Abstract
In recent years, the myosin superfamily has kept expanding at an explosive rate, but the understanding of their complex functions has been lagging. Therefore,Dictyostelium discoideum, a genetically and biochemically tractable eukaryotic amoeba, appears as a powerful model organism to investigate the involvement of the actomyosin cytoskeleton in a variety of cellular tasks. Because of the relatively high degree of functional redundancy, such studies would be greatly facilitated by the prior knowledge of the whole myosin repertoire in this organism. Here, we present a strategy based on PCR amplification using degenerate primers and followed by negative hybridization screening which led to the potentially exhaustive identification of members of the myosin family inD. discoideum. Two novel myosins were identified and their genetic loci mapped by hybridization to an ordered YAC library. Preliminary inspection ofmyoK andmyoM sequences revealed that, despite carrying most of the hallmarks of myosin motors, both molecules harbor features surprisingly divergent from most known myosins.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Mermall, V., Post, P. L., and Mooseker, M. S. (1998) Unconventional myosins in cell movement, membrane traffic, and signal transduction.Science,279, 527–533.
Probst, F. J., et al. (1998) Correction of deafness inshaker-2 mice by an unconventional myosin in BAC transgene.Science 280, 1444–1447.
Wang, A., et al. (1998) Association of unconventional myosin MYO15 mutations with human nonsyndromic deafness DFNB3.Science 280, 1447–1451.
Sweeney, F. P., Pocklington, M. J., and Orr, E. (1991) The yeast type II myosin heavy chain: analysis of its predicted polypeptide sequence.J. Muscle Res. Cell Motil. 12, 61–68.
Goodson, H. V., Anderson, B. L., Warrick, H. M., Pon, L. A., and Spudich, J. A. (1996) Synthetic lethality screen identifies a novel yeast myosin I gene (MYO5): myosin I proteins are required for polarization of the actin cytoskeleton.J. Cell Biol. 133, 1277–1291.
Govindan, B., Bowser, R., and Novick, P. (1995) The role of Myo2, a yeast class V myosin, in vesicular transport.J. Cell Biol. 128, 1055–1068.
Haarer, B. K., Petzold, A., Lillie, S. H., and Brown, S. S. (1995) Identification of MYO4, a second class V myosin gene in yeast.J. Cell Sci. 107, 1055–1064.
Bähler, M. (1996) Myosins on the move to signal transduction.Current Opin. Cell Biol. 8, 18–22.
Loomis, W. F., and Kuspa, A. (1997) The genome ofDictyostelium discoideum, in: Dictyostelium—A Model System for Cell and Developmental Biology (Maeda, Y., Inouye, K. and Takeuchi, I., eds.), Universal Academy Press, Tokyo, Japan, pp. 15–30.
Smith, D. W. and Loomis, W. F. (1997) Dicty DB—A genomic database forDictyostelium discoideum, in: Dictyostelium—A Model System for Cell and Developmental Biology. (Maeda, Y., Inouye, K. and Takeuchi, I., eds.), Universal Academy Press, Tokyo, Japan, pp. 471–477.
Lee, S. F. and Cote, G. P. (1993) Isolation and characterization of threeDictyostelium Myosin-I isozymes.J. Biol. Chem. 268, 20,923–20,929.
Jung, G. and Hammer III, J. A. (1990) Generation and characterization ofDictyostelium cells deficient in a myosin I heavy chain isoform.J. Cell Biol. 110, 1955–1964.
Wessels, D., Murray, J., Jung, G., Hammer III, J. A., and Soll, D. R. (1991) Myosin IB null mutants ofDictyostelium exhibit abnormalities in motility.Cell Motil. Cytoskel. 20, 301–315.
Peterson, M. D., Novak, K. D., Reedy, M. C., Ruman, J. I., and Titus, M. A. (1995) Molecular genetic analysis of myoC, aDictyostelium myosin I.J. Cell Sci. 108, 1093–1103.
Novak, K. D., Peterson, M. D., Reedy, M. C., and Titus, M. A. (1995)Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis.J. Cell Biol. 131, 1205–1221.
Temesvari, L. A., Bush, J. M., Peterson, M. D., Novak, K. D., Titus, M. A., and Cardelli, J. A. (1996) Examination of the endosomal and lysosomal pathways inDictyostelium discoideum myosin I mutants.J. Cell Sci. 109, 663–673.
Jung, G., Wu, X. F., and Hammer III, J. A. (1996)Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions.J. Cell Biol. 133, 305–323.
Titus, M. A. (1997) An unconventional myosin required for phagocytosis.Mol. Biol. Cell 8, 352a.
Uyeda, T. Q. and Titus, M. A. (1997) The myosins ofDictyostelium, in: Dictyosterlium-A Model System for Cell and Developmental Biology (Maeda, Y., Inouye, K. and Takeuchi, I., eds.), Universal Academy Press, Tokyo, Japan, pp. 43–64.
el-Amraoui, A., Sahly, I., Picaud, S., Sahel, J., Abitbol, M., and Petit, C. (1996) Human Usher 1B/mouse shaker-1: the retinal phenotype discrepancy explained by the presence/absence of myosin VIIA in the photoreceptor cells.Hum. Mol. Gen. 5, 1171–1178.
Liu, X., Vansant, G., Udovichenko, I. P., Wolfrum, U., and Williams, D. S. (1997) Myosin Vlla, the product of the Usher 1B syndrome gene, is concentrated in the connecting cilia of photoreceptor cells.Cell Motil. Cytosk. 37, 240–252.
Titus, M. A., Kuspa, A., and Loomis, W. F. (1994) Discovery of myosin genes by physical mapping inDictyostelium.Proc. Natl. Acad. Sci. USA 91, 9446–9450.
Hammer III, J. A. and Jung, G. (1996) The sequence of theDictyostelium myo J heavy chain gene predicts a novel, dimeric, unconventional myosin with a heavy chain molecular mass of 258 kDa.J. Biol. Chem. 271, 7120–7127.
Sussman, M. (1987) Cultivation and synchronous morphogenesis ofDictyostelium under controlled experimental conditions.Meth. Cell Biol. 28, 9–29.
Manstein, D. J., Titus, M. A., De Lozanne, A. and Spudich, J. A. (1989) Gene replacement inDictyostelium: generation of myosin null mutants.EMBO J. 8, 923–932.
Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 162, 156–159.
Nellen, W., Datta, S., Reymond, C., Sivertsen, A., Mann, S., Crowley, T. and Firtel, R. A. (1987) Molecular biology inDictyostelium: tools and applications.Meth. Cell Biol. 28, 67–100.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1997) Preparation and analysis of RNA, in:Current Protocols in Molecular Biology,1:4.2.2.
Greener, A. (1990) E. coli SURETM Strain: Clone “unclonable” DNA.Strategies 3, 5–6.
Bullock, W. O., Fernandez, J. M., and Short, J. M. (1987) XL1-blue: A High Efficiency Plasmid Transforming Reca ES.Biotechniques 5, 376–379.
Jerpseth, B., Greener, A., Short, J. M., Viola, J., and Kretz, P.L. (1992) XL1-blue MRF′E. coli Cells: McrA−, McrCB−, Mrr−, HsdR− derivative of XL1-Blue cells.Strategies 5, 81–83.
Sanger, F., Nicklen, S., and Coulson A. R. (1977) DNA sequencing with chain-terminating inhibitors.Proc. Natl. Acad. Sci. USA 74, 5463–5467.
Warrick, H. M. (1987) Codon frequency inDictyostelium discoideum.Meth. Cell Biol. 28, 497–500.
Bement, W. M. and Mooseker, M. S. (1995) TEDS rule: a molecular rationale for differential regulation of myosins by phosphorylation of the heavy chain head.Cell Motil. Cytosk. 31,87–92.
Albanesi, J. P., Hammer III, J. A., and Korn, E. D. (1983) The interaction of F-actin with phosphorylated and unphosphorylated myosins IA and IB fromAcanthamoeba castellanii.J. Biol. Chem. 258, 10,176–10,181.
Albanesi, J. P., Fujisaki, H., Hammer III, J. A., Korn, E. D., Jones, R., and Sheetz, M. P. (1985) MonomericAcanthamoeba myosins I support movement in vitro.J. Biol. Chem. 260, 8649–8652.
Cote, G. P., Albanesi, J. P., Ueno, T., Hammer III, J. A., and Korn, E. D. (1985) Purification fromDictyostelium discoideum of a low-molecular weight myosin that resembles myosin I fromAcanthamoeba castellanii.J. Biol. Chem. 260, 4543–4546.
Lee, S. F., Egelhoff, T. T., Mahasneh, A., and Cote, G. P. (1996) Cloning and characterization of aDictyostelium myosin I heavy chain kinase activated by Cdc42 and Rac.J. Biol. Chem. 271, 27,044–27,048.
Brzeska, H., Lynch, T. J., Martin, B., and Korn, E. D. (1989) The localization and sequence of the phosphorylation sites ofAcanthamoeba myosins I. An improved method for locating the phosphorylated amino acid.J. Biol. Chem. 264, 19,340–19,348.
Brzeska, H., Lynch, T. J., Martin, B., Corigliano-Murphy, A., and Korn, E. D. (1990) Substrate specificity ofAcanthamoeba myosin I heavy chain kinase as determined with synthetic peptides.J. Biol. Chem. 265, 16,138–16,144.
Wu, C., Lee, S. F., Furmaniak-Kazmierczak, E., Cote, G. P., Thomas, D. Y., and Leberer, E. (1996) Activation of myosin-I by members of the Ste20p protein kinase family.J. Biol. Chem. 271, 31,787–31,790.
Su, X. Z., Wu, Y., Sifri, C. D., and Wellems, T. E. (1996) Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA.Nucl. Acids Res. 24, 1574–1575.
Bement, W. M., Hasson, T., Wirth, J. A., Cheney, R. E., and Mooseker, M. S. (1994) Identification and overlapping expression of multiple unconventional myosin genes in vertebrate cell types.Proc. Natl. Acad. Sci. USA 91, 6549–6553.
Baker, J. P. and Titus, M. A. (1997) A family of unconventional myosins from the nematodeCaenorhabditis elegans.J. Mol. Biol. 272, 523–535.
Dobrowolski, J. M. and Sibley, L. D. (1996)Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite.Cell 84, 933–939.
Pinder, J. C., Fowler, R. E., Dluzewski, A. R., Bannister, L. H., Lavin, F. M., Mitchell, G. H., Wilson, R. J. M., and Gratzer, W. B. (1998) Actomyosin motor in the merozoite of the malaria parasite,Plasmodium falciparum: implication for red cell invasion.J. Cell Sci. 111, 1831–1839.
Titus, M. A., Wessels, D., Spudich, J. A., and Soll, D. (1993) The unconventional myosin encoded by the myoA gene plays a role inDictyostelium motility.Mol. Biol. Cell 4, 233–246.
Jung, G., Saxe III, C. L., Kimmel, A. R., and Hammer III, J. A. (1989)Dictyostelium discoideum contains a gene encoding a myosin I heavy chain.Proc. Natl. Acad. Sci. USA 86, 6186–6190.
Titus, M. A., Warrick, H. M., and Spudich, J. A. (1989) Multiple actin based motor genes inDictyostelium.Cell Regulation 1, 55–63.
Urrutia, R. A., Jung, G., and Hammer III, J. A. (1993) TheDictyostelium myosin-IE heavy chain gene encodes a truncated isoform that lacks sequences corresponding to the actin binding site in the tail.Biochim. Biophys. Acta 1173, 225–229.
Titus, M. A., Novak, K. D., Hanes, G. P., and Urioste, A. S. (1995) Molecular genetic analysis of myoF, a newDictyostelium myosin I gene.Biophys. J. 68, S152-S157.
Clarke, M. and Spudich, J. (1974) Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. Isolation and characterization of myosin from amoebae ofDictyostelium discoideum.J. Mol. Biol. 86, 209–222.
Warrick, H. M., De Lozanne, A., Leinwand, L. A., and Spudich, J. A. (1986) Conserved protein domains in a myosin heavy chain gene fromDictyostelium discoideum.Proc. Natl. Acad. Sci. USA 83, 9433–9437.
Peterson, M. D., Urioste, A. S., and Titus, M. A. (1996)Dictyostelium discoideum myoJ: A member of a broadly defined myosin V class or a class XI unconventional myosin?.J. Muscle Res. Cell Motil. 17, 411–424.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schwarz, E.C., Geissler, H. & Soldati, T. A potentially exhaustive screening strategy reveals two novel divergent myosins inDictyostelium . Cell Biochem Biophys 30, 413–435 (1999). https://doi.org/10.1007/BF02738122
Issue Date:
DOI: https://doi.org/10.1007/BF02738122