Malaria Parasite Actin Polymerization and Filament Structure*

A novel form of acto-myosin regulation has been proposed in which polymerization of new actin filaments regulates motility of parasites of the apicomplexan class of protozoa. In vivo and in vitro parasite F-actin is very short and unstable, but the structural basis and details of filament dynamics remain unknown. Here, we show that long actin filaments can be obtained by polymerizing unlabeled rabbit skeletal actin (RS-actin) onto both ends of the short rhodamine-phalloidin-stabilized Plasmodium falciparum actin I (Pf-actin) filaments. Following annealing, hybrid filaments of micron length and “zebra-striped” appearance are observed by fluorescence microscopy that are stable enough to move over myosin class II motors in a gliding filament assay. Using negative stain electron microscopy we find that pure Pf-actin stabilized by jasplakinolide (JAS) also forms long filaments, indistinguishable in length from RS-actin filaments, and long enough to be characterized structurally. To compare structures in near physiological conditions in aqueous solution we imaged Pf-actin and RS-actin filaments by atomic force microscopy (AFM). We found the monomer stacking to be distinctly different for Pf-actin compared with RS-actin, such that the pitch of the double helix of Pf-actin filaments was 10% larger. Our results can be explained by a rotational angle between subunits that is larger in the parasite compared with RS-actin. Modeling of the AFM data using high-resolution actin filament models supports our interpretation of the data. The structural differences reported here may be a consequence of weaker inter- and intra-strand contacts, and may be critical for differences in filament dynamics and for regulation of parasite motility.

[1]  K. Matuschewski,et al.  A Plasmodium actin-depolymerizing factor that binds exclusively to actin monomers. , 2005, Molecular Biology of the Cell.

[2]  T. Pollard,et al.  Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments. , 1991, Biochemistry.

[3]  R. Fowler,et al.  The cytoskeleton and motility in apicomplexan invasion. , 2004, Advances in parasitology.

[4]  B. Somogyi,et al.  A simple model for the cooperative stabilisation of actin filaments by phalloidin and jasplakinolide , 2005, FEBS letters.

[5]  Christopher J. Tonkin,et al.  A malaria parasite formin regulates actin polymerization and localizes to the parasite-erythrocyte moving junction during invasion. , 2008, Cell host & microbe.

[6]  M. Bartoo,et al.  The stiffness of rabbit skeletal actomyosin cross-bridges determined with an optical tweezers transducer. , 1998, Biophysical journal.

[7]  M. Smits,et al.  Extremely diverged actin proteins in Plasmodium falciparum. , 1988, Molecular and biochemical parasitology.

[8]  Plasmodium motility: actin not actin' like actin. , 2006, Trends in parasitology.

[9]  S. Kano,et al.  Effect of jasplakinolide on the growth, invasion, and actin cytoskeleton of Plasmodium falciparum , 2002, Parasitology Research.

[10]  H. Schüler ATPase activity and conformational changes in the regulation of actin. , 2001, Biochimica et biophysica acta.

[11]  E. Egelman,et al.  A change in actin conformation associated with filament instability after Pi release. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[12]  L. Sibley,et al.  Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii , 1997, Molecular microbiology.

[13]  I. Coppens,et al.  Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites , 2003, Journal of Cell Science.

[14]  T D Pollard,et al.  Nucleotide exchange, structure, and mechanical properties of filaments assembled from ATP-actin and ADP-actin. , 1992, The Journal of biological chemistry.

[15]  H. Faulstich,et al.  Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms. , 1978, CRC critical reviews in biochemistry.

[16]  A. Knight,et al.  Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy. , 2003, Methods.

[17]  Hemantha K. Wickramasinghe,et al.  Atomic force microscope–force mapping and profiling on a sub 100‐Å scale , 1987 .

[18]  C. Schmidt,et al.  Resolving the molecular structure of microtubules under physiological conditions with scanning force microscopy , 2004, European Biophysics Journal.

[19]  J. Gómez‐Herrero,et al.  WSXM: a software for scanning probe microscopy and a tool for nanotechnology. , 2007, The Review of scientific instruments.

[20]  R. Cooke The role of the bound nucleotide in the polymerization of actin. , 1975, Biochemistry.

[21]  Edward H. Egelman,et al.  A New Internal Mode in F-Actin Helps Explain the Remarkable Evolutionary Conservation of Actin's Sequence and Structure , 2002, Current Biology.

[22]  D. DeRosier,et al.  The variable twist of actin and its modulation by actin-binding proteins , 1987, The Journal of cell biology.

[23]  D. DeRosier,et al.  F-actin is a helix with a random variable twist , 1982, Nature.

[24]  L. Sibley,et al.  Toxoplasma Invasion of Mammalian Cells Is Powered by the Actin Cytoskeleton of the Parasite , 1996, Cell.

[25]  David Baker,et al.  Prediction of the structure of symmetrical protein assemblies , 2007, Proceedings of the National Academy of Sciences.

[26]  T. Pollard Polymerization of ADP-actin , 1984, The Journal of cell biology.

[27]  K C Holmes,et al.  Refinement of the F-actin model against X-ray fiber diffraction data by the use of a directed mutation algorithm. , 1993, Journal of molecular biology.

[28]  A Leith,et al.  SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. , 1996, Journal of structural biology.

[29]  L. Sibley,et al.  Comparative genome analysis reveals a conserved family of actin-like proteins in apicomplexan parasites , 2005, BMC Genomics.

[30]  M. Carlier,et al.  Probing the mechanism of ATP hydrolysis on F-actin using vanadate and the structural analogs of phosphate BeF-3 and A1F-4. , 1988, The Journal of biological chemistry.

[31]  S. Howell,et al.  Malaria parasite actin filaments are very short. , 2005, Journal of molecular biology.

[32]  P Dancker,et al.  Phalloidin reduces the release of inorganic phosphate during actin polymerization. , 1990, Biochimica et biophysica acta.

[33]  W. Chiu,et al.  Structure of the acrosomal bundle , 2004, Nature.

[34]  S. Lowey,et al.  [7] Preparation of myosin and its subfragments from rabbit skeletal muscle , 1982 .

[35]  W Chiu,et al.  EMAN: semiautomated software for high-resolution single-particle reconstructions. , 1999, Journal of structural biology.

[36]  D. Weibel,et al.  Reconstitution of DNA Segregation Driven by Assembly of a Prokaryotic Actin Homolog , 2007, Science.

[37]  Ikuko Fujiwara,et al.  Polymerization kinetics of ADP- and ADP-Pi-actin determined by fluorescence microscopy , 2007, Proceedings of the National Academy of Sciences.

[38]  Edward H. Egelman,et al.  Actin Structure and Function: What We Still Do Not Understand* , 2007, Journal of Biological Chemistry.

[39]  L. Sibley,et al.  Actin in the parasite Toxoplasma gondii is encoded by a single copy gene, ACT1 and exists primarily in a globular form. , 1997, Cell motility and the cytoskeleton.

[40]  T. Pollard,et al.  Kinetics and thermodynamics of phalloidin binding to actin filaments from three divergent species. , 1996, Biochemistry.

[41]  T. Speed,et al.  Regulation of apicomplexan actin-based motility , 2006, Nature Reviews Microbiology.

[42]  J. Hanson Axial Period of Actin Filaments , 1967, Nature.

[43]  K. Namba,et al.  Distinct structural changes detected by X-ray fiber diffraction in stabilization of F-actin by lowering pH and increasing ionic strength. , 2001, Biophysical journal.

[44]  E. Sausville,et al.  Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin. , 1994, The Journal of biological chemistry.

[45]  J. Spudich,et al.  Fluorescent actin filaments move on myosin fixed to a glass surface. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[46]  D. Sept,et al.  Unusual kinetic and structural properties control rapid assembly and turnover of actin in the parasite Toxoplasma gondii. , 2005, Molecular biology of the cell.

[47]  L. Sibley,et al.  Actin filament polymerization regulates gliding motility by apicomplexan parasites. , 2003, Molecular biology of the cell.

[48]  Wah Chiu,et al.  Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function , 1997, The Journal of cell biology.

[49]  K. Matuschewski,et al.  Unusual properties of Plasmodium falciparum actin: new insights into microfilament dynamics of apicomplexan parasites , 2005, FEBS letters.

[50]  O. Poupel,et al.  Toxoplasma gondii motility and host cell invasiveness are drastically impaired by jasplakinolide, a cyclic peptide stabilizing F-actin. , 1999, Microbes and infection.

[51]  Patricia De la Vega,et al.  Discovery of Gene Function by Expression Profiling of the Malaria Parasite Life Cycle , 2003, Science.

[52]  J. Villarrubia Algorithms for Scanned Probe Microscope Image Simulation, Surface Reconstruction, and Tip Estimation , 1997, Journal of research of the National Institute of Standards and Technology.

[53]  Carl Frieden,et al.  Effect of pH on the mechanism of actin polymerization. , 1988, Biochemistry.

[54]  H. Boleti,et al.  Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments. , 2000, Molecular biology of the cell.

[55]  M. Smits,et al.  Nucleotide sequence and deduced amino acid sequence of a Plasmodium falciparum actin gene. , 1988, Molecular and biochemical parasitology.

[56]  J. D. Pardee,et al.  [18] Purification of muscle actin , 1982 .

[57]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[58]  K. Matuschewski,et al.  Regulation of Apicomplexan Microfilament Dynamics by a Minimal Set of Actin‐Binding Proteins , 2006, Traffic.

[59]  G. Mashanov,et al.  Automatic detection of single fluorophores in live cells. , 2007, Biophysical journal.

[60]  L. G. Tilney,et al.  Induction of an acrosomal process in Toxoplasma gondii: visualization of actin filaments in a protozoan parasite. , 1999, Proceedings of the National Academy of Sciences of the United States of America.