As a member of the highly conserved Ena/VASP family, UNC-34 possesses an N-terminal EVH1 (Ena Vasp Homology) domain, a central proline-rich domain, and a C-terminal EVH2 domain.
unc-34 loss-of-function mutants display defects in cell migration and in axon guidance.
unc-34 mutants also show a synthetic embryonic lethality with a
was-1 (the WASp ortholog) mutant. To determine how
unc-34 is functioning, we are characterizing the domain requirements for UNC-34 activity in cell migration, axon outgrowth, and embryogenesis.Western blots using an antibody directed against the C-terminal EVH2 domain of UNC-34 reveal that most
unc-34 alleles lack detectable protein. Currently, we have identified six alleles of
unc-34 that produce UNC-34 protein, and will therefore provide information about UNC-34 domains. One of the alleles,
unc-34(
gm114), is a missense mutation in the EVH1 domain.
unc-34(
gm114) mutants display the same cell migration and axon outgrowth defects as genetic nulls, indicating that the EVH1 domain is required for these UNC-34 functions. Another allele,
unc-34(
e315), is a nonsense mutation just upstream of the EVH2 domain. While we detect no protein in
unc-34(
e315) mutants with the EVH2 antibody, we believe the mutants produce a functional truncated protein because they display minor cell migration and axon outgrowth defects at 15 degrees C. This observation indicates that the EVH2 domain does not play a major role in cell migration and axon outgrowth. Both alleles, however, show complete embryonic lethality with a
was-1 mutant, suggesting that both EVH1 and EVH2 domains are essential for this function. We are currently analyzing the other four alleles of
unc-34.We are also interested in determining which domains are required for UNC-34 localization. Antibody staining shows that UNC-34 localizes to adherens junctions and axons. In
unc-34(
gm114) mutants, UNC-34 fails to localize to adherens junctions and axons but is visible in neuronal cell bodies, suggesting that the EVH1 domain is required for localization. UNC-34 localization in other
unc-34 mutants is currently being analyzed. We also plan to create
unc-34 domain fusions to GFP that are driven by hypodermal and neuronal promoters to test which UNC-34 domain/s are sufficient for localization.