In many secretory cells actin and myosin are specifically recruited to the surface of secretory granules following their fusion with the plasma membrane. Actomyosin-dependent compression of fused granules is essential to promote active extrusion of cargo. However, little is known about molecular mechanisms regulating actin coat formation and contraction. Here, we provide a detailed kinetic analysis of the molecules regulating actin coat contraction on fused lamellar bodies in primary alveolar type II cells. We demonstrate that ROCK1 and myosin light chain kinase 1 (MLCK1, also known as MYLK) translocate to fused lamellar bodies and activate myosin II on actin coats. However, myosin II activity is not sufficient for efficient actin coat contraction. In addition, cofilin-1 and α-actinin translocate to actin coats. ROCK1-dependent regulated actin depolymerisation by cofilin-1 in cooperation with actin crosslinking by α-actinin is essential for complete coat contraction. In summary, our data suggest a complementary role for regulated actin depolymerisation and crosslinking, and myosin II activity, to contract actin coats and drive secretion.

Trafficking and regulation of the epithelial brush border membrane (BBM) Na+ /H + exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal-dependent and possibly segment-specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger-mediated and receptor-mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid-activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin-induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca 2+ ionophore-or carbachol-mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal-and segment-specific fashion, and is therefore an important regulator of intestinal fluid transport.

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Key points• Cystic fibrosis (CF) is a lethal disease characterized by low rates of epithelial Cl − and HCO 3 − secretion and obstruction of the airways and gastrointestinal and reproductive organs by sticky mucus. HCO 3 − secretion has recently been demonstrated to be necessary for mucus hydration.• The most frequent CF mutation is F508del. This mutant protein is usually degraded in the proteasome. New therapeutic strategies have been developed which deliver F508del to the plasma membrane.• Utilizing transgenic F508del mutant and cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice, apical membrane expression of F508del protein was found to be associated with enhanced stimulation of intestinal HCO 3 − secretion.• The predominant molecular mechanism for enhanced F508del HCO 3 − stimulation appeared to be the activation of a Cl − recycling pathway, with Cl − exit via membrane-resident F508del protein and Cl − entry in exchange for HCO 3 − by apical Cl − /HCO 3 − exchange. In contrast, the predominant molecular mechanism for cAMP-activated HCO 3 − secretion in WT intestine appears to be HCO 3 − exit via CFTR itself.Abstract This study investigated whether expression of the common cystic fibrosis transmembrane conductance regulator (CFTR) mutant F508del in the apical membrane of enterocytes confers increased bicarbonate secretory capacity on the intestinal epithelium of F508del mutant mice compared to that of CFTR knockout (KO) mice. CFTR KO mice, F508del mutant mice (F508del) and wild-type (WT) littermates were bred on the FVB/N background. F508del isolated brush border membrane (BBM) contained approximately 5-10% fully glycosylated band C protein compared to WT BBM. Similarly, the forskolin (FSK)-induced, CFTR-dependent short-circuit current ( I sc ) of F508del mucosa was approximately 5-10% of WT, whereas the HCO 3 − secretory response ( J HCO F508del, but significantly more slowly in CFTR KO intestine. In conclusion, the data demonstrate that low levels of F508del membrane expression in the intestine of F508del mice significantly increased FSK-induced HCO 3 − secretion mediated by Cl − /HCO 3 − exchange. However, in WT mucosa FSK elicited strong SPAK/OSR1 phosphorylation and Cl − -independent HCO 3 − efflux. This suggests that therapeutic strategies which deliver F508del to the apical membrane have the potential to significantly enhance epithelial HCO 3 − secretion.