Actin depolymerizing factor but not cofilin‐1 is necessary to have a normal intestinal brush border (1109.16)

Abstract

Two highly conserved isoforms of the cofilin gene family (actin severing proteins expressed in intestinal epithelial cells) are cofilin‐1 (COF) and actin depolymerizing factor (ADF). COF and ADF take part in Arp2/3 remodeling and seem to play overlapping roles since both rescue the knockdown (KD) phenotypes of the other. Their precise role in the intestine is unknown. We hypothesized that COF and/or ADF change local actin polymerization at the base of epithelial cell microvilli by terminal web remodeling. COF and ADF were KD using lentivirus/puromycin in Caco‐2 cells, a polarized intestinal Na‐absorptive cell line. Both were normally subapical, as well as diffusely cytosolic in the cells. Structural studies were performed using confocal and electron microscopy with brush border (BB) characterization by phalloidin and wheat germ agglutinin binding. ADF KD, but not COF, significantly changed epithelial cell structure with a) shorter cell heights; b) greater cell diameter; c) shorter/sparser BB. In addition, Western blot showed increased B‐actin and villin amount in ADF KD cells, normalized to GAPDH. In contrast, COF KD had less dramatic changes in intestinal cell structure: a) slight increase in cell height; b) taller/denser microvilli; c) minimally increased B‐actin and villin. We conclude that in spite of similar sub‐apical localization, ADF but not COF is necessary to have a normal intestinal BB, including the microvilli.Grant Funding Source: Supported by APS UGSRF, NIH NIDDK R01DK26523, R01DK61765, P01DK72084, P30DK89502