Abstract
Whereas it has been demonstrated that muscle and nonmuscle isoactins are segregated into distinct cytoplasmic domains, the mechanism regulating subcellular sorting is unknown (Herman, 1993a). To reveal whether isoform-specific actin-binding proteins function to coordinate these events, cell extracts derived from motile (Em) versus stationary (Es) cytoplasm were selectively and sequentially fractionated over filamentous isoactin affinity columns prior to elution with a KCl step gradient. A polypeptide of interest, which binds specifically to beta- actin filament columns, but not to muscle actin columns has been conclusively identified as the ERM family member, ezrin. We studied ezrin-beta interactions in vitro by passing extracts (Em) over isoactin affinity matrices in the presence of Ca(2+)-containing versus Ca(2+)- free buffers, with or without cytochalasin D. Ezrin binds and can be released from beta-actin Sepharose-4B in the presence of Mg2+/EGTA and 100 mM NaCl (at 4 degrees C and room temperature), but not when affinity fractionation of Em is carried out in the presence of 0.2 mM CaCl2 or 2 microM cytochalasin D. N-acetyl-(leucyl)2-norleucinal and E64, two specific inhibitors of the calcium-activated protease, calpain I, protect ezrin binding to beta actin in the presence of calcium. Moreover, biochemical analysis of endothelial lysates reveals that a calpain I cleavage product of ezrin emerges when cell locomotion is stimulated in response to monolayer injury. Immunofluorescence analysis of leading lamellae reveals that anti-ezrin and anti-beta-actin IgGs can be simultaneously co-localized, extending the results of isoactin affinity fractionation of Em-derived extracts and suggesting that ezrin and beta-actin interact in vivo. To test the hypothesis that ezrin binds directly to beta-actin, we performed three sets of studies under a wide range of physiological conditions (pH 7.0-8.5) using purified pericyte ezrin and either alpha- or beta-actin. These included co- sedimentation, isoactin affinity fractionation, and co- immunoprecipitation. Results of these experiments reveal that purified ezrin does not directly bind to beta-actin filaments, either in solution or while isoactins are covalently cross-linked to Sepharose- 4B. This is in contrast to our finding that ezrin and beta-actin could be co-immunoprecipitated or co-sedimented from Em-derived cell lysates. To explore whether calcium transients occur in cellular domains enriched in ezrin and beta-actin, we mapped cellular free calcium in endothelial monolayers crawling in response to injury.(ABSTRACT TRUNCATED AT 400 WORDS)
Full Text
The Full Text of this article is available as a PDF (2.6 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Algrain M., Turunen O., Vaheri A., Louvard D., Arpin M. Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker. J Cell Biol. 1993 Jan;120(1):129–139. doi: 10.1083/jcb.120.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Askey D. B., Herman I. M. Computer-assisted analysis of the vascular endothelial cell motile response to injury. Comput Biomed Res. 1988 Dec;21(6):551–561. doi: 10.1016/0010-4809(88)90011-0. [DOI] [PubMed] [Google Scholar]
- Barrett A. J., Kembhavi A. A., Brown M. A., Kirschke H., Knight C. G., Tamai M., Hanada K. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J. 1982 Jan 1;201(1):189–198. doi: 10.1042/bj2010189. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bennett V. The membrane skeleton of human erythrocytes and its implications for more complex cells. Annu Rev Biochem. 1985;54:273–304. doi: 10.1146/annurev.bi.54.070185.001421. [DOI] [PubMed] [Google Scholar]
- Birgbauer E., Solomon F. A marginal band-associated protein has properties of both microtubule- and microfilament-associated proteins. J Cell Biol. 1989 Oct;109(4 Pt 1):1609–1620. doi: 10.1083/jcb.109.4.1609. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bretscher A. Microfilament structure and function in the cortical cytoskeleton. Annu Rev Cell Biol. 1991;7:337–374. doi: 10.1146/annurev.cb.07.110191.002005. [DOI] [PubMed] [Google Scholar]
- Bretscher A. Purification of the intestinal microvillus cytoskeletal proteins villin, fimbrin, and ezrin. Methods Enzymol. 1986;134:24–37. doi: 10.1016/0076-6879(86)34072-2. [DOI] [PubMed] [Google Scholar]
- Bretscher A. Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor. J Cell Biol. 1989 Mar;108(3):921–930. doi: 10.1083/jcb.108.3.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brundage R. A., Fogarty K. E., Tuft R. A., Fay F. S. Calcium gradients underlying polarization and chemotaxis of eosinophils. Science. 1991 Nov 1;254(5032):703–706. doi: 10.1126/science.1948048. [DOI] [PubMed] [Google Scholar]
- DeNofrio D., Hoock T. C., Herman I. M. Functional sorting of actin isoforms in microvascular pericytes. J Cell Biol. 1989 Jul;109(1):191–202. doi: 10.1083/jcb.109.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fox J. E., Reynolds C. C., Morrow J. S., Phillips D. R. Spectrin is associated with membrane-bound actin filaments in platelets and is hydrolyzed by the Ca2+-dependent protease during platelet activation. Blood. 1987 Feb;69(2):537–545. [PubMed] [Google Scholar]
- Gough A. H., Taylor D. L. Fluorescence anisotropy imaging microscopy maps calmodulin binding during cellular contraction and locomotion. J Cell Biol. 1993 Jun;121(5):1095–1107. doi: 10.1083/jcb.121.5.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gould K. L., Bretscher A., Esch F. S., Hunter T. cDNA cloning and sequencing of the protein-tyrosine kinase substrate, ezrin, reveals homology to band 4.1. EMBO J. 1989 Dec 20;8(13):4133–4142. doi: 10.1002/j.1460-2075.1989.tb08598.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hahn K., DeBiasio R., Taylor D. L. Patterns of elevated free calcium and calmodulin activation in living cells. Nature. 1992 Oct 22;359(6397):736–738. doi: 10.1038/359736a0. [DOI] [PubMed] [Google Scholar]
- Hayashi M., Inomata M., Saito Y., Ito H., Kawashima S. Activation of intracellular calcium-activated neutral proteinase in erythrocytes and its inhibition by exogenously added inhibitors. Biochim Biophys Acta. 1991 Sep 24;1094(3):249–256. doi: 10.1016/0167-4889(91)90083-a. [DOI] [PubMed] [Google Scholar]
- Herman I. M. Actin isoforms. Curr Opin Cell Biol. 1993 Feb;5(1):48–55. doi: 10.1016/s0955-0674(05)80007-9. [DOI] [PubMed] [Google Scholar]
- Herman I. M., D'Amore P. A. Microvascular pericytes contain muscle and nonmuscle actins. J Cell Biol. 1985 Jul;101(1):43–52. doi: 10.1083/jcb.101.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Herman I. M., Pollard T. D. Comparison of purified anti-actin and fluorescent-heavy meromyosin staining patterns in dividing cells. J Cell Biol. 1979 Mar;80(3):509–520. doi: 10.1083/jcb.80.3.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hill M. A., Gunning P. Beta and gamma actin mRNAs are differentially located within myoblasts. J Cell Biol. 1993 Aug;122(4):825–832. doi: 10.1083/jcb.122.4.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hill M. A., Schedlich L., Gunning P. Serum-induced signal transduction determines the peripheral location of beta-actin mRNA within the cell. J Cell Biol. 1994 Sep;126(5):1221–1229. doi: 10.1083/jcb.126.5.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoock T. C., Newcomb P. M., Herman I. M. Beta actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury. J Cell Biol. 1991 Feb;112(4):653–664. doi: 10.1083/jcb.112.4.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kislauskis E. H., Li Z., Singer R. H., Taneja K. L. Isoform-specific 3'-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. J Cell Biol. 1993 Oct;123(1):165–172. doi: 10.1083/jcb.123.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kislauskis E. H., Zhu X., Singer R. H. Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype. J Cell Biol. 1994 Oct;127(2):441–451. doi: 10.1083/jcb.127.2.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
- Leavitt J., Bushar G., Kakunaga T., Hamada H., Hirakawa T., Goldman D., Merril C. Variations in expression of mutant beta actin accompanying incremental increases in human fibroblast tumorigenicity. Cell. 1982 Feb;28(2):259–268. doi: 10.1016/0092-8674(82)90344-0. [DOI] [PubMed] [Google Scholar]
- Leavitt J., Ng S. Y., Aebi U., Varma M., Latter G., Burbeck S., Kedes L., Gunning P. Expression of transfected mutant beta-actin genes: alterations of cell morphology and evidence for autoregulation in actin pools. Mol Cell Biol. 1987 Jul;7(7):2457–2466. doi: 10.1128/mcb.7.7.2457. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lenk R., Ransom L., Kaufmann Y., Penman S. A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. Cell. 1977 Jan;10(1):67–78. doi: 10.1016/0092-8674(77)90141-6. [DOI] [PubMed] [Google Scholar]
- Lewis A. K., Bridgman P. C. Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity. J Cell Biol. 1992 Dec;119(5):1219–1243. doi: 10.1083/jcb.119.5.1219. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lubit B. W., Schwartz J. H. An antiactin antibody that distinguishes between cytoplasmic and skeletal muscle actins. J Cell Biol. 1980 Sep;86(3):891–897. doi: 10.1083/jcb.86.3.891. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Luna E. J., Hitt A. L. Cytoskeleton--plasma membrane interactions. Science. 1992 Nov 6;258(5084):955–964. doi: 10.1126/science.1439807. [DOI] [PubMed] [Google Scholar]
- Ohmori H., Toyama S., Toyama S. Direct proof that the primary site of action of cytochalasin on cell motility processes is actin. J Cell Biol. 1992 Feb;116(4):933–941. doi: 10.1083/jcb.116.4.933. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Otey C. A., Kalnoski M. H., Bulinski J. C. Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle. Cell Motil Cytoskeleton. 1988;9(4):337–348. doi: 10.1002/cm.970090406. [DOI] [PubMed] [Google Scholar]
- Pinder J. C., Gratzer W. B. Structural and dynamic states of actin in the erythrocyte. J Cell Biol. 1983 Mar;96(3):768–775. doi: 10.1083/jcb.96.3.768. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Puszkin S., Maimon J., Puszkin E. Erythrocyte actin and spectrin. Interactions with muscle contractile and regulatory proteins. Biochim Biophys Acta. 1978 Nov 2;513(2):205–220. doi: 10.1016/0005-2736(78)90174-8. [DOI] [PubMed] [Google Scholar]
- Rubenstein P. A. Differential behavior of gizzard isoactins. Arch Biochem Biophys. 1981 Sep;210(2):598–608. doi: 10.1016/0003-9861(81)90226-5. [DOI] [PubMed] [Google Scholar]
- Rubenstein P. A. The functional importance of multiple actin isoforms. Bioessays. 1990 Jul;12(7):309–315. doi: 10.1002/bies.950120702. [DOI] [PubMed] [Google Scholar]
- Saido T. C., Nagao S., Shiramine M., Tsukaguchi M., Sorimachi H., Murofushi H., Tsuchiya T., Ito H., Suzuki K. Autolytic transition of mu-calpain upon activation as resolved by antibodies distinguishing between the pre- and post-autolysis forms. J Biochem. 1992 Jan;111(1):81–86. doi: 10.1093/oxfordjournals.jbchem.a123723. [DOI] [PubMed] [Google Scholar]
- Saido T. C., Suzuki H., Yamazaki H., Tanoue K., Suzuki K. In situ capture of mu-calpain activation in platelets. J Biol Chem. 1993 Apr 5;268(10):7422–7426. [PubMed] [Google Scholar]
- Schevzov G., Lloyd C., Gunning P. High level expression of transfected beta- and gamma-actin genes differentially impacts on myoblast cytoarchitecture. J Cell Biol. 1992 May;117(4):775–785. doi: 10.1083/jcb.117.4.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spudich J. A., 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] [Google Scholar]
- Sundell C. L., Singer R. H. Actin mRNA localizes in the absence of protein synthesis. J Cell Biol. 1990 Dec;111(6 Pt 1):2397–2403. doi: 10.1083/jcb.111.6.2397. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sundell C. L., Singer R. H. Requirement of microfilaments in sorting of actin messenger RNA. Science. 1991 Sep 13;253(5025):1275–1277. doi: 10.1126/science.1891715. [DOI] [PubMed] [Google Scholar]
- Symons M. H., Mitchison T. J. Control of actin polymerization in live and permeabilized fibroblasts. J Cell Biol. 1991 Aug;114(3):503–513. doi: 10.1083/jcb.114.3.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsujinaka T., Kajiwara Y., Kambayashi J., Sakon M., Higuchi N., Tanaka T., Mori T. Synthesis of a new cell penetrating calpain inhibitor (calpeptin). Biochem Biophys Res Commun. 1988 Jun 30;153(3):1201–1208. doi: 10.1016/s0006-291x(88)81355-x. [DOI] [PubMed] [Google Scholar]
- Tsukita S., Itoh M., Nagafuchi A., Yonemura S., Tsukita S. Submembranous junctional plaque proteins include potential tumor suppressor molecules. J Cell Biol. 1993 Dec;123(5):1049–1053. doi: 10.1083/jcb.123.5.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Turunen O., Wahlström T., Vaheri A. Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family. J Cell Biol. 1994 Sep;126(6):1445–1453. doi: 10.1083/jcb.126.6.1445. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wuestehube L. J., Luna E. J. F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes. J Cell Biol. 1987 Oct;105(4):1741–1751. doi: 10.1083/jcb.105.4.1741. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yao X., Thibodeau A., Forte J. G. Ezrin-calpain I interactions in gastric parietal cells. Am J Physiol. 1993 Jul;265(1 Pt 1):C36–C46. doi: 10.1152/ajpcell.1993.265.1.C36. [DOI] [PubMed] [Google Scholar]
- Yost J. C., Herman I. M. Substratum-induced stress fiber assembly in vascular endothelial cells during spreading in vitro. J Cell Sci. 1990 Mar;95(Pt 3):507–520. doi: 10.1242/jcs.95.3.507. [DOI] [PubMed] [Google Scholar]