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J Cell Biol. Jul 1, 1983; 97(1): 112–124.
PMCID: PMC2112487

Actin from Thyone sperm assembles on only one end of an actin filament: a behavior regulated by profilin

Abstract

Thyone sperm were demembranated with Triton X-100 and, after washing, extracted with 30 mM Tris at pH 8.0 and 1 mM MgCl2. After the insoluble contaminants were removed by centrifugation, the sperm extract was warmed to 22 degrees C. Actin filaments rapidly assembled and aggregated into bundles when KCl was added to the extract. When we added preformed actin filaments, i.e., the acrosomal filament bundles of Limulus sperm, to the extract, the actin monomers rapidly assembled on these filaments. What was unexpected was that assembly took place on only one end of the bundle--the end corresponding to the preferred end for monomer addition. We showed that the absence of growth on the nonpreferred end was not due to the presence of a capper because exogenously added actin readily assembled on both ends. We also analyzed the sperm extract by SDS gel electrophoresis. Two major proteins were present in a 1:1 molar ratio: actin and a 12,500-dalton protein whose apparent isoelectric point was 8.4. The 12,500-dalton protein was purified by DEAE chromatography. We concluded that it is profilin because of its size, isoelectric point, molar ratio to actin, inability to bind to DEAE, and its effect on actin assembly. When profilin was added to actin in the presence of Limulus bundles, addition of monomers on the nonpreferred end of the bundle was inhibited, even though actin by itself assembled on both ends. Using the Limulus bundles as nuclei, we determined the critical concentration for assembly off each end of the filament and estimated the Kd for the profilin-actin complex (approximately 10 microM). We present a model to explain how profilin may regulate the extension of the Thyone acrosomal process in vivo: The profilin-actin complex can add to only the preferred end of the filament bundle. Once the actin monomer is bound to the filament, the profilin is released, and is available to bind to additional actin monomers. This mechanism accounts for the rapid rate of filament elongation in the acrosomal process in vivo.

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Selected References

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  • Bergen LG, Borisy GG. Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly. J Cell Biol. 1980 Jan;84(1):141–150. [PMC free article] [PubMed]
  • Bonder EM, Mooseker MS. Direct electron microscopic visualization of barbed end capping and filament cutting by intestinal microvillar 95-kdalton protein (villin): a new actin assembly assay using the Limulus acrosomal process. J Cell Biol. 1983 Apr;96(4):1097–1107. [PMC free article] [PubMed]
  • Carlsson L, Nyström LE, Sundkvist I, Markey F, Lindberg U. Actin polymerizability is influenced by profilin, a low molecular weight protein in non-muscle cells. J Mol Biol. 1977 Sep 25;115(3):465–483. [PubMed]
  • DeRosier DJ, Tilney LG. How actin filaments pack into bundles. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):525–540. [PubMed]
  • DeRosier D, Mandelkow E, Silliman A. Structure of actin-containing filaments from two types of non-muscle cells. J Mol Biol. 1977 Jul 15;113(4):679–695. [PubMed]
  • Harris HE, Weeds AG. Platelet actin: sub-cellular distribution and association with profilin. FEBS Lett. 1978 Jun 1;90(1):84–88. [PubMed]
  • Inoué S, Tilney LG. Acrosomal reaction of thyone sperm. I. Changes in the sperm head visualized by high resolution video microscopy. J Cell Biol. 1982 Jun;93(3):812–819. [PMC free article] [PubMed]
  • Korn ED. Actin polymerization and its regulation by proteins from nonmuscle cells. Physiol Rev. 1982 Apr;62(2):672–737. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • MacLean-Fletcher S, Pollard TD. Identification of a factor in conventional muscle actin preparations which inhibits actin filament self-association. Biochem Biophys Res Commun. 1980 Sep 16;96(1):18–27. [PubMed]
  • Markey F, Larsson H, Weber K, Lindberg U. Nucleation of actin polymerization from profilactin. Opposite effects of different nuclei. Biochim Biophys Acta. 1982 May 21;704(1):43–51. [PubMed]
  • Matsudaira PT, Burgess DR. SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal Biochem. 1978 Jul 1;87(2):386–396. [PubMed]
  • O'Farrell PZ, Goodman HM, O'Farrell PH. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. [PubMed]
  • Pollard TD, Mooseker MS. Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores. J Cell Biol. 1981 Mar;88(3):654–659. [PMC free article] [PubMed]
  • Reichstein E, Korn ED. Acanthamoeba profilin. A protein of low molecular weight from Acanpthamoeba castellanii that inhibits actin nucleation. J Biol Chem. 1979 Jul 10;254(13):6174–6179. [PubMed]
  • Schackmann RW, Shapiro BM. A partial sequence of ionic changes associated with the acrosome reaction of Strongylocentrotus purpuratus. Dev Biol. 1981 Jan 15;81(1):145–154. [PubMed]
  • Spudich JA, Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed]
  • Tilney LG. Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments. J Cell Biol. 1975 Feb;64(2):289–310. [PMC free article] [PubMed]
  • Tilney LG. The polymerization of actin. III. Aggregates of nonfilamentous actin and its associated proteins: a storage form of actin. J Cell Biol. 1976 Apr;69(1):73–89. [PMC free article] [PubMed]
  • Tilney LG, Inoué S. Acrosomal reaction of Thyone sperm. II. The kinetics and possible mechanism of acrosomal process elongation. J Cell Biol. 1982 Jun;93(3):820–827. [PMC free article] [PubMed]
  • Tilney LG, Kallenbach N. Polymerization of actin. VI. The polarity of the actin filaments in the acrosomal process and how it might be determined. J Cell Biol. 1979 Jun;81(3):608–623. [PMC free article] [PubMed]
  • Tilney LG, Bonder EM, DeRosier DJ. Actin filaments elongate from their membrane-associated ends. J Cell Biol. 1981 Aug;90(2):485–494. [PMC free article] [PubMed]
  • Tilney LG, Kiehart DP, Sardet C, Tilney M. Polymerization of actin. IV. Role of Ca++ and H+ in the assembly of actin and in membrane fusion in the acrosomal reaction of echinoderm sperm. J Cell Biol. 1978 May;77(2):536–550. [PMC free article] [PubMed]
  • Tobacman LS, Korn ED. The regulation of actin polymerization and the inhibition of monomeric actin ATPase activity by Acanthamoeba profilin. J Biol Chem. 1982 Apr 25;257(8):4166–4170. [PubMed]
  • Tseng PC, Pollard TD. Mechanism of action of Acanthamoeba profilin: demonstration of actin species specificity and regulation by micromolar concentrations of MgCl2. J Cell Biol. 1982 Jul;94(1):213–218. [PMC free article] [PubMed]
  • Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed]
  • Woodrum DT, Rich SA, Pollard TD. Evidence for biased bidirectional polymerization of actin filaments using heavy meromyosin prepared by an improved method. J Cell Biol. 1975 Oct;67(1):231–237. [PMC free article] [PubMed]

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