Myosin IIB deficiency in embryonic fibroblasts affects regulators and core members of the par polarity complex

Abstract

Wild-type (WT) and myosin heavy chain IIB null [MHCIIB (-/-)] embryonic fibroblasts were used as an experimental model to assess the role of the isoform B of myosin II (MII) in the regulation of the cell shape and intrinsic polarity. Genetic ablation of MHCIIB causes a persistent albeit, unstable protrusive activity in embryonic fibroblasts (Lo et al. in Nonmuscle myosin IIB is involved in the guidance of fibroblast migration. Mol Biol Cell 15:982-989, 2004). Here, we show that MHCIIB-deficient fibroblasts are characterized by a sustained guanine nucleotide exchange factor (GEF)-dependent activation of the small GTPase Rac-1 that is responsible for the continual lamellipodium formation. Moreover, we observed a sustained PKC-ζ activation and an increased association of cortactin with the plasma membrane in the MHCIIB (-/-) cells that were also dependent on GEF-mediated Rac-1 activation. Rac-1 activation and its downstream effects were induced in WT fibroblasts by inhibiting MII ATPase and crosslinking activities, suggesting that an altered actin-MII interaction favours Rac-1 activation, regardless of the MII isoform implicated. In addition, we found MIIB isoform-specific effects that were independent of Rac-1 activation. MHCIIA interacts with cortactin whereas MHCIIB does not. By contrast, MHCIIB interacts with Lgl1, a member of the Scribble/Dlg/Lgl polarity complex, whereas MHCIIA does not. MHCIIB (-/-) fibroblasts exhibited deregulated endogenous levels of the Par polarity complex members, Par3 and Par6. Together, the data show that MHCIIB deficiency causes imbalances in signalling pathways that are responsible for cell polarity determination. The results suggest that these pathways are targets of MIIB in the regulation of the cell's shape and polarity.

The heavy chain has its day

“…87 Lgl1 has been reported to associate with both myosin-IIA and myosin-IIB. 88,89 Consistent with Lgl binding to the coiled coil region between ACD1 and ACD2, Lg1 blocks the assembly of myosin-IIA filaments in vitro and regulates myosin-IIA localization in vivo. 89,90 S100A4.…”

Distinctive actions of connexin 46 and connexin 50 in anterior pituitary folliculostellate cells

“…Isolation of the membrane- and cytosol-enriched subcellular fractions was carried out as previously described [ 10 ; 57 ]. Briefly, cells were washed with PBS containing protein phosphatase and protease inhibitors, collected by scraping, and homogenized to obtain a total cell lysate.…”

Mammalian nonmuscle myosin II comes in three flavors

“…This study also showed that ectopically expressed Lgl1 and NMIIA could be coimmunoprecipitated from cells. However, another study revealed an interaction of Lgl1 only with NMIIB, but not NMIIA, using coimmunoprecipitation of endogenous proteins [44]. This discrepancy remains to be resolved, especially because all charged residues within the proposed Lgl1-binding site of NMIIA are conserved in NMIIB.…”