Worm Breeder's Gazette 15(1): 70 (October 1, 1997)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Departments of Pathology and Cell Biology, Emory University, Atlanta, GA 30322|
|2||Institute of Molecular Biology and Biochemistry, Simon Fraser Univeristy, Burnaby, British Columbia, CANADA V5A1S6|
Mutations in the C. elegans unc-60 gene result in slow-moving or paralyzed nematodes having large aggregates of thin filaments in body wall muscle cells. The gene encodes two actin depolymerizing factor (ADF)/cofilin proteins which are generated by alternative splicing, suggesting that ADF/cofilin is required for actin assembly in muscle (McKim et al.,1994, Mol Gen Genet 242: 346-357). We bacterially expressed and purified recombinant UNC-60A and B without any extra sequences, and found that they had different activities in regulating actin dynamics. A co-pelleting assay with rabbit muscle F-actin showed that UNC-60A depolymerized but did not bind to F-actin, while UNC-60B bound to but did not depolymerize F-actin. In the pH range of 6.8 to 8.0, the apparent activities of UNC-60A and B did not change, although UNC-60A showed greater actin-depolymerizing activity at higher pH. The effects of these proteins on actin polymerization were quite different. UNC-60A inhibited polymerization in a dose-dependent manner. On the other hand, UNC-60B strongly inhibited the nucleation process but accelerated the following elongation step. These biochemical studies show that UNC-60A primarily binds to G-actin and depolymerizes F-actin, while UNC-60B binds to both G- and F-actin and regulates the rate of actin assembly, suggesting that they are functionally distinct proteins in vivo. Northern blot analyses revealed that the expression of unc-60A and B was differentially regulated during development. The expression of unc-60A was highest in embryos, dropped at L1, gradually increased through young adult, and remarkably increased in gravid adults, implying that unc-60A has a role in early development. In contrast, the level of unc-60B mRNA was highest in embryos and gradually decreased. Microinjection of synthetic RNA into the gonad of C. elegans can often result in a mutant phenotype for the corresponding gene in the next generation. We showed that injection of RNA from A, but not from B, resulted primarily in embryos which arrested without morphogenesis, suggesting that A is essential for early development. We have determined the sequence alterations in seven of ten available homozygous viable unc-60 mutants, and found that all seven mutations were located within the unc-60B coding region. This suggests that unc-60B has a muscle-specific function. We found that one missense mutation occurred near the N-terminus. Four additional missense mutations occurred near the actin-binding site which has been identified for porcine cofilin (Yonezawa et al., 1991, JBC, 266: 10485-10489), and thus, these alterations are likely to interfere with the actin-binding activity of UNC-60B. We plan to examine the biochemical activities of these mutant proteins in order to understand how the mutations result in the phenotypes. We expect the remaining unc-60 mutations will also reside in unc-60B because fine structure genetic mapping has shown these mutations lie between e677 and m35, which we have already sequenced. We have recently generated antibodies which specifically recognize UNC-60A or B proteins. Using these antibodies, we will determine the tissue distribution during development, intracellular locations, and levels of the UNC-60 proteins in each of the unc-60 mutants.