The single-cell excretory canal of C. elegans is a powerful model for understanding molecular mechanisms of biological tube development. Coordination of vesicle trafficking, apical-basal polarity, and cytoskeletal support are all required for the development and maintenance of canal integrity. Previously, our lab showed that inactivation of the cerebral cavernous malformation 3 (CCM3) gene
ccm-3 caused canal truncations through effects on CDC-42 signaling and recycling endosomes [1]. To gain more insight into the mechanistic basis by which CCM-3 regulates canal development we conducted a genome-wide RNAi screen for genes that caused similar canal truncations as
ccm-3 and identified the myotonic dystrophy-related Cdc42-binding kinase homolog 1 (
mrck-1). Inactivation of
mrck-1 causes the same canal truncation phenotype as loss of
ccm-3, as well as reduction of markers for active CDC-42 and recycling endosomes [1]. MRCK-1 is also required for the phosphorylation of MLC-4 during embryogenesis, which regulates actomyosin to promote body elongation [2,3]. Overexpression of a phosphomimetic form of MLC-4 rescues canal truncations in
mrck-1 mutants, but only partially rescues truncations in
ccm-3 mutants. This suggests that
mrck-1 either functions downstream in collaboration with other genes, or independently of
ccm-3 to regulate actomyosin function in the canal. Using CRISPR genome editing I generated a series of mutations in the domains of MRCK-1 to better elucidate its function in canal development. Mutations in the kinase domain of MRCK-1 completely phenocopy a null mutation in
mrck-1, whereas deletion of the Cdc42 and Rac1-interactive (CRIB) domain caused very modest canal truncations, indicating that its kinase activity likely drives canal extension. Given the known role of MRCK-1 in promoting the phosphorylation of MLC-4, and the ability of a phosphomimetic form of MLC-4 to rescue canal truncations in
mrck-1 mutants, I am currently investigating the role of MLC-4 and non-muscle myosin in the excretory canal. Through this research I aim to establish how
mrck-1 regulates excretory canal extension, which may provide insights into general mechanisms of biological tube development. 1. Lant et al. (2015) Nature Comm 6:6449 2. Gally et al. (2009) Development 136:3109-3119 3. Marston et al. (2016) Current Biol 26:2079-2089