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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.


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Ezrin and EBP50 coalesce with SMase-induced cortical F-actin structures. T84 monolayers incubated apically with or without SMase were fixed and immunostained for ezrin (A), EBP50 (B), or myosin 1a (C), and F-actin was stained with labeled phalloidin in all cases. Arrows denote colocalization of ezrin and EBP50 with SMase-induced F-actin bundle structures. Scale bars, 5 μm (A, B), 10 μm (C).
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Figure 7: Ezrin and EBP50 coalesce with SMase-induced cortical F-actin structures. T84 monolayers incubated apically with or without SMase were fixed and immunostained for ezrin (A), EBP50 (B), or myosin 1a (C), and F-actin was stained with labeled phalloidin in all cases. Arrows denote colocalization of ezrin and EBP50 with SMase-induced F-actin bundle structures. Scale bars, 5 μm (A, B), 10 μm (C).

Mentions: The ezrin/radixin/moesin (ERM) family of actin-regulating proteins plays an important role in cortical F-actin and brush border membrane dynamics in polarized epithelia (Bretscher, 1999; Saotome et al., 2004). Because the ERM proteins are coregulated by membrane PIP2 (Bretscher et al., 2002), we tested whether they also might be involved in the SMase-induced phenotype by localizing them in relation to the SMase-induced actin bundles. Immunolocalization studies in untreated T84 monolayers showed that ezrin and ezrin-binding protein 50 (EBP50) distributed with cortical F-actin across the apical PM, but they were excluded from TJ regions (Figure 7, A and B, top). In SMase-treated monolayers, however, the location of both ERM proteins was altered. Each colocalized with the F-actin bundles induced by SMase (Figure 7, A and B, bottom). In contrast to the ERM proteins, immunofluorescence of Myosin 1a showed no change in the lateral distribution of Myo1a across the apical PM, indicating that only certain cortical F-actin and microvillar effectors are affected by SMase treatment (Figure 7C).


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)

Ezrin and EBP50 coalesce with SMase-induced cortical F-actin structures. T84 monolayers incubated apically with or without SMase were fixed and immunostained for ezrin (A), EBP50 (B), or myosin 1a (C), and F-actin was stained with labeled phalloidin in all cases. Arrows denote colocalization of ezrin and EBP50 with SMase-induced F-actin bundle structures. Scale bars, 5 μm (A, B), 10 μm (C).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 7: Ezrin and EBP50 coalesce with SMase-induced cortical F-actin structures. T84 monolayers incubated apically with or without SMase were fixed and immunostained for ezrin (A), EBP50 (B), or myosin 1a (C), and F-actin was stained with labeled phalloidin in all cases. Arrows denote colocalization of ezrin and EBP50 with SMase-induced F-actin bundle structures. Scale bars, 5 μm (A, B), 10 μm (C).
Mentions: The ezrin/radixin/moesin (ERM) family of actin-regulating proteins plays an important role in cortical F-actin and brush border membrane dynamics in polarized epithelia (Bretscher, 1999; Saotome et al., 2004). Because the ERM proteins are coregulated by membrane PIP2 (Bretscher et al., 2002), we tested whether they also might be involved in the SMase-induced phenotype by localizing them in relation to the SMase-induced actin bundles. Immunolocalization studies in untreated T84 monolayers showed that ezrin and ezrin-binding protein 50 (EBP50) distributed with cortical F-actin across the apical PM, but they were excluded from TJ regions (Figure 7, A and B, top). In SMase-treated monolayers, however, the location of both ERM proteins was altered. Each colocalized with the F-actin bundles induced by SMase (Figure 7, A and B, bottom). In contrast to the ERM proteins, immunofluorescence of Myosin 1a showed no change in the lateral distribution of Myo1a across the apical PM, indicating that only certain cortical F-actin and microvillar effectors are affected by SMase treatment (Figure 7C).

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