scholarly journals Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro.

1976 ◽  
Vol 71 (1) ◽  
pp. 136-158 ◽  
Author(s):  
L A Staehelin
Keyword(s):  
Plasma Membranes ◽  
Structural Changes ◽  
Freeze Fracture ◽  
Chloroplast Membranes ◽  
Ps Ii ◽  
Membrane Particles ◽  
Intramembranous Particles ◽  
Membrane Surfaces ◽  
Ps Ii Activity

Freeze-fracture and freeze-etch techniques have been employed to study the supramolecular structure of isolated spinach chloroplast membranes and to monitor structural changes associated with in vitro unstacking and restacking of these membranes. High-resolution particle size histograms prepared from the four fracture faces of normal chloroplast membranes reveal the presence of four distinct categories of intramembranous particles that are nonrandomly distributed between grana and stroma membranes. The large surface particles show a one to one relationship with the EF-face particles. Since the distribution of these particles between grana and stroma membranes coincides with the distribution of photosystem II (PS II) activity, it is argued that they could be structural equivalents of PS II complexes. An interpretative model depicting the structural relationship between all categories of particles is presented. Experimental unstacking of chloroplast membranes in low-salt medium for at least 45 min leads to a reorganization of the lamellae and to a concomitant intermixing of the different categories of membrane particles by means of translational movements in the plane of the membrane. In vitro restacking of such experimentally unstacked chloroplast membranes can be achieved by adding 2-20 mM MgCl2 or 100-200 mM NaCl to the membrane suspension. Membranes allowed to restack for at least 1 h at room temperature demonstrate a resegregation of the EF-face particles into the newly formed stacked membrane regions to yield a pattern and a size distribution nearly indistinguishable from the normally stacked controls. Restacking occurs in two steps: a rapid adhesion of adjoining stromal membrane surfaces with little particle movement, and a slower diffusion of additional large intramembranous particles into the stacked regions where they become trapped. Chlorophyll a:chlorophyll b ratios of membrane fraction obtained from normal, unstacked, and restacked membranes show that the particle movements are paralleled by movements of pigment molecules. The directed and reversible movements of membrane particles in isolated chloroplasts are compared with those reported for particles of plasma membranes.

Cellulose microfibrils, cell motility, and plasma membrane protein organization change in parallel during culmination in Dictyostelium discoideum

1996 ◽  
Vol 109 (13) ◽  
pp. 3079-3087 ◽  
Author(s):  
M.J. Grimson ◽  
C.H. Haigler ◽  
R.L. Blanton
Keyword(s):  
Cell Motility ◽  
Dictyostelium Discoideum ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Cellulose Microfibrils ◽  
Stalk Cell ◽  
Cellulose Synthesis ◽  
Particle Aggregates ◽  
Terminal Complexes

Prestalk cells of Dictyostelium discoideum contribute cellulose to two distinct structures, the stalk tube and the stalk cell wall, during culmination. This paper demonstrates by freeze fracture electron microscopy that two distinct types of intramembrane particle aggregates, which can be characterized as cellulose microfibril terminal complexes, occur in the plasma membranes of cells synthesizing these different forms of cellulose. The same terminal complexes were observed in situ in developing culminants and in vitro in monolayer cells induced to synthesize the two types of cellulose. We propose that cessation of cell motility is associated with a change in packing and intramembrane mobility of the particle aggregates, which cause a change in the nature of the cellulose synthesized. The terminal complexes are compared to those described in other organisms and related to the previous hypothesis of two modes of cellulose synthesis in Dictyostelium.

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 Membrane interactions between secretion granules and plasmalemma in three exocrine glands

1980 ◽  
Vol 84 (2) ◽  
pp. 438-453 ◽  
Author(s):  
Y Tanaka ◽  
P De Camilli ◽  
J Meldolesi
Keyword(s):  
Plasma Membranes ◽  
Intact Cell ◽  
Freeze Fracture ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Cells ◽  
Membrane Interactions ◽  
Thin Sections ◽  
Secretion Granules

Three types of membrane interactions were studied in three exocrine systems (the acinar cells of the rat parotid, rat lacrimal gland, and guinea pig pancrease) by freeze- fracture and thin-section electron microscopy: exocytosis, induced in vivo by specific pharmacological stimulations; the mutual apposition of secretory granule membranes in the intact cell; membrane appositions induced in vitro by centrifugation of the isolated granules. In all three glandular cells, the distribution of intramembrane particles (IMP) on the fracture faces of the luminal plasmagranule membrane particles (IMP) on the fracture faces of the lumenal plasmalemma appeared random before stimulation. However, after injection of secretagogues, IMP were rapidly clearly from the areas of granule- plasmalemma apposition in the parotid cells and, especially, in lacrimocytes. In the latter, the cleared areas appeared as large bulges toward the lumen, whereas in the parotid they were less pronounced. Exocytotic openings were usually large and the fracture faces of their rims were covered with IMP. In contrast, in stimulated pancreatic acinar cells, the IMP distribution remained apparently random after stimulation. Exocytoses were established through the formation of narrown necks, and no images which might correspond to early stages of membrane fusion were revealed. Within the cytoplasm of parotid and lacrimal cells (but not in the pancreas), both at rest and after stimulation, secretion granules were often closely apposed by means of flat, circular areas, also devoid of IMP. In thin sections, the images corresponding to IMP-free areas were close granule-granule and granule-plasmalemma appositions, sometimes with focal merging of the membrane outer layers to yield pentalaminar structures. Isolated secretion granules were forced together in vitro by centrifugation. Under these conditions, increasing the centrifugal force from 1,600 to 50,000 g for 10 min resulted in a progressive, statistically significant increase of the frequency of IMP-free flat appositions between parotid granules. In contrast, no such areas were seen between freeze-fractured pancreatic granules, although some focal pentalaminar appositions appeared in section after centrifugation at 50 and 100,000 g for 10 min. On the basis of the observation that, in secretory cells, IMP clearing always develops in deformed membrane areas (bulges, depressions, flat areas), it is suggested that it might result from the forced mechanical apposition of the interacting membranes. This might be a preliminary process not sufficient to initiate fusion. In the pancreas, IMP clearing could occur over surface areas too small to be detected. In stimulated parotid and lacrimal glands they were exceptional. These structures were either attached at the sites of continuity between granule and plasma membranes, or free in the acinar lumen, with a preferential location within exocytotic pockets or in their proximity. Experiments designed to investigate the nature of these blisters and vesicles revealed that they probably arise artifactually during glutaraldehyde fixation. In fact, (a) they were large and numerous in poorly fixed samples but were never observed in thin sections of specimens fixed in one step with glutaraldehyde and OsO(4); and (b) no increase in concentration of phospholipids was observed in the parotid saliva and pancreatic juice after stimulation of protein discharge, as was to be expected if release of membrane material were occurring after exocytosis.

scholarly journals Phospholipase C activation by ethanol in rat hepatocytes is unaffected by chronic ethanol feeding

1990 ◽  
Vol 272 (1) ◽  
pp. 59-64 ◽  
Author(s):  
J B Hoek ◽  
T F Taraschi ◽  
K Higashi ◽  
E Rubin ◽  
A P Thomas
Keyword(s):  
Phospholipase C ◽  
Order Parameter ◽  
Plasma Membranes ◽  
Structural Changes ◽  
Rat Hepatocytes ◽  
Maximal Response ◽  
Molecular Order ◽  
Transient Activation ◽  
Ethanol Feeding

The activation of phosphoinositide-specific phospholipase C by ethanol was compared in hepatocytes isolated from ethanol-fed rats and from pair-fed control animals. Ethanol (100-300 mM) caused a dose-dependent transient increase in cytosolic free Ca2+ levels in indo-1-loaded hepatocytes from both groups of animals. The rate of Ca2+ increase was similar in hepatocytes from control and ethanol-fed rats, but the decay of the Ca2+ increase was somewhat slower in the latter preparation. The ethanol-induced Ca2+ increase caused activation of glycogen phosphorylase, with 50% response at 50 mM-ethanol and a maximal response at 150-200 mM-ethanol, not significantly different in hepatocytes from control and ethanol-fed animals. Ins(1,4,5)P3 formation in response to ethanol (300 mM) or vasopressin (2 nM or 40 nM) was also similar in the two preparations. It is concluded that long-term ethanol feeding does not lead to an adaptive response with respect to the ethanol-induced phospholipase C activation in rat hepatocytes. The ability of ethanol in vitro to decrease membrane molecular order in liver plasma membranes from ethanol-fed and control rats was measured by e.s.r. Membranes from ethanol-fed animals had a significantly lower baseline order parameter compared with control preparations (0.313 and 0.327 respectively), indicative of decreased membrane molecular order. Addition of 100 mM-ethanol significantly decreased the order parameter in control preparations by 2.1%, but had no effect on the order parameter of plasma membranes from ethanol-fed rats, indicating that the plasma membranes had developed tolerance to ethanol, similar to other membranes in the liver. Thus the membrane structural changes associated with this membrane tolerance do not modify the ethanol-induced activation of phospholipase C. The transient activation of phospholipase C by ethanol in hepatocytes may play a role in maintaining an adaptive phenotype in rat liver.

scholarly journals Ultrastructure of the sodium pump: Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+, K+)-ATPase

1977 ◽  
Vol 75 (3) ◽  
pp. 619-634 ◽  
Author(s):  
N Deguchi ◽  
PL Jorgensen ◽  
AB Maunsbach
Keyword(s):  
Membrane Surface ◽  
Freeze Fracture ◽  
Negative Staining ◽  
Intramembranous Particle ◽  
Freeze Fracturing ◽  
Intramembranous Particles ◽  
Membrane Surfaces ◽  
Thin Sectioning ◽  
Surface Particle ◽  
Protein Components

Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 μm. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per μm(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per μm(2) and 390 +/- 170 per μm(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.

An unusual intramembranous structure in the renal tubule of a snake

1978 ◽  
Vol 56 (3) ◽  
pp. 516-518
Author(s):  
William D. Peek
Keyword(s):  
Gap Junctions ◽  
Plasma Membranes ◽  
Renal Tubule ◽  
Freeze Fracture ◽  
Unknown Origin ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Intramembranous Particles ◽  
Relative Paucity ◽  
Proximal Tubular

Freeze–fracture revealed unusual structures of unknown origin in plasma membranes of proximal tubular cells in the nephron of the garter snake. These areas are closely associated with gap junctions and are characterized primarily by the relative paucity of intramembranous particles.

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.

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 Rearrangements of integral membrane components during in vitro aging of sheep erythrocyte membranes

1977 ◽  
Vol 74 (2) ◽  
pp. 389-398 ◽  
Author(s):  
HU Lutz ◽  
AJ Lomant ◽  
P McMillan ◽  
E Wehrli
Keyword(s):  
Membrane Proteins ◽  
Particle Density ◽  
Sheep Erythrocyte ◽  
Freeze Fracture ◽  
Erythrocyte Membranes ◽  
Intramembranous Particles ◽  
In Vitro Aging ◽  
Membrane Components ◽  
Fracture Electron

In vitro aged sheep erythrocytes and sheep erythrocyte ghosts spontaneously release vesicles that consist of long protrusions affixed to flattened headlike structures. The intramembranous particles seen on the protoplasmic face of freeze fracture electron micrographs of vesicle protrusions are arranged in paired particle rows. On the equivalent fracture face of headlike structures, the particle density is low; if particles are present, they are clustered along the rim of the flattened headlike structure and at the junction with the protrusion. The released vesicles are depleted of the intramembranous particles seen on the exoplasmic face of ghost but retain almost exclusively particles of the protoplasmic face. Correspondingly, the exoplasmic face of ghosts that have released vesicles reveals a 28 percent higher density of intramembranous particles than that of fresh ghosts. Purified vesicles are depleted of spectrin but retain integral membrane proteins, with one of an apparent mol wt of 160,000 accounting for nearly 50 percent of the total protein (Lutz, H.U.,R. Barber, and R.F. McGuire. 1976. J. Biol. Chem. 251:3500-3510). When vesicles are modified with the cleavable cross-linking reagent [(35)S]dithiobis (succinimidyl propionate)at 0 degrees C, the 160,000 mol wt protein is rapidly converted to disulfide-linked dimers and higher oligomers. Exposure of intact ghosts to the reagent in the same way fails to yield equivalent polymers. A comparison of the morphological and biochemical aspects of ghosts and vesicles suggest that a marked rearrangement of membrane proteins accompanies the supramolecular redistribution of intramembranous particles during spontaneous vesiculation. The results also suggest that the paired particles of the protoplasmic face of vesicle protrusions are arranged in paired helices and contain the 160,000 mol wt protein as dimers.

Structural changes of spores of tree fungal pathogens after treatment with the designed antimicrobial peptide D2A21

2000 ◽  
Vol 78 (4) ◽  
pp. 462-471 ◽  
Author(s):  
D Rioux ◽  
V Jacobi ◽  
M Simard ◽  
R C Hamelin
Keyword(s):  
Plasma Membrane ◽  
Spore Germination ◽  
Fungal Pathogens ◽  
Plasma Membranes ◽  
Structural Changes ◽  
Cronartium Ribicola ◽  
Ophiostoma Ulmi ◽  
Nectria Galligena ◽  
In Vitro Effects

In vitro effects of the antimicrobial synthetic D2A21 peptide on the structure of spores of four fungal pathogens causing important tree diseases were examined by microscopy in parallel with tests to measure inhibition of spore germination. With light microscopy, the use of SYTOX®green stain indicated that the peptide rapidly altered the plasma membrane of conidia of three Ascomycetes: Gremmeniella abietina (Lagerberg) Morelet var. abietina Petrini et al., Ophiostoma ulmi (Buism.) Nannf., and Nectria galligena Bres. With basidiospores of Cronartium ribicola J.C. Fisch., a difference between control and treated spores was also found, but it was less pronounced than with conidia of the Ascomycetes. In transmission electron microscopy, untreated conidia showed typical cytoplasmic contents with the regular presence of mitochondria, ribosomes, and nuclei, at times accompanied by vacuoles of various sizes. At concentrations of the peptide inhibitory to spore germination, plasma membranes, as well as nuclear and mitochondrial envelopes, were either generally difficult to discern or were distorted and swollen. At more advanced stages, the cytoplasm of treated spores contained numerous vesicles and was in places more electron-dense than in controls. Cytoplasm leakage was also regularly observed. Present observations strongly suggest that the primary site of action of this peptide is located at the plasma membrane level.Key words: Cronartium ribicola, Gremmeniella abietina, Nectria galligena, Ophiostoma ulmi, D2A21, peptide.

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