The N-terminal domain of the type 1 Ins(1,4,5)P3 receptor stably expressed in MDCK cells interacts with myosin IIA and alters epithelial cell morphology

Abstract

Summary Cytosolic Ca2+ controls a wide range of cellular events. The versatility of this second messenger depends on its ability to form diverse spatial and temporal patterns, including waves and oscillations. Ca 2+ -signaling patterns are thought to be determined in part by the subcellular distribution of inositol (1,4,5)-trisphosphate receptors [Ins(1,4,5)P 3 Rs] but little is currently known about how the localization of the Ins(1,4,5)P 3 R itself is regulated. Here, we report that the recruitment of GFP-tagged Ins(1,4,5)P 3 Rs in the vicinity of tight junctions in Madin-Darby canine kidney (MDCK) cells requires the N-terminal domain. Stable expression of this domain in polarized MDCK cells induced a flattened morphology, affected cytokinesis, accelerated cell migration in response to monolayer wounding and interfered with the cortical targeting of myosin IIA. In addition, downregulation of myosin IIA in polarized MDCK cells was found to mimic the effects of stable expression of the N-terminal part of Ins(1,4,5)P 3 R on cell shape and to alter localization of endogenous Ins(1,4,5)P 3 Rs. Taken together, these results support a model in which the recruitment of Ins(1,4,5)P 3 Rs at the apex of the lateral membrane in polarized MDCK cells, involves myosin IIA and might be important for the regulation of cortical actin dynamics.

Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1

“…Myosin II-mediated contractility is the main force-generating cellular component required for DC fast migration in confined environments (Faure-Andre et al , 2008; Lammermann et al , 2008). Furthermore, myosin IIA and IP 3 R1 were shown to physically interact in MDCK cells (Hours & Mery, 2010). We therefore investigated whether myosin II activity was altered in IP 3 R knockdown DCs by analyzing the phosphorylation levels of its regulatory light chain MLC.…”

Vimentin and the K-Ras-induced actin-binding protein control inositol-(1,4,5)-trisphosphate receptor redistribution during MDCK cell differentiation.

“…We have previously shown that the recruitment of InsP 3 R1-GFP near the tight junctions (TJ) in polarized MDCK cells depends on its N-terminal region (Hours and Mery, 2010). In order to more precisely define the domain which directs InsP 3 R to the perijunctional area, a GFP-fused InsP 3 R1 deletion mutant lacking most of the regulatory domain (amino acids 650 to 1949) and referred to as DRD-GFP was generated (supplementary material Fig.…”

“…Transfection of plasmid DNA into MDCK cells was performed by the calcium phosphate coprecipitation method as previously described (Hours and Mery, 2010). Approximately 16 hours after transfection, cells were washed twice with Ca 2+ /Mg 2+ -free phosphate-buffered saline (PBS) and placed in fresh growth medium containing 600 mg/ml of active G418.…”

“…The Triton X-100-soluble and insoluble fractions were prepared as previously described (Hours and Mery, 2010). The Triton X-100-insoluble material was solubilized in ice-cold buffer A (PBS supplemented with 0.5% SDS, 2 mM EDTA and protease inhibitors) or buffer B (containing 50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitors) and then gently sonicated on ice for 10 s. Protein concentrations were determined with the Bio Rad DC kit using BSA as standard.…”

“…In hepatocytes, InsP 3 R2 is exclusively found underneath the luminal membrane whereas InsP 3 R1 is distributed relatively uniformly throughout the cell (Hirata et al, 2002;Shibao et al, 2003;Cruz et al, 2010). In polarized Madin-Darby canine kidney (MDCK) cells, InsP 3 R3 accumulates in the vicinity of TJ (Colosetti et al, 2003;Hours and Mery, 2010). It has been hypothesized that the differential expression and subcellular localization of InsP 3 R subtypes largely contribute to the spatial patterning of Ca 2+ signals in epithelial cells (Vermassen et al, 2004).…”