Freeze-Fracture and Cytoehemical Studies on the in Vitro Cyst Form of Reptilian Blastocystis pythoni

2003 ◽  
Vol 50 (1) ◽  
pp. 70-75 ◽  
Author(s):  
HISAO YOSHIKAWA ◽  
MINAKO NAGASHIMA ◽  
KEIKO MORIMOTO ◽  
YASUKO YAMANOUTI ◽  
EU HIAN YAP ◽  
...  
Keyword(s):  
Author(s):  
P.L. Moore

Previous freeze fracture results on the intact giant, amoeba Chaos carolinensis indicated the presence of a fibrillar arrangement of filaments within the cytoplasm. A complete interpretation of the three dimensional ultrastructure of these structures, and their possible role in amoeboid movement was not possible, since comparable results could not be obtained with conventional fixation of intact amoebae. Progress in interpreting the freeze fracture images of amoebae required a more thorough understanding of the different types of filaments present in amoebae, and of the ways in which they could be organized while remaining functional.The recent development of a calcium sensitive, demembranated, amoeboid model of Chaos carolinensis has made it possible to achieve a better understanding of such functional arrangements of amoeboid filaments. In these models the motility of demembranated cytoplasm can be controlled in vitro, and the chemical conditions necessary for contractility, and cytoplasmic streaming can be investigated. It is clear from these studies that “fibrils” exist in amoeboid models, and that they are capable of contracting along their length under conditions similar to those which cause contraction in vertebrate muscles.


Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.


1989 ◽  
Vol 25 (9) ◽  
pp. 791-799 ◽  
Author(s):  
Debra J. Hazen-Martin ◽  
Donald A. Sens ◽  
John G. Blackburn ◽  
Mary C. Flath ◽  
Mary Ann Sens

1996 ◽  
Vol 109 (13) ◽  
pp. 3079-3087 ◽  
Author(s):  
M.J. Grimson ◽  
C.H. Haigler ◽  
R.L. Blanton

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.


Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 583-594 ◽  
Author(s):  
N Dainiak ◽  
CM Cohen

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.


1981 ◽  
Vol 88 (3) ◽  
pp. 660-663 ◽  
Author(s):  
D M Landis ◽  
T S Reese

Membranes of the astrocytic processes investing small blood vessels and the surface of the brain contain numerous arrays of orthogonally packed particles as revealed by the freeze-fracture technique. The structure of these particle arrays, which we have termed "assemblies," is the same whether tissue is prepared for freeze-fracture by conventional fixation or by quick excision and rapid freezing. However, assemblies are progressively replaced by amorphous clumps and then disappear as the interval between decapitation and rapid freezing increases. Nearly normal numbers of assemblies may be maintained in cerebellar slices in vitro, but there too they disappear at low PO2 or in the presence of dinitrophenol. No other neuronal or glial membrane specialization exhibits a comparable lability.


1980 ◽  
Vol 84 (2) ◽  
pp. 438-453 ◽  
Author(s):  
Y Tanaka ◽  
P De Camilli ◽  
J Meldolesi

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.


1978 ◽  
Vol 235 (5) ◽  
pp. C168-C179 ◽  
Author(s):  
R. E. Garfield ◽  
S. M. Sims ◽  
M. S. Kannan ◽  
E. E. Daniel

Gap junctions between smooth muscle cells of the myometrium of pregnant rats were found only immediately prior to, during and immediately after parturition by quantitative thin-section and freeze-fracture microscopy. Ovariectomy of 16- to 17-days-pregnant rats resulted in premature termination of pregnancy and the appearance of gap junctions. Methods that prolonged normal pregnancy in rats or maintained pregnancy in ovariectomized animals (progesterone treatment) prevented the appearance of gap junctions. Gap junctions formed in tissues incubated for 24--96 h in vitro without any hormonal influence. We propose that gap junctions are essential for normal labor and delivery for synchronous contraction of the muscle of the uterus. We present a model for control of parturition that may apply to other animals including humans. The model proposes: 1) the possible roles progesterone, prostaglandins, or estrogens may play in initiating gap-junction formation; 2) that the formation of gap junctions is a necessary step in activation of the myometrium leading to labor; and 3) that agents used to stimulate or inhibit labor may do so by affecting gap junctions.


2000 ◽  
Vol 6 (S2) ◽  
pp. 854-855
Author(s):  
B. Sternberg-Papahadjopoulos ◽  
K. Hong ◽  
W. Zheng ◽  
D. Papahadjopoulos

Complexes formed during interaction of cationic liposomes with polynucleotides such as DNA (CLDC) self-assemble into a variety of polymorphic structures. They display bilayer (FIG. 1-5) and non-bilayer structures (FIG. 6). We have recorded bilayer structures such as spaghetti/meatball-type structures (FIG. I), map-pins (FIG. 2) spherical particles and invaginated liposomes (FIG. 3, 4) and oligolamellar structures (FIG. 5). The non-bilayer lipid arrangements include honeycombtype structure (Hn, FIG. 6) and cubic phase lipids.We have chosen mainly freeze-fracture electron microscopy (FIG. 1-3, 5,6) but also cryo-electron microscopy (FIG.4) for recording polymorphic structures, and for studying factors and conditions triggering the formation and stabilization of specific structure types. Furthermore, we took microscopically snapshots of the interaction of specific structure types with cultured cells. In order to find out the “active” structure in terms of transfection, we investigated the transfection activity both in vivo and in vitro of CLDC, and studied in parallel their morphology in serum as well as in cell medium.


1976 ◽  
Vol 71 (1) ◽  
pp. 136-158 ◽  
Author(s):  
L A Staehelin

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.


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