, 2010). Thus, we hypothesized that DAF-21/Hsp90 and

EBAX-1 may be involved in suppressing the level of endogenous aberrant misfolded proteins during axon guidance. Numerous human diseases have been associated with amino acid mutations, resulting in metastable proteins with temperature-sensitive (ts) misfolding defects (Gelsthorpe et al., 2008, Kjaer and Ibáñez, 2003, Pedersen et al., 2003, Singh et al., 1997 and Vollrath and Liu, 2006). One well-studied selleck inhibitor example is the ΔF508 mutant of cystic fibrosis transmembrane conductance regulator (CFTR) identified in cystic fibrosis patients (Lukacs and Verkman, 2012). The advance in therapeutic treatment of cystic fibrosis has heavily relied on PQC studies of the CFTR ΔF508 mutant. Being susceptible targets of the PQC system, such metastable mutant proteins can serve as sensitized probes to examine the function of PQC regulators. To identify in vivo targets of EBAX-1 and DAF-21, we searched for temperature-sensitive mutants with protein misfolding defects in the slt-1/sax-3 pathway. ISRIB order We found that a previously reported ts mutation of sax-3 (ky200) caused striking temperature-dependent misfolding and mislocalization of the SAX-3 receptor in touch neurons. sax-3(ky200) contains a missense mutation at a conserved proline residue (P37S) in the first immunoglobin-like domain (Ig1) ( Figure 5A) ( Zallen et al., 1998). In the vertebrate Robo1, this amino acid is close to

the contact regions between Slit2 and Robo1 but does not directly mediate their interaction ( Morlot et al., 2007). sax-3(ky200) mutant animals Sodium butyrate showed marginal penetrance of AVM guidance defects at the permissive temperature (20°C), suggesting that most mutant SAX-3 is functional under this condition. At the restrictive temperature (22.5°C), the level of defects significantly increased in sax-3(ky200) mutants ( Figure 5B). As a control, the guidance defects in sax-3(ky123) null mutants did not show temperature dependence. To test whether the temperature sensitivity of sax-3(ky200) mutants is caused by protein misfolding, we examined the expression patterns

of GFP-tagged SAX-3(WT) and SAX-3(P37S) in touch neurons. SAX-3(WT)::GFP and SAX-3(P37S)::GFP were expressed in the wild-type and sax-3(ky200) mutant backgrounds, respectively, to ensure the homogeneity of endogenous and exogenous proteins. In touch neurons at late L1 to early L2 stages, SAX-3(WT)::GFP was predominantly localized on the cell surface at both 20°C and 22.5°C ( Figure 5C). In contrast, SAX-3(P37S)::GFP showed a mixture of cell-surface and cytosolic localization with a mild degree of cytosolic aggregation at 20°C ( Figure 5C). The cytosolic mislocalization and aggregation of SAX-3(P37S)::GFP were exacerbated at 22.5°C and accompanied with a reduction of surface signals that correlated with the aggravated AVM guidance defects in sax-3(ky200) mutants ( Figures 5B and 5C).