The single-cell excretory canal of C. elegans provides a model system to interrogate molecular mechanisms of biological tube development. Previously our lab has used the excretory canal to investigate mechanisms of tube biogenesis as they relate to the human neurovascular disease cerebral cavernous malformations (CCM). CCM is characterized by formation of blood-filled cysts in the capillaries of the central nervous system and is caused by loss-of-function mutations in one of three genes: CCM1, CCM2, or CCM3. The nematode homolog of CCM3, called
ccm-3, is required for the proper development of the unicellular excretory canal. Ablation of the nematode homolog of CCM3, called
ccm-3, causes reduced CDC-42 signaling, recycling endosomes, and Golgi in the developing canal, ultimately leading to morphological defects and severe truncation. Although we have been able to characterize some of the pathways that are perturbed by loss of
ccm-3 we do not have a complete understanding of how CCM-3 promotes the proper development of the excretory canal. A full genome RNAi screen for
ccm-3-like genes identified a serine/threonine protein kinase called
mrck-1 as a possible component of
ccm-3 signaling. Loss of
mrck-1 causes the same canal truncation phenotype as loss of
ccm-3, as well as the same reduction of markers for active CDC-42, recycling endosomes, and Golgi. In cell culture MRCK-1 was shown to physically interact with components of the STRIPAK complex, where CCM-3 has also been shown to reside. MRCK-1 is required for the phosphorylation of MLC-4 and regulation of actomyosin contractions during embryogenesis. Overexpression of a constitutively active MLC-4 rescued canal truncations in
mrck-1 mutants, and partially rescued canal truncations in
ccm-3 mutants. This suggests that
mrck-1 functions downstream of
ccm-3 in the canal to activate actomyosin and promote endocytic recycling. To fully elucidate the role of
mrck-1 in the excretory canal I am using classic genetics approaches complimented by biochemistry techniques. To determine if there is a cell-autonomous role for MRCK-1 am using canal-specific expression of MRCK-1 to rescue truncations in
mrck-1 and
ccm-3 mutant worms. In parallel, I am conducting epistasis experiments comparing canal truncations in
ccm-3,
mrck-1, and
ccm-3;
mrck-1 double mutants to establish if these genes do function in the same pathway. We have identified binding partners of MRCK-1 by co-immunoprecipitation followed by mass spectrometry, which I am screening by RNAi for roles in excretory canal development. Through this research I will define the mechanism by which
mrck-1 regulates excretory canal extension in the context of
ccm-3, which should provide new insights mechanisms of biological tube development and CCM disease.