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Suppression of villin expression by antisense RNA impairs brush border assembly in polarized epithelial intestinal
M A Costa de Beauregard1, E Pringault, S Robine
1Institut Pasteur, Département de Biologie Moléculaire, Paris, France.
Abstract:
We have used an antisense RNA strategy to investigate the role of the actin-associated protein, villin, in the brush-border morphogenesis of human intestinal CaCO2 cells. Stable expression of a cDNA encoding antisense villin RNA resulted in the permanent down-regulation of the endogenous villin message and dramatically affected brush-border assembly. Ultrastructural and immunolocalization studies revealed that epithelial cell polarity was largely maintained. However, in contrast to brush-border markers such as dipeptidyl-peptidase IV, the apical localization of sucrase-isomaltase was specifically impaired. Retransfection of the villin antisense-expressing cell line with a cDNA encoding a partial sense villin RNA restored both brush-border assembly and sucrase-isomaltase apical expression. The suggestion that brush-border morphogenesis may be important for the trafficking of certain proteins is discussed.
Insights
Villin protein is crucial for proper brush border assembly in human intestinal cells. Down-regulating villin disrupts protein trafficking to the cell surface, impacting cell function.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The brush border is a critical apical specialization of differentiated epithelial cells.
- Villin is an actin-associated protein essential for brush border assembly.
Purpose of the Study:
- To investigate the role of villin in brush border morphogenesis using an antisense RNA strategy.
- To determine villin's specific function in protein trafficking within intestinal epithelial cells.
Main Methods:
- Antisense RNA strategy to down-regulate villin expression in CaCO2 cells.
- Ultrastructural analysis and immunolocalization to assess brush border structure and protein localization.
- Functional rescue experiments using sense villin RNA.
Main Results:
- Down-regulation of villin message significantly impaired brush border assembly.
- Epithelial cell polarity was maintained, but apical localization of sucrase-isomaltase was specifically affected.
- Restoration of villin expression rescued brush border assembly and sucrase-isomaltase localization.
Conclusions:
- Villin plays a critical role in brush border morphogenesis.
- Brush border assembly is essential for the proper apical trafficking of specific proteins like sucrase-isomaltase.
- Villin's function extends beyond structural support to protein trafficking regulation.