scholarly journals Membrane differentiations at sites specialized for cell fusion.

1977 ◽  
Vol 72 (1) ◽  
pp. 144-160 ◽  
Author(s):  
R L Weiss ◽  
D A Goodenough ◽  
U W Goodenough
Keyword(s):  
Plasma Membrane ◽  
Cell Fusion ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Active Role ◽  
Central Dome ◽  
Thin Sections ◽  
Mating Structure ◽  
Putative Site ◽  
Freeze Fracture Electron Microscopy

Fusion of plasma membranes between Chlamydomonas reinhardtii gametes has been studied by freeze-fracture electron microscopy of unfixed cells. The putative site of cell fusion developes during gametic differentiation and is recognized in thin sections of unmated gametes as a plaque of dense material subjacent to a sector of the anterior plasma membrane (Goodenough, U.W., and R.L. Weiss. 1975.J. Cell Biol. 67:623-637). The overlying membrane proves to be readily recognized in replicas of unmated gametes as a circular region roughly 500 nm in diameter which is relatively free of "regular" plasma membrane particles on both the P and E fracture faces. The morphology of this region is different for mating-type plus (mt+) and mt- gametes: the few particles present in the center of the mt+ region are distributed asymmetrically and restricted to the P face, while the few particles present in the center of the mt- region are distributed symmetrically in the E face. Each gamete type can be activated for cell fusion by presenting to it isolated flagella of opposite mt. The activated mt+ gamete generates large expanses of particle-cleared membrane as it forms a long fertilization tubule from the mating structure region. In the activated mt- gamete, the E face of the mating structure region is transformed into a central dome of densely clustered particles surrounded by a particle-cleared zone. When mt+ and mt- gametes are mixed together, flagellar agglutination triggeeeds to fuse with an activated mt- region. The fusion lip is seen to develop within the particle-dense central dome. We conclude that these mt- particles play an active role in membrane fusion.

scholarly journals Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 583-594 ◽  
Author(s):  
N Dainiak ◽  
CM Cohen
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Mononuclear Cells ◽  
Surface Membrane ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Target Cells ◽  
Freeze Fracture Electron Microscopy

Abstract In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

scholarly journals Thrombocytopoiesis--analysis by membrane tracer and freeze-fracture studies on fresh human and cultured mouse megakaryocytes.

1984 ◽  
Vol 99 (2) ◽  
pp. 390-402 ◽  
Author(s):  
D Zucker-Franklin ◽  
S Petursson
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Precursor Cell ◽  
Membrane Domains ◽  
Thin Sections ◽  
Energy Dependent ◽  
First Time ◽  
Later Phase ◽  
Peripheral Cytoplasm

The origin of platelets (Pt) from megakaryocytes (MK) is beyond question, but the mechanism whereby Pts are released from the precursor cell is still debated. A widely-held theory claims that the MK plasma membrane invaginates to form demarcation membranes (DMS), which delineate Pt territories. Accordingly, Pts would be derived mostly from the periphery of the MK, and the MK and Pt plasma membranes would have to be virtually identical. Since, on morphologic grounds, this theory is untenable, several aspects of thrombocytopoiesis were reexamined with the help of membrane tracer and freeze-fracture analyses of freshly-collected human and cultured mouse MK. To our surprise, freeze-cleavage of the MK plasma membrane revealed that the vast majority of intramembranous particles (IMP) remained associated with the protoplasmic leaflet (P face), whereas the partition coefficient of IMPs of the platelet membrane was the reverse. This is the first time that any difference between MK and Pt membranes has been determined. Replicas of freeze-fractured MK that were in the process of thrombocytopoiesis revealed an additional novel phenomenon, i.e., numerous areas of membrane discontinuity that appeared to be related to Pt discharge. When such areas were small, the IMP were lined up along the margin of the crevice. At a later phase, a labyrinth of fenestrations was observed. Thin sections of MK at various stages of differentiation showed that Pt territories were fully demarcated before connections of the DMS with the surface could be found. Therefore, the Pt envelope is probably not derived from invaginations of the MK plasma membrane. When living, MK were incubated with cationic ferritin or peroxidase at 37 degrees C, the tracers entered into the DMS but did not delineate all membranes with which the DMS was in continuity, suggesting the existence of distinctive membrane domains. Interiorization of tracer was not energy-dependent, but arrested at low temperatures. At 4 degrees C the DMS remained empty, unless there was evidence that Pts had been released. In such instances, the tracers outlined infoldings of peripheral cytoplasm that was devoid of organelles. Thus, the majority of Pts seem to originate from the interior of the MK, and the surface membranes of the two cells differ in origin and structure. The observations do not only throw new light on the process of thrombocytopoiesis, but also strengthen the possibility that MKs and Pts may be subject to different stimuli.

Evidence for septate junctions in the syzygy of the protozoon Gregarina polymorpha (Protozoa, Apicomplexa)

1988 ◽  
Vol 89 (2) ◽  
pp. 217-224
Author(s):  
ROMANO DALLAI ◽  
MARIA VEGNI TALLURI
Keyword(s):  
Plasma Membrane ◽  
Intercellular Space ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Septate Junction ◽  
Fracture Face ◽  
Septate Junctions ◽  
Thin Sections ◽  
Intramembranous Particles ◽  
Lanthanum Tracer

A septate junction is described in reproductive pairs of the protozoon Gregarina polymorpha, using conventional thin sections, lanthanum tracer and freeze-fracture techniques. The septate junction is established between the plasma membranes at the tips of the joined epicytic folds. It is characterized by an intercellular space of 14–17 nm traversed by septa with a repeat of 15–25 nm. Lanthanum-treated material exhibits transparent curves forming a meshwork. Freeze-fracture replicas show membrane modifications in the shape of short rows of intramembranous particles on the E fracture face of the plasma membrane. The significance of the finding of such a septate junction between protozoan cells is discussed.

Freeze-fracture study of phagocytosis in Dictyostelium discoideum

1981 ◽  
Vol 51 (1) ◽  
pp. 63-84
Author(s):  
C. Favard-Sereno ◽  
M.A. Ludosky ◽  
A. Ryter
Keyword(s):  
Plasma Membrane ◽  
Dictyostelium Discoideum ◽  
Freeze Fracture ◽  
Thin Sections ◽  
Latex Beads ◽  
Fracture Study ◽  
Lateral Phase Separation ◽  
Membrane Changes ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

The plasma membrane and its derivative, the phagosome membrane, were studied during and after ingestion of yeast of latex beads in Dictyostelium discoideum. Freeze-fracture electron microscopy, which provides information on the internal architecture of the membranes, and observation of thin sections of cells treated by cytochemical methods were used in parallel. For visualization of membrane sterols in the replicas, the cells were fixed in the presence of digitonin or the antibiotic filipin. No lateral phase separation occurred during yeast engulfment: the intramembranous particles (IMPs), phospholipids and sterols remained distributed at random in the forming phagosome membrane. In contrast architectural modifications of the membrane were observed upon phagosome internalization. Compared to the plasma membrane, the phagosome membrane displayed 2–3 times more IMPs a shift in the IMP size distribution and a higher sterol content. These changes were completed soon after phagosome closure; they were not related either to the nature of the ingested particles (yeast, latex beads) or to the pH in the membrane environment. The membrane changes too place when the phagosomes began to fuse with pre-existing digestive or autophagic vacuoles and lysosomes. Some of the experimental evidence suggests that the restructuring of the membrane may be related to the presence of hydrolases.

scholarly journals ACROSOMAL DISRUPTION IN SPERM

1974 ◽  
Vol 63 (2) ◽  
pp. 466-479 ◽  
Author(s):  
Daniel S. Friend ◽  
Irene Rudolf
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Direct Consequence ◽  
Thin Sections ◽  
Geometric Patterns ◽  
Acrosomal Membrane ◽  
Principal Piece

"Capacitation" is a physiological event which alters sperm to permit rapid penetration through oocyte investments and fusion between gametes. Acrosomal "reaction," the physiological release of acrosomal contents, occurs after this facilitating process. In this study, acrosomal "disruption" of guinea pig and rat sperm was achieved in vitro by incubating sperm together with the follicular contents of superovulated mice. The samples contained both "reacted" and "disrupted" sperm. Thin sections of affected sperm revealed rupture and vesiculation of the plasma membrane overlying the acrosome, as well as loss of both the outer acrosomal membrane and the acrosomal content. Freeze-fracture revealed disintegration of the characteristic geometric patterns in regions of the acrosomal and plasma membranes thus disrupted and major modifications in particle distribution in the sperm tail. In the guinea pig, strands of 6–8-nm particles, usually confined to the plasma membrane of the midpiece, which overlies mitochondria, also appeared in the principal piece. Likewise, in rat sperm, bands of similarly small particles formed acute angles throughout the membrane of the principal piece. Compared with the membranes of control preparations, these membrane alterations are apparently a direct consequence of incubation with ovarian follicular contents.

scholarly journals Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 583-594
Author(s):  
N Dainiak ◽  
CM Cohen
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Mononuclear Cells ◽  
Surface Membrane ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Target Cells ◽  
Freeze Fracture Electron Microscopy

In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

scholarly journals Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes.

1978 ◽  
Vol 78 (2) ◽  
pp. 577-596 ◽  
Author(s):  
P M Elias ◽  
J Goerke ◽  
D S Friend ◽  
B E Brown
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Lipid Vesicles ◽  
Cholesterol Content ◽  
Erythrocyte Membranes ◽  
Membrane Domains ◽  
Thin Sections ◽  
Multilamellar Liposomes ◽  
Fracture Identification

To advance our understanding of the organization of cholesterol within cell membranes, we used digitonin in freeze-fracture investigations of model lipid vesicles and tissues. Cholesterol suspensions or multilamellar liposomes composed of phosphatidylcholine with and without cholesterol were exposed to digitonin. Freeze-fracture replicas of those multilamellar liposomes containing cholesterol displayed either 50--60-nm wide intramembrane corrugations or extramembrane tubular complexes. Comparable intramembrane hemitubular scallops and extra-cellular free tubular complexes were observed in thin sections. Exposure of sperm, erythrocytes (whole and ghosts), and intact tissues (skin, liver, adrenal gland, epididymis) to digitonin produced the same types of intra- and extramembrane complexes or furrows as were formed in liposomes. The plasma membrane of guinea pig serum tail had two unfurrowed regions: the annulus and the zipper. Incubating erythrocyte membranes with digitonin resulted in rapid displacement of cholesterol, accompanied by intramembrane particle clustering and membrane faceting, a feature which we did not see in the intact epithelia studied. In freeze-fractured epithelia, we found that plasma membranes, lysosomes, and some vesicular organelles commonly furrowed, but that mitochondrial membranes and nuclear envelopes were generally spared, correlating well with their known cholesterol content. Finally, plasma membrane corrugations approached but did not impinge on either gap or tight junctions, or on coated vesicles. We conclude that freeze-fracture of membranes exposed to digitonin: (a) reveals distinctive cholesterol-digitonin structural complexes; (b) distinguishes cholesterol-rich and -poor organelle membranes; and (c) demonstrates membrane domains rich or poor in cholesterol.

scholarly journals Redistribution of alpha-granules and their contents in thrombin-stimulated platelets.

1984 ◽  
Vol 98 (2) ◽  
pp. 748-760 ◽  
Author(s):  
P E Stenberg ◽  
M A Shuman ◽  
S P Levine ◽  
D F Bainton
Keyword(s):  
Plasma Membrane ◽  
Tannic Acid ◽  
Extracellular Space ◽  
Plasma Membranes ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Thin Sections ◽  
Immunocytochemical Techniques ◽  
Morphologic Changes ◽  
Stimulation Of

The redistribution of beta-thromboglobulin (beta TG), platelet Factor 4 (PF4), and fibrinogen from the alpha granules of the platelet after stimulation with thrombin was studied by morphologic and immunocytochemical techniques. The use of tannic acid stain and quick-freeze techniques revealed several thrombin-induced morphologic changes. First, the normally discoid platelet became rounder in form, with filopodia, and the granules clustered in its center. The granules then fused with one another and with elements of the surface-connected canalicular system (SCCS) to form large vacuoles in the center of the cell and near the periphery. Neither these vacuoles nor the alpha granules appeared to fuse with the plasma membrane, but the vacuoles were connected to the extracellular space by wide necks, presumably formed by enlargement of the narrow necks connecting the SCCS to the surface of the unstimulated cell. The presence of fibrinogen, beta TG, and PF4 in corresponding large intracellular vacuoles and along the platelet plasma membrane after thrombin stimulation was demonstrated by immunocytochemical techniques in saponin-permeabilized and nonpermeabilized platelets. Immunocytochemical labeling of the three proteins on frozen thin sections of thrombin-stimulated platelets confirmed these findings and showed that all three proteins reached the plasma membrane by the same pathway. We conclude that thrombin stimulation of platelets causes at least some of the fibrinogen, beta TG, and PF4 stored in their alpha granules to be redistributed to their plasma membranes by way of surface-connected vacuoles formed by fusion of the alpha granules with elements of the SCCS.

scholarly journals The Chlamydomonas Mating Type Plus Fertilization Tubule, a Prototypic Cell Fusion Organelle: Isolation, Characterization, and In Vitro Adhesion to Mating Type Minus Gametes

1997 ◽  
Vol 137 (7) ◽  
pp. 1537-1553 ◽  
Author(s):  
Nedra F. Wilson ◽  
Mary J. Foglesong ◽  
William J. Snell
Keyword(s):  
Cell Fusion ◽  
Mating Type ◽  
Plasma Membranes ◽  
Surface Proteins ◽  
Filamentous Actin ◽  
Mating Structure ◽  
Cell Protrusion ◽  
Isolation And Characterization ◽  
In Vitro Adhesion

In the biflagellated alga Chlamydomonas, adhesion and fusion of the plasma membranes of gametes during fertilization occurs via an actin-filled, microvillus-like cell protrusion. Formation of this ∼3-μm-long fusion organelle, the Chlamydomonas fertilization tubule, is induced in mating type plus (mt+) gametes during flagellar adhesion with mating type minus (mt−) gametes. Subsequent adhesion between the tip of the mt+ fertilization tubule and the apex of a mating structure on mt− gametes is followed rapidly by fusion of the plasma membranes and zygote formation. In this report, we describe the isolation and characterization of fertilization tubules from mt+ gametes activated for cell fusion. Fertilization tubules were detached by homogenization of activated mt+ gametes in an EGTA-containing buffer and purified by differential centrifugation followed by fractionation on sucrose and Percoll gradients. As determined by fluorescence microscopy of samples stained with a fluorescent probe for filamentous actin, the method yielded 2–3 × 106 fertilization tubules/μg protein, representing up to a 360-fold enrichment of these organelles. Examination by negative stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated mt+ gametes, retaining both the extracellular fringe and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins identified by biotinylation studies, copurified with the fertilization tubules. Most importantly, the isolated mt+ fertilization tubules bound to the apical ends of activated mt− gametes between the two flagella, the site of the mt− mating structure; a single fertilization tubule bound per cell, binding was specific for gametes, and fertilization tubules isolated from trypsin-treated, activated mt+ gametes did not bind to activated mt− gametes.

scholarly journals Initial events during phagocytosis by macrophages viewed from outside and inside the cell: membrane-particle interactions and clathrin.

1982 ◽  
Vol 94 (3) ◽  
pp. 613-623 ◽  
Author(s):  
J Aggeler ◽  
Z Werb
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cell Attachment ◽  
High Resolution Electron Microscopy ◽  
Peritoneal Macrophages ◽  
Coated Pits ◽  
Thin Sections ◽  
Particle Uptake ◽  
Freeze Dried

The initial events during phagocytosis of latex beads by mouse peritoneal macrophages were visualized by high-resolution electron microscopy of platinum replicas of freeze-dried cells and by conventional thin-section electron microscopy of macrophages postfixed with 1% tannic acid. On the external surface of phagocytosing macrophages, all stages of particle uptake were seen, from early attachment to complete engulfment. Wherever the plasma membrane approached the bead surface, there was a 20-nm-wide gap bridged by narrow strands of material 12.4 nm in diameter. These strands were also seen in thin sections and in replicas of critical-point-dried and freeze-fractured macrophages. When cells were broken open and the plasma membrane was viewed from the inside, many nascent phagosomes had relatively smooth cytoplasmic surfaces with few associated cytoskeletal filaments. However, up to one-half of the phagosomes that were still close to the cell surface after a short phagocytic pulse (2-5 min) had large flat or spherical areas of clathrin basketwork on their membranes, and both smooth and clathrin-coated vesicles were seen fusing with or budding off from them. Clathrin-coated pits and vesicles were also abundant elsewhere on the plasma membranes of phagocytosing and control macrophages, but large flat clathrin patches similar to those on nascent phagosomes were observed only on the attached basal plasma membrane surfaces. These resulted suggest that phagocytosis shares features not only with cell attachment and spreading but also with receptor-mediated pinocytosis.

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