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Abstract
The Caenorhabditis elegans unc-60 gene encodes two actin depolymerizing factor/cofilin proteins which are implicated in the regulation of actin filament assembly in body wall muscle. We examined the interaction of recombinant UNC-60A and B proteins with actin and found that they differentially regulate actin filament dynamics. Co-pelleting assays with F-actin showed that UNC-60A depolymerized but did not remain bound to F-actin, whereas UNC-60B bound to but did not depolymerize F-actin. In the pH range of 6.8-8.0, the apparent activities of UNC-60A and B did not change although UNC-60A showed greater actin-depolymerizing activity at higher pH. These activities were further confirmed by a light scattering assay and electron microscopy. The effects of these proteins on actin polymerization were quite different. UNC-60A inhibited polymerization in a concentration-dependent manner. On the other hand, UNC-60B strongly inhibited the nucleation process but accelerated the following elongation step. However, an excess amount of UNC-60B increased the amount of unpolymerized actin. These results indicate that UNC-60A depolymerizes actin filaments and inhibits actin polymerization, whereas UNC-60B strongly binds to F-actin without depolymerizing it and, through binding to G-actin, changes the rate of actin polymerization depending on the UNC-60B:actin ratio. These data suggest that the two UNC-60 isoforms play differential roles in regulating actin filament dynamics in vivo.
Highlights
Quantitative analysis has shown that the concentrations of these three proteins are sufficient for sequestering most of G-actin at a late stage of embryonic muscle [5], suggesting that they are responsible for regulating actin filament assembly
Unc-60 mutant muscle has large accumulations of thin filaments especially at the ends of muscle cells but only a few thin filaments scattered among thick filaments that are present in normal numbers and roughly organized into A-bands [12]
We studied the biochemical characteristics of two UNC-60 proteins, and found that they regulate actin filament dynamics in different manners
Summary
Quantitative analysis has shown that the concentrations of these three proteins are sufficient for sequestering most of G-actin at a late stage of embryonic muscle [5], suggesting that they are responsible for regulating actin filament assembly. In embryonic chicken skeletal muscle, proteins that bind to G-actin to prevent them from polymerization have been identified as profilin [2], actin depolymerizing factor (ADF)1 [3], and cofilin [4]. Biochemical studies on members of the ADF/cofilin family in other organisms suggest that the UNC-60 proteins regulate actin polymerization.
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