Uneven Distribution of Surface Antigens During Antigenic Variation in Paramecium Primaurelia

1989 ◽  
Vol 92 (2) ◽  
pp. 205-215
Author(s):  
CLAUDE ANTONY ◽  
YVONNE CAPDEVILLE
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Antigenic Variation ◽  
Temperature Shift ◽  
Surface Antigens ◽  
Immunogold Electron Microscopy ◽  
Membrane Domains ◽  
Phosphatidylinositol Phospholipase C

In Paramecium primaurelia surface antigen (SAg) expression can be experimentally controlled by temperature-shift-induced antigenic variation. As only one SAg is usually expressed at the cell surface under stable environmental conditions, we used the temperature-shift-induced change in SAg to follow the newly expressed antigen and the disappearing one, by both immunofluorescence and immunogold electron microscopy. The new SAg initially appeared scattered at the cell surface, over the ciliary and interciliary membrane domains, without any readily identifiable specific site of insertion into the plasma membrane. The concentration of the newly incorporated molecules then increased gradually on the plasma membrane. In contrast, the surface of the previously expressed SAg was not complementary to the pattern of the appearing SAg. The loss of the old SAg is delayed after the temperature shift and seems to occur more suddenly the appearance of new SAg. This loss is characterized by a subpopulation of cilia bearing old SAg coexisting with other cilia and a pellicle almost devoid of the old SAg molecules. The topological distribution of the new and old SAgs is discussed in relation to the lipidic nature of the SAg anchor and to a possible role of an Paramecium phosphatidylinositol phospholipase C.

Compartmentalization, processing and redistribution of the plasma membrane protein CE9 on rodent spermatozoa. Relationship of the annulus to domain boundaries in the plasma membrane of the tail

1994 ◽  
Vol 107 (2) ◽  
pp. 561-570 ◽  
Author(s):  
M.M. Cesario ◽  
J.R. Bartles
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Domain Boundary ◽  
Immunogold Electron Microscopy ◽  
Membrane Domains ◽  
Tail Domain ◽  
Membrane Domain ◽  
Plasma Membrane Protein ◽  
Plasma Membrane Domain

Western blotting, immunofluorescence and immunogold electron microscopy were used to examine the compartmentalization, processing and redistribution of the integral plasma membrane protein CE9 on the spermatozoa of rats, mice and hamsters. In each species examined, spermatozoal CE9 was found to undergo endoproteolytic processing followed by a net redistribution from the posterior-tail domain into the anterior-tail domain of the plasma membrane during epididymal maturation. Compared to spermatozoa of the rat and mouse, those of the hamster were found to express a greater proportion of their CE9 within the anterior-tail plasma membrane domain at all stages of maturation. As a consequence, CE9 was judged to be a suitable marker for two different spermatozoal plasma membrane domains: the posterior-tail plasma membrane domain (spermatozoa from the testis and caput epididymidis of the rat and mouse) and the anterior-tail domain (spermatozoa from the cauda epididymidis of the hamster). Immunogold electron microscopy was used to pinpoint the positions of the boundaries of these CE9-containing plasma membrane domains at a high level of resolution. In each case, the position of the CE9 domain boundary was found to be strongly correlated with that of the subplasmalemmal electron-dense ring known as the annulus. The precise spatial relationship between the CE9 domain boundary and the annulus was, however, found to differ significantly among species and/or as a function of maturation.

Immunogold electron microscopy of surface antigens of oral bacteria

1984 ◽  
Vol 30 (8) ◽  
pp. 1008-1013 ◽  
Author(s):  
C. Mouton ◽  
L. Lamonde
Keyword(s):  
Electron Microscopy ◽  
Surface Antigens ◽  
Oral Bacteria ◽  
Immunogold Electron Microscopy ◽  
Bacterial Surface ◽  
Outer Aspect ◽  
Bacteroides Gingivalis ◽  
Cell Envelopes ◽  
Inexpensive Procedure ◽  
Monospecific Antibodies

Colloidal gold particles 3–6 nm in diameter were prepared and stabilized with the IgG fraction of polyspecific rabbit antisera produced against four different oral bacteria. The immunogold markers were used in homologous reactions to label the bacteria in a preembedding procedure for electron microscopy. An indirect immunofluorescence procedure was concurrently used to optimize the labelling conditions before observation with the electron microscope. The immunogold markers labelled fibrillar structures extending outward 50–275 nm from the Gram-positive cell envelopes and a fuzzy 5–10 nm thick capsulelike layer on the outer aspect of Bacteroides gingivalis. The immunogold method appears to be a simple, rapid, and inexpensive procedure suitable for the study of bacterial surface antigens and can be upgraded with the use of monospecific antibodies.

The GTPase Rac1 selectively regulates Salmonella invasion at the apical plasma membrane of polarized epithelial cells

2001 ◽  
Vol 114 (7) ◽  
pp. 1331-1341 ◽  
Author(s):  
A.K. Criss ◽  
D.M. Ahlgren ◽  
T.S. Jou ◽  
B.A. McCormick ◽  
J.E. Casanova
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Bacterial Pathogen ◽  
Small Gtpases ◽  
Apical Plasma Membrane ◽  
Membrane Domains ◽  
Intestinal Epithelial ◽  
Actin Structure

The bacterial pathogen Salmonella typhimurium colonizes its animal hosts by inducing its internalization into intestinal epithelial cells. This process requires reorganization of the actin cytoskeleton of the apical plasma membrane into elaborate membrane ruffles that engulf the bacteria. Members of the Ρ family of small GTPases are critical regulators of actin structure, and in nonpolarized cells, the GTPase Cdc42 has been shown to modulate Salmonella entry. Because the actin architecture of epithelial cells is organized differently from that of nonpolarized cells, we examined the role of two ‘Rgr; family GTPases, Cdc42 and Rac1, in invasion of polarized monolayers of MDCK cells by S. typhimurium. Surprisingly, we found that endogenous Rac1, but not Cdc42, was activated during bacterial entry at the apical pole, and that this activation required the bacterial effector protein SopE. Furthermore, expression of dominant inhibitory Rac1 but not Cdc42 significantly inhibited apical internalization of Salmonella, indicating that Rac1 activation is integral to the bacterial entry process. In contrast, during basolateral internalization, both Cdc42 and Rac1 were activated; however, neither GTPase was required for entry. These findings, which differ significantly from previous observations in nonpolarized cells, indicate that the host cell signaling pathways activated by bacterial pathogens may vary with cell type, and in epithelial tissues may further differ between plasma membrane domains.

Species-specific difference in distribution of voltage-gated L-type Ca2+ channels of cardiac myocytes

2000 ◽  
Vol 279 (6) ◽  
pp. C1963-C1969 ◽  
Author(s):  
Yoshiko Takagishi ◽  
Kenji Yasui ◽  
Nicholas J. Severs ◽  
Yoshiharu Murata
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cardiac Myocytes ◽  
Immunogold Electron Microscopy ◽  
Surface Plasma ◽  
Gold Particles ◽  
Intracellular Ca ◽  
Rat Myocytes ◽  
T Tubules ◽  
Species Specific

Ca2+influx via sarcolemmal voltage-dependent Ca2+ channels (L-type Ca2+ channels) is the fundamental step in excitation-contraction (E-C) coupling in cardiac myocytes. Physiological and pharmacological studies reveal species-specific differences in E-C coupling resulting from a difference in the contribution of Ca2+ influx and intracellular Ca2+ release to activation of contraction. We investigated the distribution of L-type Ca2+ channels in isolated cardiac myocytes from rabbit and rat ventricle by correlative immunoconfocal and immunogold electron microscopy. Immunofluorescence labeling revealed discrete spots in the surface plasma membrane and transverse (T) tubules in rabbit myocytes. In rat myocytes, labeling appeared more intense in T tubules than in the surface sarcolemma. Immunogold electron microscopy extended these findings, showing that the number of gold particles in the surface plasma membrane was significantly higher in rabbit than rat myocytes. In rabbit myocyte plasma membrane, the gold particles were distributed as clusters in both regions that were associated with junctional sarcoplasmic reticulum and those that were not. The findings are consistent with the idea that influx of Ca2+ via surface sarcolemmal Ca2+ channels contributes to intracellular Ca2+ to a greater degree in rabbit than in rat myocytes.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

scholarly journals Phospholipase D2 Mediates Acute Aldosterone Secretion in Response to Angiotensin II in Adrenal Glomerulosa Cells

Endocrinology ◽  
2010 ◽  
Vol 151 (5) ◽  
pp. 2162-2170 ◽  
Author(s):  
Haixia Qin ◽  
Michael A. Frohman ◽  
Wendy B. Bollag
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Cell Surface ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Cell Interior ◽  
Aldosterone Secretion ◽  
Phospholipase D2 ◽  
Glomerulosa Cells

In primary bovine adrenal glomerulosa cells, the signaling enzyme phospholipase D (PLD) is suggested to mediate priming, the enhancement of aldosterone secretion after pretreatment with and removal of angiotensin II (AngII), via the formation of persistently elevated diacylglycerol (DAG). To further explore PLD’s role in priming, glomerulosa cells were pretreated with an exogenous bacterial PLD. Using this approach, phosphatidic acid (PA) is generated on the outer, rather than the inner, leaflet of the plasma membrane. Although PA is not readily internalized, the PA is nonetheless rapidly hydrolyzed by cell-surface PA phosphatases to DAG, which efficiently flips to the inner leaflet and accesses the cell interior. Pretreatment with bacterial PLD resulted in priming upon subsequent AngII exposure, supporting a role of DAG in this process, because the increase in DAG persisted after exogenous PLD removal. To determine the PLD isoform mediating aldosterone secretion, and presumably priming, primary glomerulosa cells were infected with adenoviruses expressing GFP, PLD1, PLD2, or lipase-inactive mutants. Overexpressed PLD2 increased aldosterone secretion by approximately 3-fold over the GFP-infected control under basal conditions, with a significant enhancement to about 16-fold over the basal value upon AngII stimulation. PLD activity was also increased basally and upon stimulation with AngII. In contrast, PLD1 overexpression had little effect on aldosterone secretion, despite the fact that PLD activity was enhanced. In both cases, the lipase-inactive PLD mutants showed essentially no effect on PLD activity or aldosterone secretion. Our results suggest that PLD2 is the isoform that mediates aldosterone secretion and likely priming.

scholarly journals Surface redistribution and release of antibody-induced caps in entamoebae.

1980 ◽  
Vol 151 (1) ◽  
pp. 184-193 ◽  
Author(s):  
J Calderón ◽  
M de Lourdes Muñoz ◽  
H M Acosta
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Segregation ◽  
Surface Membrane ◽  
Single Layer ◽  
Surface Antigens ◽  
Spontaneous Release ◽  
Plasma Membrane Regeneration ◽  
Membrane Components ◽  
Radiolabeled Antibodies

Polyspecific antibodies bound to Entamoeba induced surface redistribution of membrane components toward the uroid region. Capping of surface antigens was obtained with a single layer of antibodies in E. histolytica and E. invadens. This surface segregation progressed to a large accumulation of folded plasma membrane that extruded as a defined vesicular cap. A spontaneous release of the cap at the end of the capping process took place. These released caps contained most of the antibodies that originally bound to the whole cell surface. Two-thirds of radiolabeled antibodies bound to the surface of E. histolytica were released into the medium in 2 h. Successive capping induced by repeated exposure of E. invadens to antibodies produced conglomerates of folded surface membrane, visualized as stacked caps, in proportion to the number of antibody exposures. These results indicate the remarkable ability of Entamoeba to rapidly regenerate substantial amounts of plasma membbrane. The properties of surface redistribution, liberation of caps, and plasma membrane regeneration, may contribute to the survival of the parasite in the host during infection.

scholarly journals Neisseria gonorrhoeae Porin P1.B Induces Endosome Exocytosis and a Redistribution of Lamp1 to the Plasma Membrane

2002 ◽  
Vol 70 (11) ◽  
pp. 5965-5971 ◽  
Author(s):  
Patricia Ayala ◽  
Brandi Vasquez ◽  
Lee Wetzler ◽  
Magdalene So
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Neisseria Gonorrhoeae ◽  
Bacterial Infection ◽  
Immunoglobulin A ◽  
Late Endosomes ◽  
A Cell ◽  
Iga Protease

ABSTRACT The immunoglobulin A (IgA) protease secreted by pathogenic Neisseria spp. cleaves Lamp1, thereby altering lysosomes in a cell and promoting bacterial intracellular survival. We sought to determine how the IgA protease gains access to cellular Lamp1 in order to better understand the role of this cleavage event in bacterial infection. In a previous report, we demonstrated that the pilus-induced Ca2+ transient triggers lysosome exocytosis in human epithelial cells. This, in turn, increases the level of Lamp1 at the plasma membrane, where it can be cleaved by IgA protease. Here, we show that porin also induces a Ca2+ flux in epithelial cells. This transient is similar in nature to that observed in phagocytes exposed to porin. In contrast to the pilus-induced Ca2+ transient, the porin-induced event does not trigger lysosome exocytosis. Instead, it stimulates exocytosis of early and late endosomes and increases Lamp1 on the cell surface. These results indicate that Neisseria pili and porin perturb Lamp1 trafficking in epithelial cells by triggering separate and distinct Ca2+-dependent exocytic events, bringing Lamp1 to the cell surface, where it can be cleaved by IgA protease.

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