Polarization of the C. elegans Embryo by RhoGAP-Mediated Exclusion of PAR-6 from Cell Contacts

Abstract

Early embryos of some metazoans polarize radially to facilitate critical patterning events such as gastrulation and asymmetric cell division; however, little is known about how radial polarity is established. C. elegans early embryos polarize radially when cell contacts restrict the polarity protein PAR-6 to contact-free cell surfaces, where PAR-6 regulates gastrulation movements. Here, we identify a Rho GTPase activating protein (RhoGAP), PAC-1, which mediates C. elegans radial polarity and gastrulation by excluding PAR-6 from contacted cell surfaces. We show that PAC-1 is recruited to cell contacts, and we suggest that PAC-1 controls radial polarity by restricting active CDC-42 to contact-free surfaces, where CDC-42 binds and recruits PAR-6. Thus, PAC-1 provides a dynamic molecular link between cell contacts and PAR proteins that polarizes embryos radially.

Repurposing an endogenous degradation system for rapid and targeted depletion ofC. elegansproteins

“…For example, we have shown previously that both cytoplasmic (Anderson et al, 2008;Chan and Nance, 2013;Nance et al, 2003) and transmembrane proteins (Chihara and Nance, 2012;Wehman et al, 2011) are effectively degraded. However, a few types of proteins would be difficult to analyze, including those essential for cell viability or division; these proteins would be removed in the early embryo by endogenous ZIF-1 protein, causing cell death or division defects.…”

“…We envision that this approach will be feasible for most genes, especially given the small size of the ZF1 tag (36 amino acids), which is unlikely to interfere with the function of most tagged proteins. Moreover, the ZF1 tag itself appears robust, as we have used it to tag proteins at the N-terminus (ZF1-GFP-CDC-42) or C-terminus (SEC-5-ZF1-YFP) (see also Anderson et al, 2008;Chihara and Nance, 2012;Nance et al, 2003).…”

“…We and others showed previously that fusing the ZF1 domain to heterologous cytosolic or transmembrane proteins is sufficient to trigger their rapid degradation within early embryonic somatic cells (Achilleos et al, 2010;Anderson et al, 2008;Chan and Nance, 2013;Chihara and Nance, 2012;Nance et al, 2003;Reese et al, 2000;Totong et al, 2007;Wehman et al, 2011), mimicking loss-of-function phenotypes. For example, degradation of the RhoGAP PAC-1 produces cell polarity defects in somatic cells of the early embryo that are identical to those of pac-1 null mutants (Anderson et al, 2008), and degradation of the RhoGEF ECT-2 causes cytokinesis defects, similar to ect-2(RNAi) early embryos (Chan and Nance, 2013). ZF1-mediated degradation is thought to occur when ZIF-1, a maternally expressed SOCS-box adaptor protein that binds to ZF1 domains, recruits ZF1-containing protein to an ECS (Elongin-C, Cul2, SOCS-box family) E3 ubiquitin ligase complex for subsequent proteasome-mediated destruction (DeRenzo et al, 2003) (Fig.…”

“…Freeze-cracked slides were fixed in methanol for 20 min and 4% paraformaldehyde with salts for 5 min, as described previously (Anderson et al, 2008). Slides were incubated with rabbit anti-GFP (1:2000, Abcam), washed, incubated in Alexa-488 conjugated antirabbit secondary antibodies, incubated in DAPI, washed and mounted in DABCO (Sigma) as described previously (Anderson et al, 2008).…”

Widely Conserved Signaling Pathways in the Establishment of Cell Polarity

“…Spatially restricted GAPs could also function to exclude Cdc42-GTP from certain regions of the cell, by converting it to the GDP-bound state. Indeed, during radial polarization in the early C. elegans embryo, a GAP called PAC-1 localizes to the basolateral membranes, whereas Par-6 is restricted to the contact-free cell surfaces, most likely because Cdc42-GTP is destroyed by the PAC-1 at other surfaces (Anderson et al 2008;see Munro and Bowerman 2009). In MDCK cells, annexin2, a Ca-dependent regulator of actin dynamics, has been proposed to recruit Cdc42-GTP to the apical surface (Martin-Belmonte et al 2007), but it remains unclear how the annexin-bound Cdc42 could also bind Par-6.…”