The eukaryotic cytoskeleton is composed of three major filament systems: actin microfilaments, microtubules, and intermediate filaments (IFs). In C. elegans embryogenesis, elongation of the epidermis involves cell shape changes that require functional actin and microtubule networks (Priess and Hirsh 1986). We here show that epidermal IFs also are required for elongation. We found that a mutation,
ju71, disrupts the IF gene
ifb-1 and causes defects in epidermal morphogenesis (Woo and Chisholm, 2002 West Coast Worm Meeting abstract 91).
ifb-1 encodes two isoforms, IFB-1A and IFB-1B (previously IFB-1S and IFB-1L). The
ju71 mutation specifically affects the IFB-1A isoform. A functional IFB-1A::GFP is expressed in epidermal cells, within which it localizes to attachment structures. Attachment structures are IF-containing structures found in regions of the epidermis subject to mechanical stress, especially regions overlying muscle. Thus, IFB-1 functions within the epidermis to promote cell shape changes during elongation. RNAi of both IFB-1 isoforms resulted in embryonic arrest (our lab and Karabinos et al., 2001). Using 4-D analysis we find that such
ifb-1(RNAi) embryos arrest during elongation and also display muscle attachment (Mua) defects. This
ifb-1(RNAi) phenotype is stronger than the phenotype of
ju71 or
ju71/Df, suggesting that the IFB-1B isoform might also play a role in epidermal morphogenesis. Using isoform-specific RNAi we found that
ifb-1A and
ifb-1B both contribute to epidermal elongation but have diverging roles in larval development.
ifb-1A(RNAi) larvae are Mua, while
ifb-1B(RNAi) larvae display detachment of epidermis from cuticle. Epidermal attachment structures contain at least two other IFs, including IFA-2/MUA-6 (Hresko et al. 2000 Midwest Worm Meeting). Interestingly,
ifa-2(null) mutants display normal embryonic elongation. We find that
ifb-1(
ju71);
ifa-2(null) mutants embryos resemble
ifb-1(
ju71) embryos, suggesting that lack of IFA-2 is not compensated for by IFB-1, nor vice versa. In addition,
ifb-1(
ju71) enhances the Mua phenotype of
ifa-2(null), indicating that
ifb-1 and
ifa-2 have distinct functions in muscle attachment. In summary, our data show that the IF cytoskeleton plays a key role in morphogenesis of the epidermis and that different epidermal IFs have distinct functions. To explore how IFB-1 functions in development, we have screened for IFB-1-binding proteins using a yeast two-hybrid approach. We also plan to screen for mutants that affect IFB-1A::GFP localization. Results of these screens will be reported.