Myosin II regulation duringC. elegansembryonic elongation:LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles

Abstract

Myosin II plays a central role in epithelial morphogenesis; however, its role has mainly been examined in processes involving a single cell type. Here we analyze the structure, spatial requirement and regulation of myosin II during C. elegans embryonic elongation, a process that involves distinct epidermal cells and muscles. We developed novel GFP probes to visualize the dynamics of actomyosin remodeling, and found that the assembly of myosin II filaments, but not actin microfilaments, depends on the myosin regulatory light chain (MLC-4) and essential light chain (MLC-5, which we identified herein). To determine how myosin II regulates embryonic elongation, we rescued mlc-4 mutants with various constructs and found that MLC-4 is essential in a subset of epidermal cells. We show that phosphorylation of two evolutionary conserved MLC-4 serine and threonine residues is important for myosin II activity and organization. Finally, in an RNAi screen for potential myosin regulatory light chain kinases, we found that the ROCK, PAK and MRCK homologs act redundantly. The combined loss of ROCK and PAK, or ROCK and MRCK, completely prevented embryonic elongation, but a constitutively active form of MLC-4 could only rescue a lack of MRCK. This result, together with systematic genetic epistasis tests with a myosin phosphatase mutation, suggests that ROCK and MRCK regulate MLC-4 and the myosin phosphatase. Moreover, we suggest that ROCK and PAK regulate at least one other target essential for elongation, in addition to MLC-4.

The making of hemidesmosome structures in vivo

“…Newly formed hemidesmosomes on both sides could later reorganize and align with each other during development to form mature fibrous organelles. Indeed, as discussed in the next section, recent studies have found a mechanism that specifically regulates basal hemidesmosome formation in C. elegans (Zahreddine et al, 2009). However, the detailed process of hemidesmosome assembly into fibrous organelles still awaits further investigation.…”

“…Genetic analysis has revealed that embryonic elongation depends on proper establishment of epithelial polarity, adherens junction assembly, hemide smosome assembly, and cytoskeleton remodeling (Chisholm and Hardin, 2005;Labouesse, 2006). The initial phase of elongation depends mainly on the lateral epidermal cells (Diogon et al, 2007;Gally et al, 2009). The second part of elongation also requires muscle activity, although it is not known what kind of input muscles provide to the epidermis (Waterston, 1989;Williams and Waterston, 1994).…”

“…Recent data have suggested that the folding of UNC-52 and its subsequent secretion to the ECM depends on the function of the molecular chaperone CRT-1/calreticulin. In crt-1 mutants, UNC-52 protein level is reduced by 50% (Zahreddine et al, 2009). This minor reduction is detrimental to fibrous organelle formation in a background with compromised VAB-10A function, resulting in detachment of muscles from the cuticle, and disorganized fibrous organelle patterns in areas with detached muscles (Fig.…”

“…This minor reduction is detrimental to fibrous organelle formation in a background with compromised VAB-10A function, resulting in detachment of muscles from the cuticle, and disorganized fibrous organelle patterns in areas with detached muscles (Fig. 3A,B) (Zahreddine et al, 2009). This finding, together with UNC-52 distribution and unc-52 mutant phenotype, suggests that UNC-52 function is essential for fibrous organelle formation, and that UNC-52 directly interacts with the membrane receptor LET-805/myotactin (Fig.…”

“…Recently, a genetic screen for genes acting together with vab-10A to promote fibrous organelle formation identified two protein kinases, the p21-activated kinase PAK-1, and the Leucine zipper kinase PIG-1. Their loss of function combined with a weak vab-10A mutation affects fibrous organelle stability (Zahreddine et al, 2009). It would be interesting to explore the functions of these kinases in fibrous organelle assembly, and identify their phosphorylation targets.…”