Cerebral Cavernous Malformation (CCM) is a vascular disease affecting approximately 1 in 500 individuals. A mutation in one of three genes (CCM1/2/3) can cause seizures, strokes and other neurological defects; CCM3 mutations being the most severe. In C. elegans the two CCM homologues CCM1/kri-1 and CCM3/ccm-3 (C14A4.11) are expressed in the intestine, with
ccm-3 also present in the excretory cell. Ablation of both genes causes synthetic lethality. The nematode excretory cell body branches into two canals along the length of the worm, and extends throughout worm development. Like multicellular mammalian tubules, such as blood vessels, the canal has apical and basal surfaces, and employs similar developmental signaling. As such, we used the canal as a model of tubulogenesis to elucidate CCM-3 function. Loss of
ccm-3 gene function causes severe canal truncation and frequent surface cyst formation. Orthologs of the STRIPAK complex, which bind mammalian CCM3 in cultured cells, similarly regulate nematode canal extension. Ablation of the CCM-3 interacting kinase GCK-1 and striatin CASH-1 caused truncation and cysts (like
ccm-3(lf)), supporting mammalian and nematode STRIPAK conservation. Overexpression of GCK-1 restored canal length in both
ccm-3(lf) and
gck-1(lf) mutants, suggesting
gck-1 acts downstream of
ccm-3 to promote canal growth. Subsequently, a pilot RNAi screen to find
ccm-3 interactors identified exocyst gene
exoc-8, and myotonic dystrophy kinase-related Cdc42 binding kinase ortholog
mrck-1. As with STRIPAK members, ablation of these genes caused canal truncation and cysts, suggesting they also operate in the CCM-3 pathway. Given the importance of Cdc42 and exocyst complexes in vesicle transport we asked whether the CCM-3 pathway regulates transport or recycling to promote canal growth. Using fluorescent markers for endosomes, CDC-42 and the Golgi, we found that reduction of STRIPAK and
mrck-1 decreased CDC-42 and Golgi signals. Preliminary TEM work also suggests that
ccm-3 (lf) mutants show altered vesicular aggregation and positioning. Collectively, these results suggest that a major function of the CCM-3 pathway is to regulate tube growth through vesicle transport and endosomal recycling.