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Microbial sphingomyelinase induces RhoA-mediated reorganization of the apical brush border membrane and is protective against invasion.

Saslowsky DE, Thiagarajah JR, McCormick BA, Lee JC, Lencer WI - Mol. Biol. Cell (2016)

Bottom Line: How the BBM dynamically responds to pathogenic and commensal bacterial signals can define intestinal homeostasis and immune function.We previously found that in model intestinal epithelium, the conversion of apical membrane sphingomyelin to ceramide by exogenous bacterial sphingomyelinase (SMase) protected against the endocytosis and toxicity of cholera toxin.Here we elucidate a mechanism of action by showing that SMase induces a dramatic, reversible, RhoA-dependent alteration of the apical cortical F-actin network.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology and Nutrition, Boston Children's Hospital, Boston, MA 02115 Harvard Digestive Diseases Center, Boston Children's Hospital, Boston, MA 02115 Harvard Medical School, Boston, MA 02115 david.saslowsky@nih.gov.

No MeSH data available.


Related in: MedlinePlus

Effects of SMase on polarized T84 subcellular structure. (A) Transmission electron microscope micrographs of T84 monolayers incubated apically with or without SMase for 50 min. Ultrathin sections are perpendicular to the monolayer support. Scale bar, 1 μm. Box insets are 2× digital zooms of apical PM region. (B) Three-dimensional reconstructions from XY-stacks of monolayers stained with phalloidin and immunostained for ZO-1 and β-tubulin. Arrow denotes lack of microtubules in the internal region of the SMase-induced F-actin bundle structure. (C) Monolayers treated with or without SMase were methanol fixed and immunostained for the ER marker calnexin (a fixation method not compatible with phalloidin staining). Enclosed areas represent single-cell outlines, and arrows denote regions of decreased calnexin immunostaining in SMase-treated monolayers. (D) Monolayers treated with or without SMase were fixed and immunostained for the Golgi marker GM130 or (E) Rab11 to mark recycling endosomes (arrows denote region of altered F-actin structure for the latter). Scale bars, 1 μm (A), 10 μm (B–E).
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Figure 5: Effects of SMase on polarized T84 subcellular structure. (A) Transmission electron microscope micrographs of T84 monolayers incubated apically with or without SMase for 50 min. Ultrathin sections are perpendicular to the monolayer support. Scale bar, 1 μm. Box insets are 2× digital zooms of apical PM region. (B) Three-dimensional reconstructions from XY-stacks of monolayers stained with phalloidin and immunostained for ZO-1 and β-tubulin. Arrow denotes lack of microtubules in the internal region of the SMase-induced F-actin bundle structure. (C) Monolayers treated with or without SMase were methanol fixed and immunostained for the ER marker calnexin (a fixation method not compatible with phalloidin staining). Enclosed areas represent single-cell outlines, and arrows denote regions of decreased calnexin immunostaining in SMase-treated monolayers. (D) Monolayers treated with or without SMase were fixed and immunostained for the Golgi marker GM130 or (E) Rab11 to mark recycling endosomes (arrows denote region of altered F-actin structure for the latter). Scale bars, 1 μm (A), 10 μm (B–E).

Mentions: The striking change in cortical F-actin structure prompted us to examine whether other subcellular features of the BBM or intracellular organelles might be affected by conversion of apical SM to ceramide. Transmission electron microscopy showed microvillar effacement in the central region of cells overlying the region of F-actin rearrangement in T84 monolayers (Figure 5A). By confocal microscopy, we found that microtubules extended deeper into the cell interior and essentially encased the SMase-induced F-actin bundles without obviously penetrating those structures (Figure 5B). Immunolocalization of the endoplasmic reticulum (ER)–resident protein calnexin showed an obvious relocalization of calnexin away from the central, subapical region of each cell in monolayers treated apically with SMase as compared with buffer controls (Figure 5C). This is the approximate area of the F-actin bundles, which, we infer, physically excluded the ER from these sites. We did not observe any changes in the intracellular distribution or morphology of the Golgi or recycling endosomes, as visualized by GM130 or Rab11 immunostaining (Figure 5, D and E, respectively), nuclei, or the early or late endosomal compartments (gauged by EEA1 and LAMP1 immunostaining, respectively; unpublished data), consistent with their more basal and perinuclear localizations within polarized epithelial cells.


Microbial sphingomyelinase induces RhoA-mediated reorganization of the apical brush border membrane and is protective against invasion.

Saslowsky DE, Thiagarajah JR, McCormick BA, Lee JC, Lencer WI - Mol. Biol. Cell (2016)

Effects of SMase on polarized T84 subcellular structure. (A) Transmission electron microscope micrographs of T84 monolayers incubated apically with or without SMase for 50 min. Ultrathin sections are perpendicular to the monolayer support. Scale bar, 1 μm. Box insets are 2× digital zooms of apical PM region. (B) Three-dimensional reconstructions from XY-stacks of monolayers stained with phalloidin and immunostained for ZO-1 and β-tubulin. Arrow denotes lack of microtubules in the internal region of the SMase-induced F-actin bundle structure. (C) Monolayers treated with or without SMase were methanol fixed and immunostained for the ER marker calnexin (a fixation method not compatible with phalloidin staining). Enclosed areas represent single-cell outlines, and arrows denote regions of decreased calnexin immunostaining in SMase-treated monolayers. (D) Monolayers treated with or without SMase were fixed and immunostained for the Golgi marker GM130 or (E) Rab11 to mark recycling endosomes (arrows denote region of altered F-actin structure for the latter). Scale bars, 1 μm (A), 10 μm (B–E).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Effects of SMase on polarized T84 subcellular structure. (A) Transmission electron microscope micrographs of T84 monolayers incubated apically with or without SMase for 50 min. Ultrathin sections are perpendicular to the monolayer support. Scale bar, 1 μm. Box insets are 2× digital zooms of apical PM region. (B) Three-dimensional reconstructions from XY-stacks of monolayers stained with phalloidin and immunostained for ZO-1 and β-tubulin. Arrow denotes lack of microtubules in the internal region of the SMase-induced F-actin bundle structure. (C) Monolayers treated with or without SMase were methanol fixed and immunostained for the ER marker calnexin (a fixation method not compatible with phalloidin staining). Enclosed areas represent single-cell outlines, and arrows denote regions of decreased calnexin immunostaining in SMase-treated monolayers. (D) Monolayers treated with or without SMase were fixed and immunostained for the Golgi marker GM130 or (E) Rab11 to mark recycling endosomes (arrows denote region of altered F-actin structure for the latter). Scale bars, 1 μm (A), 10 μm (B–E).
Mentions: The striking change in cortical F-actin structure prompted us to examine whether other subcellular features of the BBM or intracellular organelles might be affected by conversion of apical SM to ceramide. Transmission electron microscopy showed microvillar effacement in the central region of cells overlying the region of F-actin rearrangement in T84 monolayers (Figure 5A). By confocal microscopy, we found that microtubules extended deeper into the cell interior and essentially encased the SMase-induced F-actin bundles without obviously penetrating those structures (Figure 5B). Immunolocalization of the endoplasmic reticulum (ER)–resident protein calnexin showed an obvious relocalization of calnexin away from the central, subapical region of each cell in monolayers treated apically with SMase as compared with buffer controls (Figure 5C). This is the approximate area of the F-actin bundles, which, we infer, physically excluded the ER from these sites. We did not observe any changes in the intracellular distribution or morphology of the Golgi or recycling endosomes, as visualized by GM130 or Rab11 immunostaining (Figure 5, D and E, respectively), nuclei, or the early or late endosomal compartments (gauged by EEA1 and LAMP1 immunostaining, respectively; unpublished data), consistent with their more basal and perinuclear localizations within polarized epithelial cells.

Bottom Line: How the BBM dynamically responds to pathogenic and commensal bacterial signals can define intestinal homeostasis and immune function.We previously found that in model intestinal epithelium, the conversion of apical membrane sphingomyelin to ceramide by exogenous bacterial sphingomyelinase (SMase) protected against the endocytosis and toxicity of cholera toxin.Here we elucidate a mechanism of action by showing that SMase induces a dramatic, reversible, RhoA-dependent alteration of the apical cortical F-actin network.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology and Nutrition, Boston Children's Hospital, Boston, MA 02115 Harvard Digestive Diseases Center, Boston Children's Hospital, Boston, MA 02115 Harvard Medical School, Boston, MA 02115 david.saslowsky@nih.gov.

No MeSH data available.


Related in: MedlinePlus