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Redistribution of actin and fascin during fertilization in sea urchin eggs
Cell Differentiation, 11 (1982) 279--280 Elsevier/North-Holland Scientific Publishers, Ltd.
279
R E D I S T R I B U T I O N OF ACTIN AND FASCIN D U R I N G F E R T I L I Z A T I O N IN SEA U R C H I N EGGS* JOSEPH BRYAN Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, U.S.A.
egg
cortex
microvilli
calcium dependence
There is a substantial amount of morphological evidence that shows the restructuring of the echinoderm egg cortex following fertilization (Eddy and Shapiro 1976; Schroeder, 1978, 1979). These changes involve the polymerization of actin filaments and their organization into the elongating microvillar cores. Measurements on the actin content of eggs from Tripneustes gratilla demonstrate that actin is a b o u t 1.2% of the total egg protein ( O t t o et al., 1980). In unfertilized eggs approximately 10% of the actin is associated with the cortex versus 38% in fertilized eggs with elongated microvflli. Fascin, a 58,000 dalton protein, which is known to cross-link adjacent actin filaments into tight bundles in vitro (Kane, 1975, 1976; Bryan and Kane, 1978) exhibits a similar redistribution. This molecule accounts for a b o u t 0.3% of the total egg protein, cannot be localized in the unfertilized egg cortex using either indirect immunofluorescence or RIA techniques, b u t is easily detectable in a soluble fraction. Post fertilization, approximately 35% of the egg fascin is in the egg cortex and can be localized in the microvilli. We conclude that fascin organizes newly assembled actin filaments into the core structures o f the cortical microvilli. What mechanisms control the assembly of actin filaments at fertilization? Begg and
* Funded by N.I.H. Grant GM 26091.
sea urchins
Rebhun (1979) have provided morphological evidence that connects actin assembly with the transient increase in calcium and the cytoplasmic alkalinization that follow insemination. We propose that eggs have calciumdependent actin binding proteins, similar to those in platelets (Wang and Bryan, 1981) and that these molecules nucleate the assembly of actin filaments in the cortex. Using NBD-actin, a fluorescent actin probe, we have established that unfertilized eggs have nucleating activities that will initiate filament formation in 20 mM KC1 only if calcium is present in micromolar concentrations. This activity can be separated into two fractions using gel filtration. One activity elutes in the void volume of a G-150 column; the second activity co-elutes with monomeric actin. Both fractions contain calcium dependent G-actin-binding proteins which can be identified using the [ l:s I]G-Actin overlay procedure described by Snabes et al. (1981). The void volume fraction contains a major G-actin-binding activity at 100--105 kilodaltons and minor components at 90, 80 and 58 kilodaltons. The second fraction has a single G-actin binding protein at 46--48 kilodaltons. When assayed with the overlay technique, the 1 0 0 - 1 0 5 kilodalton components is the major G-actin-binding protein. We are trying to purify these molecules and establish their role in the cortical changes which occur at fertilization. Finally, since these molecules are present in the egg cytoplasmic extracts used by Kane (1975, 1976) to study actin gelation,
0045-6039/82/0000--0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publishers, Ltd.
280
they could account for the calcium sensitivity of the gelation process by restrictingthe length of the filaments formed in vitro.
References Begg, D.A. and L.I. Rebhun: J. Cell Biol. 83, 241-248 (1979). Bryan, J. and R.E. Kane: J. Mol. Biol. 125, 207-224 (1978).
Eddy, E.M. and B.M. Shapiro: J. Cell Biol. 71, 35-48 (1976). Kane, R.E.: J. Cell Biol. 66, 305--315 (1975). Kane, R.E.: J. Cell Biol. 7 1 , 7 0 4 - - 7 1 4 (1976). Otto, J.O., R.E. Kane and J. Bryan: Cell Motility 1, 31--40 (1980). Schroeder, T.S.: Dev. Biol. 64, 342--346 (1978). Schroeder, T.S.: Dev. Biol. 70, 306--326 (1979). Snabes, M.C., A.E. Boyd III and J. Bryan: J. Cell Biol 90, 809--812 (1981). Wang, L.L. and J. Bryan: Cell 25, 637--649 (1981).
279
R E D I S T R I B U T I O N OF ACTIN AND FASCIN D U R I N G F E R T I L I Z A T I O N IN SEA U R C H I N EGGS* JOSEPH BRYAN Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, U.S.A.
egg
cortex
microvilli
calcium dependence
There is a substantial amount of morphological evidence that shows the restructuring of the echinoderm egg cortex following fertilization (Eddy and Shapiro 1976; Schroeder, 1978, 1979). These changes involve the polymerization of actin filaments and their organization into the elongating microvillar cores. Measurements on the actin content of eggs from Tripneustes gratilla demonstrate that actin is a b o u t 1.2% of the total egg protein ( O t t o et al., 1980). In unfertilized eggs approximately 10% of the actin is associated with the cortex versus 38% in fertilized eggs with elongated microvflli. Fascin, a 58,000 dalton protein, which is known to cross-link adjacent actin filaments into tight bundles in vitro (Kane, 1975, 1976; Bryan and Kane, 1978) exhibits a similar redistribution. This molecule accounts for a b o u t 0.3% of the total egg protein, cannot be localized in the unfertilized egg cortex using either indirect immunofluorescence or RIA techniques, b u t is easily detectable in a soluble fraction. Post fertilization, approximately 35% of the egg fascin is in the egg cortex and can be localized in the microvilli. We conclude that fascin organizes newly assembled actin filaments into the core structures o f the cortical microvilli. What mechanisms control the assembly of actin filaments at fertilization? Begg and
* Funded by N.I.H. Grant GM 26091.
sea urchins
Rebhun (1979) have provided morphological evidence that connects actin assembly with the transient increase in calcium and the cytoplasmic alkalinization that follow insemination. We propose that eggs have calciumdependent actin binding proteins, similar to those in platelets (Wang and Bryan, 1981) and that these molecules nucleate the assembly of actin filaments in the cortex. Using NBD-actin, a fluorescent actin probe, we have established that unfertilized eggs have nucleating activities that will initiate filament formation in 20 mM KC1 only if calcium is present in micromolar concentrations. This activity can be separated into two fractions using gel filtration. One activity elutes in the void volume of a G-150 column; the second activity co-elutes with monomeric actin. Both fractions contain calcium dependent G-actin-binding proteins which can be identified using the [ l:s I]G-Actin overlay procedure described by Snabes et al. (1981). The void volume fraction contains a major G-actin-binding activity at 100--105 kilodaltons and minor components at 90, 80 and 58 kilodaltons. The second fraction has a single G-actin binding protein at 46--48 kilodaltons. When assayed with the overlay technique, the 1 0 0 - 1 0 5 kilodalton components is the major G-actin-binding protein. We are trying to purify these molecules and establish their role in the cortical changes which occur at fertilization. Finally, since these molecules are present in the egg cytoplasmic extracts used by Kane (1975, 1976) to study actin gelation,
0045-6039/82/0000--0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publishers, Ltd.
280
they could account for the calcium sensitivity of the gelation process by restrictingthe length of the filaments formed in vitro.
References Begg, D.A. and L.I. Rebhun: J. Cell Biol. 83, 241-248 (1979). Bryan, J. and R.E. Kane: J. Mol. Biol. 125, 207-224 (1978).
Eddy, E.M. and B.M. Shapiro: J. Cell Biol. 71, 35-48 (1976). Kane, R.E.: J. Cell Biol. 66, 305--315 (1975). Kane, R.E.: J. Cell Biol. 7 1 , 7 0 4 - - 7 1 4 (1976). Otto, J.O., R.E. Kane and J. Bryan: Cell Motility 1, 31--40 (1980). Schroeder, T.S.: Dev. Biol. 64, 342--346 (1978). Schroeder, T.S.: Dev. Biol. 70, 306--326 (1979). Snabes, M.C., A.E. Boyd III and J. Bryan: J. Cell Biol 90, 809--812 (1981). Wang, L.L. and J. Bryan: Cell 25, 637--649 (1981).