Most cells are exposed to mechanical stress either during development, movement or in case of harsh encounter with the environment. Failure to resist to mechanical shearing forces can lead to severe integrity problems. As part of our ongoing analysis of genes involved in epithelial morphogenesis, we have identified new mutations in the gene
vab-10 that strongly affect embryonic elongation.
vab-10 corresponds to a complex locus encoding two sets of proteins belonging to the spectraplakin/plakin family. One class of VAB-10 isoforms, VAB-10A, is strongly related to human BPAG1-e and plectin. Immuno-EM experiments and genetic analysis demonstrate that VAB-10 is a core component of fibrous organelles, and is required to maintain attachment of epidermal cells to the ECM when muscle contract. The second main class of VAB-10 isoforms, VAB-10B, is strongly related to mouse BPAG1-a and MACF. VAB-10B forms a network of circumferential bands in the epidermis distinct from fibrous organelles, which is required to protect epidermal cells against mechanical rupture when their shape changes during development. Plakins are huge proteins that can usually bind at least two different cytoskeletal networks (i.e. microfilaments, intermediate filaments, microtubules). VAB-10A and VAB-10B isoforms have a predicted actin-binding domain in their common N-terminus. The six C-terminal plectin repeats of VAB-10A can potentially bind intermediate filaments. Finally, the VAB-10B C-terminal region contains a putative GAR domain that could mediate interaction with microtubules. We tested if the conserved binding domains are functional in C. elegans by creating fusions with fluorescent reporters, under the control of epidermal promoter derived from the
lin-26 promoter study. The putative actin binding domain (ABD) fused to GFP displays a circumferentially oriented pattern fitting with the cuticular annulae and similar to that observed for actin. The putative VAB-10A IF-binding domain (IFBD) fused to GFP generates a pattern of parallel bands in areas of muscle contact, consistent with a fibrous organelle distribution. Finally, a GFP fused to the putative VAB-10B GAR domain is sufficient to reveal an intracellular network similar to that revealed by a GFP fused to alpha-tubulin. We are currently testing whether the VAB-10 ABD, IFBD and GAR do indeed colocalize with actin, intermediate filaments and microtubules. We next plan to use these constructs in different mutant backgrounds.