scholarly journals Detergent-Resistant Membrane Microdomains Facilitate Ib Oligomer Formation and Biological Activity of Clostridium perfringens Iota-Toxin

2004 ◽  
Vol 72 (4) ◽  
pp. 2186-2193 ◽  
Author(s):  
Martha L. Hale ◽  
Jean-Christophe Marvaud ◽  
Michel R. Popoff ◽  
Bradley G. Stiles
Keyword(s):  
Clostridium Perfringens ◽  
Cell Types ◽  
Cell Receptor ◽  
Vero Cells ◽  
Membrane Microdomains ◽  
Oligomer Formation ◽  
A Cell ◽  
Clostridium Perfringens Iota Toxin ◽  
Detergent Resistant Membrane ◽  
Soluble Fractions

ABSTRACT Clostridium perfringens iota-toxin consists of two separate proteins identified as a cell binding protein, iota b (Ib), which forms high-molecular-weight complexes on cells generating Na+/K+-permeable pores through which iota a (Ia), an ADP-ribosyltransferase, presumably enters the cytosol. Identity of the cell receptor and membrane domains involved in Ib binding, oligomer formation, and internalization is currently unknown. In this study, Vero (toxin-sensitive) and MRC-5 (toxin-resistant) cells were incubated with Ib, after which detergent-resistant membrane microdomains (DRMs) were extracted with cold Triton X-100. Western blotting revealed that Ib oligomers localized in DRMs extracted from Vero, but not MRC-5, cells while monomeric Ib was detected in the detergent-soluble fractions of both cell types. The Ib protoxin, previously shown to bind Vero cells but not form oligomers or induce cytotoxicity, was detected only in the soluble fractions. Vero cells pretreated with phosphatidylinositol-specific phospholipase C before addition of Ib indicated that glycosylphosphatidyl inositol-anchored proteins were minimally involved in Ib binding or oligomer formation. While pretreatment of Vero cells with filipin (which sequesters cholesterol) had no effect, methyl-β-cyclodextrin (which extracts cholesterol) reduced Ib binding and oligomer formation and delayed iota-toxin cytotoxicity. These studies showed that iota-toxin exploits DRMs for oligomer formation to intoxicate cells.

scholarly journals Binding Component of Clostridium perfringens Iota-Toxin Induces Endocytosis in Vero Cells

2002 ◽  
Vol 70 (4) ◽  
pp. 1909-1914 ◽  
Author(s):  
Masahiro Nagahama ◽  
Koichi Nagayasu ◽  
Keiko Kobayashi ◽  
Jun Sakurai
Keyword(s):  
Clostridium Perfringens ◽  
Vero Cells ◽  
Binary Toxin ◽  
Wild Type ◽  
Oligomer Formation ◽  
Clostridium Perfringens Iota Toxin ◽  
Binding Component ◽  
Pronase Treatment

ABSTRACT Clostridium perfringens iota-toxin is a binary toxin consisting of two individual proteins, the binding component (Ib) and the enzyme component (Ia). Wild-type Ib bound to Vero cells at 4 and 37°C and formed oligomers at 37°C but not at 4°C. The Ib-induced K+ release from the cells was dependent on the oligomer formation of Ib in the cells, but the oligomer formation did not induce rounding activity or cytotoxicity. After incubation of the cells with recombinant Ib (rIb) at 37°C, the Ib oligomer in the cell became resistant to pronase treatment with time, but the Ib monomer was sensitive to the treatment. Furthermore, treatment of Vero cells with rIb in the presence of bafilomycin, methylamine, or ethylamine resulted in accumulation of the oligomer in the cells but had no effect on K+ release. Moreover, incubation with Ib plus Ia in the presence of these agents caused no rounding in the cells. These observations suggest that Ib binds to Vero cells, itself oligomerizing to form ion-permeable channels and that the formation of oligomer then induces endocytosis.

scholarly journals Clostridium perfringens Iota Toxin: Binding Studies and Characterization of Cell Surface Receptor by Fluorescence-Activated Cytometry

2000 ◽  
Vol 68 (6) ◽  
pp. 3475-3484 ◽  
Author(s):  
Bradley G. Stiles ◽  
Martha L. Hale ◽  
Jean-Christophe Marvaud ◽  
Michel R. Popoff
Keyword(s):  
Cell Surface ◽  
Clostridium Perfringens ◽  
Cell Types ◽  
Vero Cells ◽  
Cell Surface Receptor ◽  
Binding Studies ◽  
Binding Characteristics ◽  
Toxin Binding ◽  
Surface Receptor ◽  
Clostridium Perfringens Iota Toxin

ABSTRACT The binding characteristics of iota toxin, a binary enterotoxin produced by Clostridium perfringens type E, were studied by fluorescence-activated cytometry. The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37°C for 10 min. The binding of Ib was inhibited by antisera against C. perfringens type E orClostridium spiroforme culture supernatants, but notC. perfringens types C or D. Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface. The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia). Ia attached to cell-bound Ib within 10 min at 37°C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min. Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane.

scholarly journals The Long-Lived Nature of Clostridium perfringens Iota Toxin in Mammalian Cells Induces Delayed Apoptosis

2009 ◽  
Vol 77 (12) ◽  
pp. 5593-5601 ◽  
Author(s):  
Hanna Hilger ◽  
Sascha Pust ◽  
Guido von Figura ◽  
Eva Kaiser ◽  
Bradley G. Stiles ◽  
...  
Keyword(s):  
Cell Death ◽  
Clostridium Perfringens ◽  
Mammalian Cells ◽  
Catalytic Domain ◽  
Vero Cells ◽  
African Green Monkey Kidney ◽  
Adp Ribosylation ◽  
Clostridium Perfringens Iota Toxin ◽  
Adp Ribosyltransferase ◽  
C2 Toxin

ABSTRACT Mono-ADP ribosylation of actin by bacterial toxins, such as Clostridium perfringens iota or Clostridium botulinum C2 toxins, results in rapid depolymerization of actin filaments and cell rounding. Here we report that treatment of African green monkey kidney (Vero) cells with iota toxin resulted in delayed caspase-dependent death. Unmodified actin did not reappear in toxin-treated cells, and enzyme-active toxin was detectable in the cytosol for at least 24 h. C2 toxin showed comparable, long-lived effects in cells, while a C2 toxin control lacking ADP-ribosyltransferase activity did not induce cell death. To address whether the remarkable stability of the iota and C2 toxins in cytosol was crucial for inducing cell death, we treated cells with C/SpvB, the catalytic domain of Salmonella enterica SpvB. Although C/SpvB also mono-ADP ribosylates actin as do the iota and C2 toxins, cells treated with a cell-permeating C/SpvB fusion toxin became rounded but recovered and remained viable. Moreover, unmodified actin reappeared in these cells, and ADP-ribosyltransferase activity due to C/SpvB was not detectable in the cytosol after 24 h, a result most likely due to degradation of C/SpvB. Repeated application of C/SpvB prevented recovery of cells and reappearance of unmodified actin. In conclusion, a complete but transient ADP ribosylation of actin was not sufficient to trigger apoptosis, implying that long-term stability of actin-ADP-ribosylating toxins, such as iota and C2, in the cytosol is crucial for inducing delayed, caspase-dependent cell death.

scholarly journals Clostridium perfringens iota toxin: characterization of the cell-associated iota b complex

2002 ◽  
Vol 367 (3) ◽  
pp. 801-808 ◽  
Author(s):  
Bradley G. STILES ◽  
Martha L. HALE ◽  
Jean Christophe MARVAUD ◽  
Michel R. POPOFF
Keyword(s):  
Cell Surface ◽  
Molecular Mass ◽  
Clostridium Perfringens ◽  
Surface Protein ◽  
Protein S ◽  
Vero Cells ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Neutralizing Monoclonal Antibody ◽  
A Cell

Clostridium perfringens type E iota toxin consists of two unlinked proteins designated as iota a (Ia; molecular mass47kDa), an ADP-ribosyltransferase and iota b (Ib; molecular mass81kDa) which binds to the cell surface and facilitates Ia entry into the cytosol. By Western-blot analysis, Ib incubated with Vero cells at 37°C generated a cell-associated, SDS-insoluble oligomer of Ib (molecular mass>220kDa) within 15s, which was still evident 110min after washing cells. Ib oligomerization was temperature, but not pH, dependent and was facilitated by a cell-surface protein(s). Within 5min at 37°C, cell-bound Ib generated Na+/K+ permeable channels that were blocked by Ia. However, Ib-induced channels or oligomers were not formed at 4°C. Two monoclonal antibodies raised against Ib that recognize unique, neutralizing epitopes within residues 632—655 either inhibited Ib binding to cells and/or oligomerization, unlike a non-neutralizing monoclonal antibody that binds within Ib residues 28—66. The Ib protoxin (molecular mass98kDa), which does not facilitate iota cytotoxicity but binds to Vero cells, did not oligomerize or form ion-permeable channels on cells, and neither trypsin nor chymotrypsin treatment of cell-bound Ib protoxin induced large complex formation. The link between Ib oligomers and iota toxicity was also apparent with a resistant cell line (MRC-5), which bound to Ib with no evidence of oligomerization. Overall, these studies revealed that the biological activity of iota toxin is dependent on a long-lived, cell-associated Ib complex that rapidly forms ion-permeable channels in cell membranes. These results further reveal the similarities of C. perfringens iota toxin with other bacterial binary toxins produced by Bacillus anthracis and C. botulinum.

scholarly journals H-Ras Distribution and Signaling in Plasma Membrane Microdomains Are Regulated by Acylation and Deacylation Events

2015 ◽  
Vol 35 (11) ◽  
pp. 1898-1914 ◽  
Author(s):  
Lorena Agudo-Ibáñez ◽  
Ana Herrero ◽  
Mariano Barbacid ◽  
Piero Crespo
Keyword(s):  
Plasma Membrane ◽  
Posttranslational Modifications ◽  
Lateral Diffusion ◽  
Membrane Microdomains ◽  
Ras Signaling ◽  
A Cell ◽  
Cell Type Specific ◽  
Plasma Membrane Microdomains ◽  
Detergent Resistant Membrane ◽  
Activation Status

H-Ras must adhere to the plasma membrane to be functional. This is accomplished by posttranslational modifications, including palmitoylation, a reversible process whereby H-Ras traffics between the plasma membrane and the Golgi complex. At the plasma membrane, H-Ras has been proposed to occupy distinct sublocations, depending on its activation status: lipid rafts/detergent-resistant membrane fractions when bound to GDP, diffusing to disordered membrane/soluble fractions in response to GTP loading. Herein, we demonstrate that H-Ras sublocalization is dictated by its degree of palmitoylation in a cell type-specific manner. Whereas H-Ras localizes to detergent-resistant membrane fractions in cells with low palmitoylation activity, it locates to soluble membrane fractions in lineages where it is highly palmitoylated. Interestingly, in both cases GTP loading results in H-Ras diffusing away from its original sublocalization. Moreover, tilting the equilibrium between palmitoylation and depalmitoylation processes can substantially alter H-Ras segregation and, subsequently, its biochemical and biological functions. Thus, the palmitoylation/depalmitoylation balance not only regulates H-Ras cycling between endomembranes and the plasma membrane but also serves as a key orchestrator of H-Ras lateral diffusion between different types of plasma membrane and thereby of H-Ras signaling.

scholarly journals A cell atlas of human thymic development defines T cell repertoire formation

2020 ◽  
Author(s):  
Jong-Eun Park ◽  
Rachel A. Botting ◽  
Cecilia Domínguez Conde ◽  
Dorin-Mirel Popescu ◽  
Marieke Lavaert ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lineage ◽  
Cell Types ◽  
Cell Receptor ◽  
Cell Repertoire ◽  
Thymic Development ◽  
Human Thymus ◽  
Human T Cell ◽  
A Cell

AbstractThe thymus provides a nurturing environment for the differentiation and selection of T cells, a process orchestrated by their interaction with multiple thymic cell types. We utilised single-cell RNA-sequencing (scRNA-seq) to create a cell census of the human thymus and to reconstruct T-cell differentiation trajectories and T-cell receptor (TCR) recombination kinetics. Using this approach, we identified and located in situ novel CD8αα+ T-cell populations, thymic fibroblast subtypes and activated dendritic cell (aDC) states. In addition, we reveal a bias in TCR recombination and selection, which is attributed to genomic position and suggests later commitment of the CD8+ T-cell lineage. Taken together, our data provide a comprehensive atlas of the human thymus across the lifespan with new insights into human T-cell development.

scholarly journals A cell atlas of human thymic development defines T cell repertoire formation

Science ◽  
2020 ◽  
Vol 367 (6480) ◽  
pp. eaay3224 ◽  
Author(s):  
Jong-Eun Park ◽  
Rachel A. Botting ◽  
Cecilia Domínguez Conde ◽  
Dorin-Mirel Popescu ◽  
Marieke Lavaert ◽  
...  
Keyword(s):  
T Cell ◽  
Life Span ◽  
Cell Types ◽  
Cell Receptor ◽  
Cell Repertoire ◽  
Thymic Development ◽  
Human Thymus ◽  
Human T Cell ◽  
A Cell

The thymus provides a nurturing environment for the differentiation and selection of T cells, a process orchestrated by their interaction with multiple thymic cell types. We used single-cell RNA sequencing to create a cell census of the human thymus across the life span and to reconstruct T cell differentiation trajectories and T cell receptor (TCR) recombination kinetics. Using this approach, we identified and located in situ CD8αα+ T cell populations, thymic fibroblast subtypes, and activated dendritic cell states. In addition, we reveal a bias in TCR recombination and selection, which is attributed to genomic position and the kinetics of lineage commitment. Taken together, our data provide a comprehensive atlas of the human thymus across the life span with new insights into human T cell development.

Dendritic cells of the human epidermis: immuno-electron microscopic studies of la antigen reactivity

1978 ◽  
Vol 36 (2) ◽  
pp. 644-645
Author(s):  
G. Rowden ◽  
M. G. Lewis ◽  
T. M. Phillips
Keyword(s):  
Dendritic Cells ◽  
Langerhans Cells ◽  
Cell Types ◽  
Cell Type ◽  
Electron Microscopic ◽  
La Antigen ◽  
Microscopic Studies ◽  
A Cell ◽  
C3 Receptors ◽  
Antigen Reactivity

Langerhans cells of mammalian stratified squamous epithelial have proven to be an enigma since their discovery in 1868. These dendritic suprabasal cells have been considered as related to melanocytes either as effete cells, or as post divisional products. Although grafting experiments seemed to demonstrate the independence of the cell types, much confusion still exists. The presence in the epidermis of a cell type with morphological features seemingly shared by melanocytes and Langerhans cells has been especially troublesome. This so called "indeterminate", or " -dendritic cell" lacks both Langerhans cells granules and melanosomes, yet it is clearly not a keratinocyte. Suggestions have been made that it is related to either Langerhans cells or melanocyte. Recent studies have unequivocally demonstrated that Langerhans cells are independent cells with immune function. They display Fc and C3 receptors on their surface as well as la (immune region associated) antigens.

Membrane microdomains: lessons from microscopy

1995 ◽  
Vol 53 ◽  
pp. 776-777
Author(s):  
J.M. Robinson ◽  
J.M Oliver
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Types ◽  
Imaging Modalities ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lateral Heterogeneity ◽  
Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

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