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Two Caenorhabditis elegans Actin Depolymerizing Factor/Cofilin Proteins, Encoded by the unc-60 Gene, Differentially Regulate Actin Filament Dynamics

1998; Elsevier BV; Volume: 273; Issue: 6 Linguagem: Inglês

10.1074/jbc.273.6.3778

1083-351X

Shoichiro Ono, Guy M. Benian,

Cellular Mechanics and Interactions

The Caenorhabditis elegans unc-60gene 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. The Caenorhabditis elegans unc-60gene 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. Myofibrils are highly differentiated forms of actin cytoskeleton that are specialized for muscle contraction, but the mechanisms by which these complex and precise structures are assembled and maintained are largely unknown. Actin, a major component of thin filaments, has an inherent tendency to polymerize into filaments in vitro. However, the assembly of actin in developing muscle is regulated, and consequently, about 40% of actin is present in a monomeric form (1Shimizu N. Obinata T. J. Biochem. 1986; 99: 751-759Crossref PubMed Scopus (38) Google Scholar). In embryonic chicken skeletal muscle, proteins that bind to G-actin to prevent them from polymerization have been identified as profilin (2Ohshima S. Abe H. Obinata T. J. Biochem. 1989; 105: 855-857Crossref PubMed Scopus (44) Google Scholar), actin depolymerizing factor (ADF) 1The abbreviations used are: ADF, actin depolymerizing factor; EDC, 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide; Pi, inorganic phosphate; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: ADF, actin depolymerizing factor; EDC, 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide; Pi, inorganic phosphate; PAGE, polyacrylamide gel electrophoresis. (3Abe H. Obinata T. J. Biochem. 1989; 106: 172-180Crossref PubMed Scopus (42) Google Scholar), and cofilin (4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar). 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 (5Nagaoka R. Minami N. Hayakawa K. Abe H. Obinata T. J. Muscle Res. Cell Motil. 1996; 17: 463-473Crossref PubMed Scopus (31) Google Scholar), suggesting that they are responsible for regulating actin filament assembly.ADF and cofilin are highly conserved proteins, are members of an ADF/cofilin family having 25–71% homology, and are found in diverse organisms. ADF/cofilin binds to both G- and F-actin at a stoichiometry of 1:1 and regulates the rate of actin polymerization (reviewed in Ref.6Moon A. Drubin D.G. Mol. Biol. Cell. 1995; 6: 1423-1431Crossref PubMed Scopus (226) Google Scholar). Recently, ADF/cofilin has been shown to affect the on/off rates at both ends of F-actin, which results in the enhancement of treadmilling (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar). This function is necessary for the actin-based motility ofListeria monocytogenes (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar, 8Rosenblatt J. Agnew B.J. Abe H. Bamburg J.R. Mitchison T.J. J. Cell Biol. 1997; 136: 1323-1332Crossref PubMed Scopus (190) Google Scholar) and for actin turnover in cortical actin patches in yeast (9Lappalainen P. Drubin D.G. Nature. 1997; 388: 78-82Crossref PubMed Scopus (363) Google Scholar).A muscle-specific function for ADF/cofilin has been suggested by two examples. These are a mammalian muscle-specific cofilin (M-cofilin) (10Ono S. Minami N. Abe H. Obinata T. J. Biol. Chem. 1994; 269: 15280-15286Abstract Full Text PDF PubMed Google Scholar) and two ADF/cofilin homologues encoded by the Caenorhabditis elegans unc-60 gene (11McKim K.S. Matheson C. Marra M.A. Wakarchuk M.F. Baillie D.L. Mol. Gen. Genet. 1994; 242: 346-357Crossref PubMed Scopus (100) Google Scholar). Mammalian M-cofilin is predominantly expressed in skeletal and cardiac muscles (10Ono S. Minami N. Abe H. Obinata T. J. Biol. Chem. 1994; 269: 15280-15286Abstract Full Text PDF PubMed Google Scholar), but its exact function is unknown. Mutations in the unc-60 gene result in slow moving or paralyzed nematodes (11McKim K.S. Matheson C. Marra M.A. Wakarchuk M.F. Baillie D.L. Mol. Gen. Genet. 1994; 242: 346-357Crossref PubMed Scopus (100) Google Scholar, 12Waterston R.H. Thomson J.N. Brenner S. Dev. Biol. 1980; 77: 271-302Crossref PubMed Scopus (183) Google Scholar, 13McKim K.S. Heschl M.F. Rosenbluth R.E. Baillie D.L. Genetics. 1988; 118: 49-59PubMed Google Scholar). By electron microscopy, 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 (12Waterston R.H. Thomson J.N. Brenner S. Dev. Biol. 1980; 77: 271-302Crossref PubMed Scopus (183) Google Scholar). Thus, unc-60 is required for proper positioning and the correct number of thin filaments in nematode striated muscle. Theunc-60 gene has been shown to encode two transcripts, sharing only a single exon encoding the initiator methionine, and two homologous proteins of the ADF/cofilin family (11McKim K.S. Matheson C. Marra M.A. Wakarchuk M.F. Baillie D.L. Mol. Gen. Genet. 1994; 242: 346-357Crossref PubMed Scopus (100) Google Scholar). These proteins, called UNC-60A and UNC-60B, are 165 and 152 amino acids long, respectively, and are 36% identical and 72% similar. Biochemical studies on members of the ADF/cofilin family in other organisms suggest that the UNC-60 proteins regulate actin polymerization. But, analysis of the primary structures of the UNC-60 isoforms does not allow us to predict how their biochemical properties might be different. We need to know the precise biochemical properties of the UNC-60 proteins to understand the role of unc-60 in muscle development. To address this question, we studied the biochemical characteristics of two UNC-60 proteins, and found that they regulate actin filament dynamics in different manners. The results suggest that two UNC-60 proteins have physiologically distinct functions.DISCUSSIONThe present study shows that the two ADF/cofilin proteins that are generated by alternative splicing of the unc-60 gene have differential functions in regulating actin dynamics. This is the first clear demonstration of functional diversity for the ADF/cofilin family in a single organism. So far, 3 and 2 ADF/cofilin proteins have been found in mammals and chickens, respectively, but their functional differences are not clear. Previously, ADF (or destrin) and cofilin had been characterized as functionally distinct proteins. ADF primarily depolymerizes F-actin and does not bind to F-actin (3Abe H. Obinata T. J. Biochem. 1989; 106: 172-180Crossref PubMed Scopus (42) Google Scholar, 21Giuliano K.A. Khatib F.A. Hayden S.M. Daoud E.W. Adams M.E. Amorese D.A. Bernstein B.W. Bamburg J.R. Biochemistry. 1988; 27: 8931-8938Crossref PubMed Scopus (43) Google Scholar, 26Bamburg J.R. Harris H.E. Weeds A.G. FEBS Lett. 1980; 121: 178-182Crossref PubMed Scopus (158) Google Scholar, 27Nishida E. Muneyuki E. Maekawa S. Ohta Y. Sakai H. Biochemistry. 1985; 24: 6624-6630Crossref PubMed Scopus (77) Google Scholar), whereas cofilin binds to both G- and F-actin and depolymerizes F-actin in a pH-sensitive manner (4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar, 16Nishida E. Maekawa S. Sakai H. Biochemistry. 1984; 23: 5307-5313Crossref PubMed Scopus (249) Google Scholar, 28Yonezawa N. Nishida E. Sakai H. J. Biol. Chem. 1985; 260: 14410-14412Abstract Full Text PDF PubMed Google Scholar). However, recent refined biochemical studies on the activities of human and chicken ADF have shown that ADF, like cofilin, binds to F-actin at pHs between 6.5 and 7.1 and shows increasing actin-depolymerizing activity at pHs between 7.1 and 8.0 (24Hawkins M. Pope B. Maciver S.K. Weeds A.G. Biochemistry. 1993; 32: 9985-9993Crossref PubMed Scopus (239) Google Scholar, 25Hayden S.M. Miller P.S. Brauweiler A. Bamburg J.R. Biochemistry. 1993; 32: 9994-10004Crossref PubMed Scopus (202) Google Scholar).Although the activities of UNC-60A and B are different, both of them are functionally related to members of the ADF/cofilin family that have been characterized previously. The actin-depolymerizing activity of UNC-60A is quite similar to that of Acanthamoeba actophorin (29Cooper J.A. Blum J.D. Williams Jr., R.C. Pollard T.D. J. Biol. Chem. 1986; 261: 477-485Abstract Full Text PDF PubMed Google Scholar), echinoderm depactin (20Mabuchi I. J. Cell Biol. 1983; 97: 1612-1621Crossref PubMed Scopus (100) Google Scholar), and most of ADF/cofilins at alkaline pH. In addition, the profile of the effect of UNC-60A on actin polymerization kinetics is similar to that of chicken ADF (3Abe H. Obinata T. J. Biochem. 1989; 106: 172-180Crossref PubMed Scopus (42) Google Scholar). These activities can be explained by its strong G-actin binding ability that results in depolymerization of F-actin and sequestering G-actin to inhibit polymerization. The ability of UNC-60B to bind to F-actin has been observed for vertebrate ADF and cofilin at neutral pH (4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar, 16Nishida E. Maekawa S. Sakai H. Biochemistry. 1984; 23: 5307-5313Crossref PubMed Scopus (249) Google Scholar, 24Hawkins M. Pope B. Maciver S.K. Weeds A.G. Biochemistry. 1993; 32: 9985-9993Crossref PubMed Scopus (239) Google Scholar,25Hayden S.M. Miller P.S. Brauweiler A. Bamburg J.R. Biochemistry. 1993; 32: 9994-10004Crossref PubMed Scopus (202) Google Scholar). However, its lack of ability to depolymerize actin is a unique feature of UNC-60B. The effect of UNC-60B on the kinetics of actin polymerization is consistent with that observed for actophorin (29Cooper J.A. Blum J.D. Williams Jr., R.C. Pollard T.D. J. Biol. Chem. 1986; 261: 477-485Abstract Full Text PDF PubMed Google Scholar), chicken cofilin (4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar), and plant ADF (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar). Our results that substoichiometric amounts of UNC-60B accelerated, but that excess UNC-60B inhibited the rate of polymerization, can be interpreted as follows. UNC-60B binds to G-actin to inhibit polymerization, whereas it preferentially binds to F-actin which causes the enhancement of the rate of actin assembly. This effect might be due to the change in the on rate at the barbed end of actin filaments as has been shown for plant ADF (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar). The mechanism of the acceleration of polymerization is still unclear. Recently, binding of cofilin to F-actin has been shown to change the twist of the filament (19McGough A. Pope B. Chiu W. Weeds A. J. Cell Biol. 1997; 138: 771-781Crossref PubMed Scopus (572) Google Scholar). Likewise, UNC-60B may cause a conformational change in the actin filament leading to a more competent state for polymerization.Our results that inorganic phosphate inhibit the activities of both UNC-60A and B strongly suggest that UNC-60A and B preferentially bind to ADP-actin rather than ATP-actin. Preferential binding to ADP-actin appears to be a common property of ADF/cofilin family members (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar, 22Maciver S.K. Zot H.G. Pollard T.D. J. Cell Biol. 1991; 115: 1611-1620Crossref PubMed Scopus (193) Google Scholar,35Maciver S.K. Weeds A.G. FEBS Lett. 1994; 347: 251-256Crossref PubMed Scopus (96) Google Scholar) and is likely to contribute to an acceleration of the off rate at the pointed end of F-actin (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar). Probably, this is the case for UNC-60A because it rapidly depolymerizes F-actin. However, because UNC-60B does not depolymerize F-actin, it implies that UNC-60B-bound F-ADP-actin is physiologically significant. Therefore, it is of interest to examine whether UNC-60B-bound F-actin behaves differently from F-actin alone. Previously, porcine cofilin has been shown to inhibit the binding of tropomyosin and myosin to F-actin (16Nishida E. Maekawa S. Sakai H. Biochemistry. 1984; 23: 5307-5313Crossref PubMed Scopus (249) Google Scholar). Thus, a future goal will be to investigate the ability of UNC-60B-bound F-actin to bind to some other actin-binding proteins.The differences in the activities of the UNC-60 proteins are likely to result from their structural differences. The sequence identity between UNC-60A and UNC-60B is 36%, which is considerably lower than the group of mammalian ADF/cofilins (three members are 70% identical). However, both UNC-60A and UNC-60B are about 30% homologous to all three members of mammalian ADF/cofilins, and no outstanding similarity to a particular protein was detected. One obvious difference is that UNC-60A is 13 amino acids longer than UNC-60B. The alignment of the two sequences shows that an extra eight amino acids exist in UNC60A from Ile-50 to Asp-57 (Fig. 1, shown by underline). The equivalent region of yeast cofilin consists of the outer most strand of β-sheet and is exposed on the surface of the molecule (30Fedorov A.A. Lappalainen P. Fedorov E.V. Drubin D.G. Almo S.C. Nat. Struct. Biol. 1997; 4: 366-369Crossref PubMed Scopus (94) Google Scholar), but the function of this region is unknown. In addition, this sequence contains four acidic residues, which are likely to affect the charge distribution on the molecular surface. Because charged amino acids on cofilin have been shown to be important in its actin-binding (31Moriyama K. Yonezawa N. Sakai H. Yahara I. Nishida E. J. Biol. Chem. 1992; 267: 7240-7244Abstract Full Text PDF PubMed Google Scholar, 32Lappalainen P. Fedorov E.V. Fedorov A.A. Almo S.C. Drubin D.G. EMBO J. 1997; 16: 5520-5530Crossref PubMed Scopus (209) Google Scholar), the extra sequence in UNC-60A may be responsible for its functional difference from UNC-60B.The differential activities of UNC-60 proteins presented here strongly suggest that the two homologous proteins have physiologically distinct functions. The fine structure genetic map (13McKim K.S. Heschl M.F. Rosenbluth R.E. Baillie D.L. Genetics. 1988; 118: 49-59PubMed Google Scholar) and our preliminary genomic sequencing of viable unc-60 alleles has revealed that all the mutations are found within the coding region for unc-60B, 2S. Ono, D. L. Baillie, and G. M. Benian, unpublished data. implying that UNC-60B has a specific role in thin filament assembly in muscle cells. Our results on the effect of UNC-60B on the polymerization kineticsin vitro suggests that, during muscle development, when actin concentration is low initially, UNC-60B inhibits actin polymerization, but later, when actin concentration is high (1Shimizu N. Obinata T. J. Biochem. 1986; 99: 751-759Crossref PubMed Scopus (38) Google Scholar), UNC-60B accelerates actin polymerization and thus the formation of thin filaments. This function of UNC-60B is directly relevant to that of ADF/cofilin in vertebrate muscle. ADF/cofilin is involved in the regulation of actin assembly in chicken embryonic muscles (3Abe H. Obinata T. J. Biochem. 1989; 106: 172-180Crossref PubMed Scopus (42) Google Scholar, 4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar). In addition, a muscle-type cofilin isoform is expressed in mammalian skeletal and cardiac muscles (10Ono S. Minami N. Abe H. Obinata T. J. Biol. Chem. 1994; 269: 15280-15286Abstract Full Text PDF PubMed Google Scholar) although its function in muscle cells is not yet clear. UNC-60A may be widely involved in the many processes which require actin dynamics. ADF/cofilin has been shown to be essential for cytokinesis (33Gunsalus K.C. Bonaccorsi S. Williams E. Verni F. Gatti M. Goldberg M.L. J. Cell Biol. 1995; 131: 1243-1259Crossref PubMed Scopus (251) Google Scholar, 34Abe H. Obinata T. Minamide L.S. Bamburg J.R. J. Cell Biol. 1996; 132: 871-885Crossref PubMed Scopus (164) Google Scholar) and endocytosis (9Lappalainen P. Drubin D.G. Nature. 1997; 388: 78-82Crossref PubMed Scopus (363) Google Scholar), which are universal events in a broad range of cells. Accordingly, C. elegans should have an ADF/cofilin which is expressed in a variety of cells in addition to a muscle-specific isoform. Currently, we are raising antibodies against UNC-60A and UNC-60B and plan to determine tissue distribution of both proteins by immunofluorescence microscopy. Myofibrils are highly differentiated forms of actin cytoskeleton that are specialized for muscle contraction, but the mechanisms by which these complex and precise structures are assembled and maintained are largely unknown. Actin, a major component of thin filaments, has an inherent tendency to polymerize into filaments in vitro. However, the assembly of actin in developing muscle is regulated, and consequently, about 40% of actin is present in a monomeric form (1Shimizu N. Obinata T. J. Biochem. 1986; 99: 751-759Crossref PubMed Scopus (38) Google Scholar). In embryonic chicken skeletal muscle, proteins that bind to G-actin to prevent them from polymerization have been identified as profilin (2Ohshima S. Abe H. Obinata T. J. Biochem. 1989; 105: 855-857Crossref PubMed Scopus (44) Google Scholar), actin depolymerizing factor (ADF) 1The abbreviations used are: ADF, actin depolymerizing factor; EDC, 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide; Pi, inorganic phosphate; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: ADF, actin depolymerizing factor; EDC, 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide; Pi, inorganic phosphate; PAGE, polyacrylamide gel electrophoresis. (3Abe H. Obinata T. J. Biochem. 1989; 106: 172-180Crossref PubMed Scopus (42) Google Scholar), and cofilin (4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar). 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 (5Nagaoka R. Minami N. Hayakawa K. Abe H. Obinata T. J. Muscle Res. Cell Motil. 1996; 17: 463-473Crossref PubMed Scopus (31) Google Scholar), suggesting that they are responsible for regulating actin filament assembly. ADF and cofilin are highly conserved proteins, are members of an ADF/cofilin family having 25–71% homology, and are found in diverse organisms. ADF/cofilin binds to both G- and F-actin at a stoichiometry of 1:1 and regulates the rate of actin polymerization (reviewed in Ref.6Moon A. Drubin D.G. Mol. Biol. Cell. 1995; 6: 1423-1431Crossref PubMed Scopus (226) Google Scholar). Recently, ADF/cofilin has been shown to affect the on/off rates at both ends of F-actin, which results in the enhancement of treadmilling (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar). This function is necessary for the actin-based motility ofListeria monocytogenes (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar, 8Rosenblatt J. Agnew B.J. Abe H. Bamburg J.R. Mitchison T.J. J. Cell Biol. 1997; 136: 1323-1332Crossref PubMed Scopus (190) Google Scholar) and for actin turnover in cortical actin patches in yeast (9Lappalainen P. Drubin D.G. Nature. 1997; 388: 78-82Crossref PubMed Scopus (363) Google Scholar). A muscle-specific function for ADF/cofilin has been suggested by two examples. These are a mammalian muscle-specific cofilin (M-cofilin) (10Ono S. Minami N. Abe H. Obinata T. J. Biol. Chem. 1994; 269: 15280-15286Abstract Full Text PDF PubMed Google Scholar) and two ADF/cofilin homologues encoded by the Caenorhabditis elegans unc-60 gene (11McKim K.S. Matheson C. Marra M.A. Wakarchuk M.F. Baillie D.L. Mol. Gen. Genet. 1994; 242: 346-357Crossref PubMed Scopus (100) Google Scholar). Mammalian M-cofilin is predominantly expressed in skeletal and cardiac muscles (10Ono S. Minami N. Abe H. Obinata T. J. Biol. Chem. 1994; 269: 15280-15286Abstract Full Text PDF PubMed Google Scholar), but its exact function is unknown. Mutations in the unc-60 gene result in slow moving or paralyzed nematodes (11McKim K.S. Matheson C. Marra M.A. Wakarchuk M.F. Baillie D.L. Mol. Gen. Genet. 1994; 242: 346-357Crossref PubMed Scopus (100) Google Scholar, 12Waterston R.H. Thomson J.N. Brenner S. Dev. Biol. 1980; 77: 271-302Crossref PubMed Scopus (183) Google Scholar, 13McKim K.S. Heschl M.F. Rosenbluth R.E. Baillie D.L. Genetics. 1988; 118: 49-59PubMed Google Scholar). By electron microscopy, 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 (12Waterston R.H. Thomson J.N. Brenner S. Dev. Biol. 1980; 77: 271-302Crossref PubMed Scopus (183) Google Scholar). Thus, unc-60 is required for proper positioning and the correct number of thin filaments in nematode striated muscle. Theunc-60 gene has been shown to encode two transcripts, sharing only a single exon encoding the initiator methionine, and two homologous proteins of the ADF/cofilin family (11McKim K.S. Matheson C. Marra M.A. Wakarchuk M.F. Baillie D.L. Mol. Gen. Genet. 1994; 242: 346-357Crossref PubMed Scopus (100) Google Scholar). These proteins, called UNC-60A and UNC-60B, are 165 and 152 amino acids long, respectively, and are 36% identical and 72% similar. Biochemical studies on members of the ADF/cofilin family in other organisms suggest that the UNC-60 proteins regulate actin polymerization. But, analysis of the primary structures of the UNC-60 isoforms does not allow us to predict how their biochemical properties might be different. We need to know the precise biochemical properties of the UNC-60 proteins to understand the role of unc-60 in muscle development. To address this question, we studied the biochemical characteristics of two UNC-60 proteins, and found that they regulate actin filament dynamics in different manners. The results suggest that two UNC-60 proteins have physiologically distinct functions. DISCUSSIONThe present study shows that the two ADF/cofilin proteins that are generated by alternative splicing of the unc-60 gene have differential functions in regulating actin dynamics. This is the first clear demonstration of functional diversity for the ADF/cofilin family in a single organism. So far, 3 and 2 ADF/cofilin proteins have been found in mammals and chickens, respectively, but their functional differences are not clear. Previously, ADF (or destrin) and cofilin had been characterized as functionally distinct proteins. ADF primarily depolymerizes F-actin and does not bind to F-actin (3Abe H. Obinata T. J. Biochem. 1989; 106: 172-180Crossref PubMed Scopus (42) Google Scholar, 21Giuliano K.A. Khatib F.A. Hayden S.M. Daoud E.W. Adams M.E. Amorese D.A. Bernstein B.W. Bamburg J.R. Biochemistry. 1988; 27: 8931-8938Crossref PubMed Scopus (43) Google Scholar, 26Bamburg J.R. Harris H.E. Weeds A.G. FEBS Lett. 1980; 121: 178-182Crossref PubMed Scopus (158) Google Scholar, 27Nishida E. Muneyuki E. Maekawa S. Ohta Y. Sakai H. Biochemistry. 1985; 24: 6624-6630Crossref PubMed Scopus (77) Google Scholar), whereas cofilin binds to both G- and F-actin and depolymerizes F-actin in a pH-sensitive manner (4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar, 16Nishida E. Maekawa S. Sakai H. Biochemistry. 1984; 23: 5307-5313Crossref PubMed Scopus (249) Google Scholar, 28Yonezawa N. Nishida E. Sakai H. J. Biol. Chem. 1985; 260: 14410-14412Abstract Full Text PDF PubMed Google Scholar). However, recent refined biochemical studies on the activities of human and chicken ADF have shown that ADF, like cofilin, binds to F-actin at pHs between 6.5 and 7.1 and shows increasing actin-depolymerizing activity at pHs between 7.1 and 8.0 (24Hawkins M. Pope B. Maciver S.K. Weeds A.G. Biochemistry. 1993; 32: 9985-9993Crossref PubMed Scopus (239) Google Scholar, 25Hayden S.M. Miller P.S. Brauweiler A. Bamburg J.R. Biochemistry. 1993; 32: 9994-10004Crossref PubMed Scopus (202) Google Scholar).Although the activities of UNC-60A and B are different, both of them are functionally related to members of the ADF/cofilin family that have been characterized previously. The actin-depolymerizing activity of UNC-60A is quite similar to that of Acanthamoeba actophorin (29Cooper J.A. Blum J.D. Williams Jr., R.C. Pollard T.D. J. Biol. Chem. 1986; 261: 477-485Abstract Full Text PDF PubMed Google Scholar), echinoderm depactin (20Mabuchi I. J. Cell Biol. 1983; 97: 1612-1621Crossref PubMed Scopus (100) Google Scholar), and most of ADF/cofilins at alkaline pH. In addition, the profile of the effect of UNC-60A on actin polymerization kinetics is similar to that of chicken ADF (3Abe H. Obinata T. J. Biochem. 1989; 106: 172-180Crossref PubMed Scopus (42) Google Scholar). These activities can be explained by its strong G-actin binding ability that results in depolymerization of F-actin and sequestering G-actin to inhibit polymerization. The ability of UNC-60B to bind to F-actin has been observed for vertebrate ADF and cofilin at neutral pH (4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar, 16Nishida E. Maekawa S. Sakai H. Biochemistry. 1984; 23: 5307-5313Crossref PubMed Scopus (249) Google Scholar, 24Hawkins M. Pope B. Maciver S.K. Weeds A.G. Biochemistry. 1993; 32: 9985-9993Crossref PubMed Scopus (239) Google Scholar,25Hayden S.M. Miller P.S. Brauweiler A. Bamburg J.R. Biochemistry. 1993; 32: 9994-10004Crossref PubMed Scopus (202) Google Scholar). However, its lack of ability to depolymerize actin is a unique feature of UNC-60B. The effect of UNC-60B on the kinetics of actin polymerization is consistent with that observed for actophorin (29Cooper J.A. Blum J.D. Williams Jr., R.C. Pollard T.D. J. Biol. Chem. 1986; 261: 477-485Abstract Full Text PDF PubMed Google Scholar), chicken cofilin (4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar), and plant ADF (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar). Our results that substoichiometric amounts of UNC-60B accelerated, but that excess UNC-60B inhibited the rate of polymerization, can be interpreted as follows. UNC-60B binds to G-actin to inhibit polymerization, whereas it preferentially binds to F-actin which causes the enhancement of the rate of actin assembly. This effect might be due to the change in the on rate at the barbed end of actin filaments as has been shown for plant ADF (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar). The mechanism of the acceleration of polymerization is still unclear. Recently, binding of cofilin to F-actin has been shown to change the twist of the filament (19McGough A. Pope B. Chiu W. Weeds A. J. Cell Biol. 1997; 138: 771-781Crossref PubMed Scopus (572) Google Scholar). Likewise, UNC-60B may cause a conformational change in the actin filament leading to a more competent state for polymerization.Our results that inorganic phosphate inhibit the activities of both UNC-60A and B strongly suggest that UNC-60A and B preferentially bind to ADP-actin rather than ATP-actin. Preferential binding to ADP-actin appears to be a common property of ADF/cofilin family members (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar, 22Maciver S.K. Zot H.G. Pollard T.D. J. Cell Biol. 1991; 115: 1611-1620Crossref PubMed Scopus (193) Google Scholar,35Maciver S.K. Weeds A.G. FEBS Lett. 1994; 347: 251-256Crossref PubMed Scopus (96) Google Scholar) and is likely to contribute to an acceleration of the off rate at the pointed end of F-actin (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar). Probably, this is the case for UNC-60A because it rapidly depolymerizes F-actin. However, because UNC-60B does not depolymerize F-actin, it implies that UNC-60B-bound F-ADP-actin is physiologically significant. Therefore, it is of interest to examine whether UNC-60B-bound F-actin behaves differently from F-actin alone. Previously, porcine cofilin has been shown to inhibit the binding of tropomyosin and myosin to F-actin (16Nishida E. Maekawa S. Sakai H. Biochemistry. 1984; 23: 5307-5313Crossref PubMed Scopus (249) Google Scholar). Thus, a future goal will be to investigate the ability of UNC-60B-bound F-actin to bind to some other actin-binding proteins.The differences in the activities of the UNC-60 proteins are likely to result from their structural differences. The sequence identity between UNC-60A and UNC-60B is 36%, which is considerably lower than the group of mammalian ADF/cofilins (three members are 70% identical). However, both UNC-60A and UNC-60B are about 30% homologous to all three members of mammalian ADF/cofilins, and no outstanding similarity to a particular protein was detected. One obvious difference is that UNC-60A is 13 amino acids longer than UNC-60B. The alignment of the two sequences shows that an extra eight amino acids exist in UNC60A from Ile-50 to Asp-57 (Fig. 1, shown by underline). The equivalent region of yeast cofilin consists of the outer most strand of β-sheet and is exposed on the surface of the molecule (30Fedorov A.A. Lappalainen P. Fedorov E.V. Drubin D.G. Almo S.C. Nat. Struct. Biol. 1997; 4: 366-369Crossref PubMed Scopus (94) Google Scholar), but the function of this region is unknown. In addition, this sequence contains four acidic residues, which are likely to affect the charge distribution on the molecular surface. Because charged amino acids on cofilin have been shown to be important in its actin-binding (31Moriyama K. Yonezawa N. Sakai H. Yahara I. Nishida E. J. Biol. Chem. 1992; 267: 7240-7244Abstract Full Text PDF PubMed Google Scholar, 32Lappalainen P. Fedorov E.V. Fedorov A.A. Almo S.C. Drubin D.G. EMBO J. 1997; 16: 5520-5530Crossref PubMed Scopus (209) Google Scholar), the extra sequence in UNC-60A may be responsible for its functional difference from UNC-60B.The differential activities of UNC-60 proteins presented here strongly suggest that the two homologous proteins have physiologically distinct functions. The fine structure genetic map (13McKim K.S. Heschl M.F. Rosenbluth R.E. Baillie D.L. Genetics. 1988; 118: 49-59PubMed Google Scholar) and our preliminary genomic sequencing of viable unc-60 alleles has revealed that all the mutations are found within the coding region for unc-60B, 2S. Ono, D. L. Baillie, and G. M. Benian, unpublished data. implying that UNC-60B has a specific role in thin filament assembly in muscle cells. Our results on the effect of UNC-60B on the polymerization kineticsin vitro suggests that, during muscle development, when actin concentration is low initially, UNC-60B inhibits actin polymerization, but later, when actin concentration is high (1Shimizu N. Obinata T. J. Biochem. 1986; 99: 751-759Crossref PubMed Scopus (38) Google Scholar), UNC-60B accelerates actin polymerization and thus the formation of thin filaments. This function of UNC-60B is directly relevant to that of ADF/cofilin in vertebrate muscle. ADF/cofilin is involved in the regulation of actin assembly in chicken embryonic muscles (3Abe H. Obinata T. J. Biochem. 1989; 106: 172-180Crossref PubMed Scopus (42) Google Scholar, 4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar). In addition, a muscle-type cofilin isoform is expressed in mammalian skeletal and cardiac muscles (10Ono S. Minami N. Abe H. Obinata T. J. Biol. Chem. 1994; 269: 15280-15286Abstract Full Text PDF PubMed Google Scholar) although its function in muscle cells is not yet clear. UNC-60A may be widely involved in the many processes which require actin dynamics. ADF/cofilin has been shown to be essential for cytokinesis (33Gunsalus K.C. Bonaccorsi S. Williams E. Verni F. Gatti M. Goldberg M.L. J. Cell Biol. 1995; 131: 1243-1259Crossref PubMed Scopus (251) Google Scholar, 34Abe H. Obinata T. Minamide L.S. Bamburg J.R. J. Cell Biol. 1996; 132: 871-885Crossref PubMed Scopus (164) Google Scholar) and endocytosis (9Lappalainen P. Drubin D.G. Nature. 1997; 388: 78-82Crossref PubMed Scopus (363) Google Scholar), which are universal events in a broad range of cells. Accordingly, C. elegans should have an ADF/cofilin which is expressed in a variety of cells in addition to a muscle-specific isoform. Currently, we are raising antibodies against UNC-60A and UNC-60B and plan to determine tissue distribution of both proteins by immunofluorescence microscopy. The present study shows that the two ADF/cofilin proteins that are generated by alternative splicing of the unc-60 gene have differential functions in regulating actin dynamics. This is the first clear demonstration of functional diversity for the ADF/cofilin family in a single organism. So far, 3 and 2 ADF/cofilin proteins have been found in mammals and chickens, respectively, but their functional differences are not clear. Previously, ADF (or destrin) and cofilin had been characterized as functionally distinct proteins. ADF primarily depolymerizes F-actin and does not bind to F-actin (3Abe H. Obinata T. J. Biochem. 1989; 106: 172-180Crossref PubMed Scopus (42) Google Scholar, 21Giuliano K.A. Khatib F.A. Hayden S.M. Daoud E.W. Adams M.E. Amorese D.A. Bernstein B.W. Bamburg J.R. Biochemistry. 1988; 27: 8931-8938Crossref PubMed Scopus (43) Google Scholar, 26Bamburg J.R. Harris H.E. Weeds A.G. FEBS Lett. 1980; 121: 178-182Crossref PubMed Scopus (158) Google Scholar, 27Nishida E. Muneyuki E. Maekawa S. Ohta Y. Sakai H. Biochemistry. 1985; 24: 6624-6630Crossref PubMed Scopus (77) Google Scholar), whereas cofilin binds to both G- and F-actin and depolymerizes F-actin in a pH-sensitive manner (4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar, 16Nishida E. Maekawa S. Sakai H. Biochemistry. 1984; 23: 5307-5313Crossref PubMed Scopus (249) Google Scholar, 28Yonezawa N. Nishida E. Sakai H. J. Biol. Chem. 1985; 260: 14410-14412Abstract Full Text PDF PubMed Google Scholar). However, recent refined biochemical studies on the activities of human and chicken ADF have shown that ADF, like cofilin, binds to F-actin at pHs between 6.5 and 7.1 and shows increasing actin-depolymerizing activity at pHs between 7.1 and 8.0 (24Hawkins M. Pope B. Maciver S.K. Weeds A.G. Biochemistry. 1993; 32: 9985-9993Crossref PubMed Scopus (239) Google Scholar, 25Hayden S.M. Miller P.S. Brauweiler A. Bamburg J.R. Biochemistry. 1993; 32: 9994-10004Crossref PubMed Scopus (202) Google Scholar). Although the activities of UNC-60A and B are different, both of them are functionally related to members of the ADF/cofilin family that have been characterized previously. The actin-depolymerizing activity of UNC-60A is quite similar to that of Acanthamoeba actophorin (29Cooper J.A. Blum J.D. Williams Jr., R.C. Pollard T.D. J. Biol. Chem. 1986; 261: 477-485Abstract Full Text PDF PubMed Google Scholar), echinoderm depactin (20Mabuchi I. J. Cell Biol. 1983; 97: 1612-1621Crossref PubMed Scopus (100) Google Scholar), and most of ADF/cofilins at alkaline pH. In addition, the profile of the effect of UNC-60A on actin polymerization kinetics is similar to that of chicken ADF (3Abe H. Obinata T. J. Biochem. 1989; 106: 172-180Crossref PubMed Scopus (42) Google Scholar). These activities can be explained by its strong G-actin binding ability that results in depolymerization of F-actin and sequestering G-actin to inhibit polymerization. The ability of UNC-60B to bind to F-actin has been observed for vertebrate ADF and cofilin at neutral pH (4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar, 16Nishida E. Maekawa S. Sakai H. Biochemistry. 1984; 23: 5307-5313Crossref PubMed Scopus (249) Google Scholar, 24Hawkins M. Pope B. Maciver S.K. Weeds A.G. Biochemistry. 1993; 32: 9985-9993Crossref PubMed Scopus (239) Google Scholar,25Hayden S.M. Miller P.S. Brauweiler A. Bamburg J.R. Biochemistry. 1993; 32: 9994-10004Crossref PubMed Scopus (202) Google Scholar). However, its lack of ability to depolymerize actin is a unique feature of UNC-60B. The effect of UNC-60B on the kinetics of actin polymerization is consistent with that observed for actophorin (29Cooper J.A. Blum J.D. Williams Jr., R.C. Pollard T.D. J. Biol. Chem. 1986; 261: 477-485Abstract Full Text PDF PubMed Google Scholar), chicken cofilin (4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar), and plant ADF (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar). Our results that substoichiometric amounts of UNC-60B accelerated, but that excess UNC-60B inhibited the rate of polymerization, can be interpreted as follows. UNC-60B binds to G-actin to inhibit polymerization, whereas it preferentially binds to F-actin which causes the enhancement of the rate of actin assembly. This effect might be due to the change in the on rate at the barbed end of actin filaments as has been shown for plant ADF (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar). The mechanism of the acceleration of polymerization is still unclear. Recently, binding of cofilin to F-actin has been shown to change the twist of the filament (19McGough A. Pope B. Chiu W. Weeds A. J. Cell Biol. 1997; 138: 771-781Crossref PubMed Scopus (572) Google Scholar). Likewise, UNC-60B may cause a conformational change in the actin filament leading to a more competent state for polymerization. Our results that inorganic phosphate inhibit the activities of both UNC-60A and B strongly suggest that UNC-60A and B preferentially bind to ADP-actin rather than ATP-actin. Preferential binding to ADP-actin appears to be a common property of ADF/cofilin family members (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar, 22Maciver S.K. Zot H.G. Pollard T.D. J. Cell Biol. 1991; 115: 1611-1620Crossref PubMed Scopus (193) Google Scholar,35Maciver S.K. Weeds A.G. FEBS Lett. 1994; 347: 251-256Crossref PubMed Scopus (96) Google Scholar) and is likely to contribute to an acceleration of the off rate at the pointed end of F-actin (7Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (820) Google Scholar). Probably, this is the case for UNC-60A because it rapidly depolymerizes F-actin. However, because UNC-60B does not depolymerize F-actin, it implies that UNC-60B-bound F-ADP-actin is physiologically significant. Therefore, it is of interest to examine whether UNC-60B-bound F-actin behaves differently from F-actin alone. Previously, porcine cofilin has been shown to inhibit the binding of tropomyosin and myosin to F-actin (16Nishida E. Maekawa S. Sakai H. Biochemistry. 1984; 23: 5307-5313Crossref PubMed Scopus (249) Google Scholar). Thus, a future goal will be to investigate the ability of UNC-60B-bound F-actin to bind to some other actin-binding proteins. The differences in the activities of the UNC-60 proteins are likely to result from their structural differences. The sequence identity between UNC-60A and UNC-60B is 36%, which is considerably lower than the group of mammalian ADF/cofilins (three members are 70% identical). However, both UNC-60A and UNC-60B are about 30% homologous to all three members of mammalian ADF/cofilins, and no outstanding similarity to a particular protein was detected. One obvious difference is that UNC-60A is 13 amino acids longer than UNC-60B. The alignment of the two sequences shows that an extra eight amino acids exist in UNC60A from Ile-50 to Asp-57 (Fig. 1, shown by underline). The equivalent region of yeast cofilin consists of the outer most strand of β-sheet and is exposed on the surface of the molecule (30Fedorov A.A. Lappalainen P. Fedorov E.V. Drubin D.G. Almo S.C. Nat. Struct. Biol. 1997; 4: 366-369Crossref PubMed Scopus (94) Google Scholar), but the function of this region is unknown. In addition, this sequence contains four acidic residues, which are likely to affect the charge distribution on the molecular surface. Because charged amino acids on cofilin have been shown to be important in its actin-binding (31Moriyama K. Yonezawa N. Sakai H. Yahara I. Nishida E. J. Biol. Chem. 1992; 267: 7240-7244Abstract Full Text PDF PubMed Google Scholar, 32Lappalainen P. Fedorov E.V. Fedorov A.A. Almo S.C. Drubin D.G. EMBO J. 1997; 16: 5520-5530Crossref PubMed Scopus (209) Google Scholar), the extra sequence in UNC-60A may be responsible for its functional difference from UNC-60B. The differential activities of UNC-60 proteins presented here strongly suggest that the two homologous proteins have physiologically distinct functions. The fine structure genetic map (13McKim K.S. Heschl M.F. Rosenbluth R.E. Baillie D.L. Genetics. 1988; 118: 49-59PubMed Google Scholar) and our preliminary genomic sequencing of viable unc-60 alleles has revealed that all the mutations are found within the coding region for unc-60B, 2S. Ono, D. L. Baillie, and G. M. Benian, unpublished data. implying that UNC-60B has a specific role in thin filament assembly in muscle cells. Our results on the effect of UNC-60B on the polymerization kineticsin vitro suggests that, during muscle development, when actin concentration is low initially, UNC-60B inhibits actin polymerization, but later, when actin concentration is high (1Shimizu N. Obinata T. J. Biochem. 1986; 99: 751-759Crossref PubMed Scopus (38) Google Scholar), UNC-60B accelerates actin polymerization and thus the formation of thin filaments. This function of UNC-60B is directly relevant to that of ADF/cofilin in vertebrate muscle. ADF/cofilin is involved in the regulation of actin assembly in chicken embryonic muscles (3Abe H. Obinata T. J. Biochem. 1989; 106: 172-180Crossref PubMed Scopus (42) Google Scholar, 4Abe H. Ohshima S. Obinata T. J. Biochem. 1989; 106: 696-702Crossref PubMed Scopus (81) Google Scholar). In addition, a muscle-type cofilin isoform is expressed in mammalian skeletal and cardiac muscles (10Ono S. Minami N. Abe H. Obinata T. J. Biol. Chem. 1994; 269: 15280-15286Abstract Full Text PDF PubMed Google Scholar) although its function in muscle cells is not yet clear. UNC-60A may be widely involved in the many processes which require actin dynamics. ADF/cofilin has been shown to be essential for cytokinesis (33Gunsalus K.C. Bonaccorsi S. Williams E. Verni F. Gatti M. Goldberg M.L. J. Cell Biol. 1995; 131: 1243-1259Crossref PubMed Scopus (251) Google Scholar, 34Abe H. Obinata T. Minamide L.S. Bamburg J.R. J. Cell Biol. 1996; 132: 871-885Crossref PubMed Scopus (164) Google Scholar) and endocytosis (9Lappalainen P. Drubin D.G. Nature. 1997; 388: 78-82Crossref PubMed Scopus (363) Google Scholar), which are universal events in a broad range of cells. Accordingly, C. elegans should have an ADF/cofilin which is expressed in a variety of cells in addition to a muscle-specific isoform. Currently, we are raising antibodies against UNC-60A and UNC-60B and plan to determine tissue distribution of both proteins by immunofluorescence microscopy. We are grateful to Dr. David Baillie (Simon Fraser University) for suggesting this project to us. We thank Dr. Harish C. Joshi (Emory University) for help, discussion, and encouragement and thank the members of the Joshi laboratory for cooperation throughout this work. We also thank Dr. James R. Bamburg (Colorado State University), for discussion and suggestions, and Dr. Laura Fox and Dr. Winfield Sale (Emory University), for help with electron microscopy.