RhoGAP RGA-8 supports morphogenesis in C. elegans by polarizing epithelia

Abstract

CDC-42 regulation of non-muscle myosin/NMY-2 is required for polarity maintenance in the one-cell embryo of Caenorhabditis elegans. CDC-42 and NMY-2 regulate polarity throughout embryogenesis, but their contribution to later events of morphogenesis are less understood. We have shown that epidermal enclosure requires the GTPase CED-10/Rac1 and WAVE/Scar complex, its effector, to promote protrusions that drive enclosure through the branch actin regulator Arp2/3. Our analysis here of RGA-8, a homolog of SH3BP1/Rich1/ARHGAP17/Nadrin, with BAR and RhoGAP motifs, suggests it regulates CDC-42, so that actin and myosin/NMY-2 promote ventral enclosure during embryonic morphogenesis. Genetic and molecular data suggest RGA-8 regulates CDC-42, and phenocopies the CDC-42 pathway regulators WASP-1/WSP-1 and the F-BAR proteins TOCA-1 and TOCA-2. Live imaging shows RGA-8 and WSP-1 enrich myosin and regulate F-actin in migrating epidermal cells during ventral enclosure. Loss of RGA-8 alters membrane recruitment of active CDC-42. We propose TOCA proteins and RGA-8 use BAR domains to localize and regenerate CDC-42 activity, thus regulating F-actin levels, through the branched actin regulator WSP-1, and myosin enrichment. RhoGAP RGA-8 thus polarizes epithelia, to promote cell migrations and cell shape changes of embryonic morphogenesis.

CED-5/CED-12 (DOCK/ELMO) can promote and inhibit F-actin formation via distinct motifs that target different GTPases

“…1F and Table 1). These results reinforced that high levels of F-actin can interfere with morphogenesis, as we previously showed for loss of vab-1 (Bernadkaya et al, 2012), hum-7 (Wallace et al, 2018) and rga-8 (Raduwan et al, 2020).…”

“…Since the effect includes rescuing embryonic lethality, this result suggests that excess levels of CDC-42 and RHO-1 are detrimental for embryonic epidermal morphogenesis. Our studies of other proteins that when missing result in dead embryos due to excess epidermal F-actin levels are in agreement with this finding that excess F-actin must be controlled for healthy cell migrations (Bernadskaya et al, 2012, Wallace et al 2018, Raduwan et al, 2020).…”

“…Staged embryos that were oriented with ventral tissues facing up (towards the cover slip) were imaged from 280 to 320 minutes after first cleavage. These 4D movies of live embryos showed that wild type embryos are enriched for F-actin at the leading edge of the migrating row of ventral cells, as previously shown (Bernadskaya et al, 2012; Raduwan et al, 2020). Timing the migration, from the first appearance of leading cell edge protrusions, to when the leading cells met at the ventral midline, took approximately 20 minutes at 23 C. Loss of the GTPase CED-10/Rac1, showed significantly reduced levels of F-actin in leading cells, and arrested migrations, as would be expected for the removal of a major activator of branched actin (Fig.…”

“…During epidermal cell migrations, CED-5/CED-12 have an important role limiting the activity of CDC-42, and this requires the proposed CED-12 GAP function. We previously published the surprising observation that in wsp-1(gm324) mutants, which are approximately 30% lethal, epidermal F-actin is too high, and the cells migrate too quickly (Raduwan et al, 2020). This resembles what we show here for loss of the CED-12 GAP function.…”

Actin and CDC-42 contribute to nuclear migration through constricted spaces in C. elegans

“…CDC-42 then activates the Arp2/3 complex to nucleate branched actin. TOCA-1 and WAVE/WASP likely also work at the level of CDC-42 and Arp2/3 in this pathway ( Chang et al, 2013 ; Giuliani et al, 2009 ; Raduwan et al, 2020 ). Once Arp2/3 nucleates actin, other proteins organize actin into networks.…”