural studies of the relationship between actin filaments and secretory granules

1990 ◽  
Vol 48 (3) ◽  
pp. 458-459
Author(s):  
Ellen Holm Nielsen
Keyword(s):  
Electron Microscopy ◽  
Colloidal Gold ◽  
Actin Filaments ◽  
Secretory Granules ◽  
Secretory Cells ◽  
Ultrastructural Studies ◽  
Transmission Electron ◽  
Granule Membrane ◽  
Ultrathin Sections ◽  
Lowicryl K4m

In secretory cells a dense and complex network of actin filaments is seen in the subplasmalemmal space attached to the cell membrane. During exocytosis this network is undergoing a rearrangement facilitating access of granules to plasma membrane in order that fusion of the membranes can take place. A filamentous network related to secretory granules has been reported, but its structural organization and composition have not been examined, although this network may be important for exocytosis.Samples of peritoneal mast cells were frozen at -70°C and thawed at 4°C in order to rupture the cells in such a gentle way that the granule membrane is still intact. Unruptured and ruptured cells were fixed in 2% paraformaldehyde and 0.075% glutaraldehyde, dehydrated in ethanol. For TEM (transmission electron microscopy) cells were embedded in Lowicryl K4M at -35°C and for SEM (scanning electron microscopy) they were placed on copper blocks, critical point dried and coated. For immunoelectron microscopy ultrathin sections were incubated with monoclonal anti-actin and colloidal gold labelled IgM. Ruptured cells were also placed on cover glasses, prefixed, and incubated with anti-actin and colloidal gold labelled IgM.

scholarly journals Fusion of liposomes with the plasma membrane of epithelial cells: fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections.

1988 ◽  
Vol 107 (6) ◽  
pp. 2511-2521 ◽  
Author(s):  
G Knoll ◽  
K N Burger ◽  
R Bron ◽  
G van Meer ◽  
A J Verkleij
Keyword(s):  
Plasma Membrane ◽  
Apical Cell ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Morphological Evidence ◽  
Epithelial Cell Line ◽  
Rapid Freezing ◽  
Intramembrane Particle ◽  
Local Point ◽  
Basolateral Plasma Membrane

The fusion of liposomes with the plasma membrane of influenza virus-infected monolayers of an epithelial cell line, Madin-Darby canine kidney cells (van Meer et al., 1985. Biochemistry. 24:3593-3602), has been analyzed by morphological techniques. The distribution of liposomal lipids over the apical and basolateral plasma membrane domains after fusion was assessed by autoradiography of liposomal [3H]dipalmitoylphosphatidylcholine after rapid freezing or chemical fixation and further processing by freeze substitution and low temperature embedding. Before fusion, radioactivity was solely detected on the apical cell surface, indicating the absence of redistribution artifacts and demonstrating the reliability of lipid autoradiography on both a light and electron microscopical level. After induction of fusion by a low pH treatment, the basolateral plasma membrane domain became progressively labeled, indicative of rapid lateral diffusion of [3H]dipalmitoylphosphatidylcholine in the plasma membrane. Analysis of individual fusion events by freeze fracture after rapid freezing confirmed the rapid diffusion of the liposomal lipids into the plasma membrane, as intramembrane particle-free lipid patches were never observed. After the induction of liposome-cell fusion, well-defined intramembrane particles were present on the otherwise smooth liposomal fracture faces and on the fracture faces of the plasma membrane. Morphological evidence thus was obtained in favor of a local point fusion mechanism with an intramembrane particle as a specific structural fusion intermediate.

scholarly journals Syntaxin-6 SNARE Involvement in Secretory and Endocytic Pathways of Cultured Pancreatic β-Cells

2004 ◽  
Vol 15 (4) ◽  
pp. 1690-1701 ◽  
Author(s):  
Regina Kuliawat ◽  
Elena Kalinina ◽  
Jason Bock ◽  
Lloyd Fricker ◽  
Timothy E. McGraw ◽  
...  
Keyword(s):  
Secretory Granules ◽  
Expression System ◽  
Secretory Cells ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Rate Limiting Step ◽  
Protein Receptor ◽  
Golgi Network ◽  
Rate Limiting

In pancreatic β-cells, the syntaxin 6 (Syn6) soluble N-ethylmaleimide-sensitive factor attachment protein receptor is distributed in the trans-Golgi network (TGN) (with spillover into immature secretory granules) and endosomes. A possible Syn6 requirement has been suggested in secretory granule biogenesis, but the role of Syn6 in live regulated secretory cells remains unexplored. We have created an ecdysone-inducible gene expression system in the INS-1 β-cell line and find that induced expression of a membrane-anchorless, cytosolic Syn6 (called Syn6t), but not full-length Syn6, causes a prominent defect in endosomal delivery to lysosomes, and the TGN, in these cells. The defect occurs downstream of the endosomal branchpoint involved in transferrin recycling, and upstream of the steady-state distribution of mannose 6-phosphate receptors. By contrast, neither acquisition of stimulus competence nor the ultimate size of β-granules is affected. Biosynthetic effects of dominant-interfering Syn6 seem limited to slowed intragranular processing to insulin (achieving normal levels within 2 h) and minor perturbation of sorting of newly synthesized lysosomal proenzymes. We conclude that expression of the Syn6t mutant slows a rate-limiting step in endosomal maturation but provides only modest and potentially indirect interference with regulated and constitutive secretory pathways, and in TGN sorting of lysosomal enzymes.

Freeze-Fracture Studies of Human Blood Platelets Activated by Thrombin Using Rapid Freezing

1985 ◽  
Vol 54 (03) ◽  
pp. 574-578 ◽  
Author(s):  
Hans Hols ◽  
Jan J Sixma ◽  
J Leunissen-Bijvelt ◽  
Arie Verkley
Keyword(s):  
Human Blood ◽  
Shape Change ◽  
Blood Platelets ◽  
Secretory Granules ◽  
Freeze Fracture ◽  
Rapid Freezing ◽  
Freeze Fracturing ◽  
Morphological Alterations ◽  
Thrombin Activation ◽  
Human Blood Platelets

SummaryIn this study the influence of thrombin activation on human blood platelets has been followed by freeze-fracturing electron microscopy using rapid freezing in order to catch the initial changes in shape and the morphological alterations during the process of exocytosis of secretory granules. We found that isolation of the platelets by itself leads to some degree of shape change, which made it impossible to study the resting discoid platelet by rapid freezing.Activation of the platelets by thrombin induced dilation of the “surface connecting system (SCS)” with formation of large vacuoles as a result of fusion of the secretory granules with SCS. No intermediary fusion stages or structures were observed even using rapid freezing. Volcano-like protrusions and the corresponding complementary pits were seen at the SCS. These structures were interpreted by us as fractures through protoplasmic channels crossing the SCS. These channels originate during the swelling of the SCS as a result of the fusion of secretory granules with the SCS.

Quick-Freezing to Catch the Membrane Changes that Occur During Exocytosis

1977 ◽  
Vol 35 ◽  
pp. 676-679
Author(s):  
J.E. Heuser
Keyword(s):  
Synaptic Vesicle ◽  
Structural Changes ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Frog Neuromuscular Junction ◽  
The Past ◽  
Synaptic Vesicle Exocytosis ◽  
Quick Freezing ◽  
Vesicle Exocytosis ◽  
Membrane Changes

The technique that we have used to capture synaptic vesicle exocytosis at the frog neuromuscular junction - that of quick-freezing muscles followed by freeze fracture (3) or freeze substitution (6) - works sufficiently well now that it may be useful in other sorts of membrane studies, or studies of fast structural changes with the electron microscope. This note reviews the quickfreezing technique we use, and describes its application to the problem of synaptic vesicle exocytosis and recycling at the synapse.Here, many of the membrane changes of interest occur during the brief delay in synaptic transmission, on a time scale of milliseconds or fractions of milliseconds, and leave only traces thereafter. In the past, we have studied these left-over traces in tissues fixed with the standard chemicals for electron microscopy (1), and have inferred from them that vesicles discharge the quanta of neurotransmitters, as the physiologists would predict.

Role of freeze-fracture technique in tumour pathology

1992 ◽  
Vol 50 (1) ◽  
pp. 616-617
Author(s):  
T. M. Mukherjee ◽  
J. G. Swift
Keyword(s):  
Plasma Membrane ◽  
Cell Membranes ◽  
Secretory Granules ◽  
Freeze Fracture ◽  
Intercellular Junctions ◽  
Structural Components ◽  
Thin Sections ◽  
Human Tumours ◽  
Tumour Pathology

The freeze-fracture technique is unique in its ability to expose extensive face views of the interior of the cell membranes. This feature is particularly useful for studies of events occurring at the plasma membrane, such as exocytosis of secretory granules and of structural components within the membrane such as the intercellular junctions. We have used freeze-fracture preparations in conjunction with conventional thin sections to study the intercellular junctions in a variety of human tumours.

Two proteins associated with secretory granule membranes identified in chicken regulated secretory cells

1994 ◽  
Vol 107 (5) ◽  
pp. 1297-1308
Author(s):  
J.L. Thomas ◽  
A. Stieber ◽  
N. Gonatas
Keyword(s):  
Secretory Granule ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Cell Types ◽  
Pancreatic Acinar Cells ◽  
Secretory Cells ◽  
Electrophoretic Patterns ◽  
Granule Membrane ◽  
The Central Nervous System ◽  
Ultrathin Frozen Sections

Lately, we have identified two polypeptides of 92–94 kDa (GRL1) and 45–60 kDa (GRL2), expressed in cytoplasmic granules of chicken granulocytes and thrombocytes. Here, we report that GRL1 and GRL2 are widely distributed in all exocrine and several endocrine cell types, but not in neurons of the central nervous system, during late stages of embryonic development, as well as in newly hatched and two-month-old chickens. Immunogold studies in ultrathin frozen sections of pancreatic acinar cells show that GRL1 and GRL2 are co-localized at the periphery of zymogen granules, in granules fused with apical acinar membranes and on apical membranes of acini, while the pregranular compartments of the secretory pathway are weakly or not labeled. Semiquantitative morphometric studies indicate that GRL1 and GRL2 are equally distributed in secretory granules. A variety of physical and metabolic studies reveal that GRL2, a highly N-glycosylated polypeptide, is an intrinsic membrane protein, while GRL1 is a peripheral membrane polypeptide released by Na2CO3 treatment of granulocyte membranes. In all hematopoietic, exocrine or endocrine cells examinated, GRL1 shows identical electrophoretic patterns, while GRL2 is identified as a diffuse band, at 40–65 kDa, in hematopoietic and pancreatic cells. Taken together, the morphological and biochemical studies indicate that GRL1 and GRL2 are components of the secretory granule membrane in chicken exocrine, endocrine and hemopoietic cell types.

scholarly journals Detection and characterization of microvesicles in the acinar lumen and in juice of unstimulated rat pancreas.

1986 ◽  
Vol 34 (8) ◽  
pp. 1079-1084 ◽  
Author(s):  
A R Beaudoin ◽  
G Grondin ◽  
A Vachereau ◽  
P St-Jean ◽  
C Cabana
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Major Protein ◽  
Zymogen Granule ◽  
Rat Pancreas ◽  
Granule Membrane ◽  
Acinar Lumen

Small vesicles were visualized in the lumen of rat pancreas acini by freeze-substitution and conventional electron microscopy. Microvesicles were subsequently isolated from pancreatic juice. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that these vesicles contain only one major protein. The major protein was identified by an immunoblot technique as GP-2, an 80 kD glycoprotein also found in the zymogen granule membrane. The immunocytochemical localization of rabbit anti-GP-2 and anti-amylase by the protein A-gold technique confirmed that GP-2 was associated with clusters of microvesicles, whereas amylase was virtually excluded. Freeze-fracture of the microvesicles revealed that their membrane was devoid of intramembrane particles. Biochemical analysis indicated also that the membrane did not contain any detectable cholesterol. These results demonstrate that GP-2 is released from the acinar cell in the gland lumen within microvesicles by a hitherto undescribed mode of secretion.

Ca-Histochemistry in slime mold plasmodia

1978 ◽  
Vol 36 (2) ◽  
pp. 130-131
Author(s):  
Ulrich Dierkes
Keyword(s):  
Physarum Polycephalum ◽  
Carbon Coating ◽  
Freeze Drying ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Slime Mold ◽  
Staining Procedure ◽  
Protoplasmic Streaming ◽  
Intracellular Ca

Calcium is supposed to play an important role in the control of protoplasmic streaming in slime mold plasmodia. The motive force for protoplasmic streaming is generated by the interaction of actin and myosin. This contraction is supposed to be controlled by intracellular Ca-fluxes similar to the triggering system in skeleton muscle. The histochemical localisation of calcium however is problematic because of the possible diffusion artifacts especially in aquous media.To evaluate this problem calcium localisation was studied in small pieces of shock frozen (liquid propane at -189°C) plasmodial strands of Physarum polycephalum, which were further processed with 3 different methods: 1) freeze substitution in ethanol at -75°C, staining in 100% ethanol with 1% uranyl acetate, and embedding in styrene-methacrylate. For comparison the staining procedure was omitted in some preparations. 2)Freeze drying at about -95°C, followed by immersion with 100% ethanol containing 1% uranyl acetate, and embedding. 3) Freeze fracture, carbon coating and SEM investigation at temperatures below -100° C.

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