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Evidence for filamentous actin in ookinetes of a malarial parasite
Molecular & Biochemical Parasitology 181 (2012) 186–189
Contents lists available at SciVerse ScienceDirect
Molecular & Biochemical Parasitology
Short communication
Evidence for filamentous actin in ookinetes of a malarial parasite Inga Siden-Kiamos a,∗ , Christos Louis a,b , Kai Matuschewski c a
Institute of Molecular Biology and Biotechnology, FORTH, N. Plastira 100, Vassilika Vouton, Heraklion 700 13, Crete, Greece Department of Biology, University of Crete, Heraklion, Greece c Parasitology Unit, Max Planck Institute for Infection Biology, Berlin, Germany b
a r t i c l e
i n f o
Article history: Received 21 September 2011 Received in revised form 13 October 2011 Accepted 2 November 2011 Available online 11 November 2011 Keywords: Malaria Ookinete Actin Motility Cytoskeleton
a b s t r a c t Extracellular stages of apicomplexan parasites utilize their own actin myosin motor machinery for gliding locomotion, penetration of cell barriers, and host cell invasion. Thus far, filamentous actin could not be visualized by standard microscopic techniques in vivo. Here, we describe the generation of a novel peptide antibody against the divergent amino-terminal portion of the major Plasmodium isoform, actin I. We show that our antiserum, termed Ab-actinI-I, is conformation-specific. In motile ookinetes it recognizes actin in rod-like structures, which are sensitive to inhibitors interfering with actin polymerization. The average size of the rods is 600 nm, which is considerably longer than what has been detected in in vitro studies of actin filaments. © 2011 Elsevier B.V. All rights reserved.
Apicomplexan parasites use an actin–myosin based motor machinery for their motility [1,2]. Although they share many features with actin–myosin based motility in higher organisms there are also substantial differences. Here, we concentrate on the ability of the major isoform of actin in Plasmodium parasites to form filaments. In most well-studied eukaryotic organisms formation of filamentous actin (F-actin) is regulated by around 150 different proteins [3], while in Apicomplexa, there are substantially fewer regulators identified [4,5]. Importantly, in vitro studies have revealed that actin from Plasmodium or Toxoplasma forms considerably shorter and more fragile filaments than actin from yeast or vertebrates [6–10]. This is also reflected by the fact that, so far, actin filaments have not been readily detected in these parasites [11,12]. Analysis of a Toxoplasma gondii mutant, in which ADF (actin depolymerizing factor) was conditionally down-regulated, revealed actin staining in a pattern consistent with filaments [13] and this pattern was also detected in jasplakinolide treated T. gondii tachyzoites expressing mutated forms of actins [10]. We are interested in understanding the role of actin in parasite motility, and for this we systematically tested antibodies recognizing actin, in order to identify one that would specifically recognize Plasmodium berghei actin I, the major actin isoform (PBANKA 145930; gi: 74991781), but not actin from animals or
Abbreviations: ADF, actin depolymerizing factor; G-actin, globular actin; F-actin, filamentous actin; IMC, inner membrane complex; ABF, actin-binding proteins. ∗ Corresponding author. Tel.: +30 2810 391118; fax: +30 2810 391104. E-mail address: [email protected] (I. Siden-Kiamos). 0166-6851/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2011.11.002
yeast. Both the antiserum (anti-PfACT1) against a specific peptide of Plasmodium falciparum actin I (peptide DEEMKTSEQSSDI, residues 224–236 in subdomain 4, conserved in Plasmodium berghei) [14] and the monoclonal antibody mAb 224–236–1, originally developed against Dictyostelium actin [15], detected the presence of actin in a well-defined region in the apical tip of the parasite, as previously described [14]. Furthermore, a similar localization was seen in transgenic parasites, expressing as a second copy YFP-tagged actin I fusion protein under the control of the circumsporozoite and TRAP-related protein (CTRP) promoter, which is active in ookinetes but not in other stages (manuscript in preparation) (Fig. 1A). Fluorescently tagged actin may interfere with the normal function of endogenous actin; although not well understood, this effect may be dependent on the ratio of non-modified to modified form (Deligianni and Siden-Kiamos, unpublished [16]). We, therefore, hypothesize that the observed accumulation of YFPactin I might reflect the fusion protein in the globular (G−) form. Clearly, we did not detect YFP-actin in a pattern consistent with F− actin. In an attempt to further characterize actin I in P. berghei we developed an antiserum against a synthetic peptide from actin I, corresponding to amino acids 16–30 in subdomain 1, named AbactinI-I; this peptide is sufficiently divergent from the Plasmodium isoform actin II [17] (Fig. 1B). The antibody specifically recognized recombinant P. berghei actin I expressed in Escherichia coli on Western blots (data not shown). Surprisingly, when we used this antibody to detect actin in ookinetes of P. berghei, we detected the protein in short rods, while staining at the apical tip was absent or less pronounced (Fig. 1C). Although the pattern of staining was
I. Siden-Kiamos et al. / Molecular & Biochemical Parasitology 181 (2012) 186–189
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Fig. 1. Rod-like structures in the cell periphery are detected in ookinetes using the Ab-actinI-I antibody. (A) YFP-actin is detected only at the apical tip. (a) Live imaging of transgenic ookinetes expressing an YFP-actin I fusion protein (b) Same ookinete surface labeled with the 13.1 antibody recognizing the Pbs21 antigen. Scale bar 5 m. (B) Sequence alignment of the amino-terminal region of P. berghei actin I in comparison to other actin proteins. Strictly conserved amino acid residues are boxed in grey. The polypeptide sequence used for immunization is shown in bold and marked by a bar. P. berghei actin I, gi: 74991781; P. berghei actin II, gi: 74989223; T. gondii actin, gi: 606857; D. melanogaster actin 5, gi: 61677879; G. gallus actin A, gi: 71894831; and S. cerevisiae actin 1, gi: 170986. (C) Ookinete immunofluorescence staining with Ab-actinI-I (red) and anti-Pb70 mAb (green) labeling the IMC. Shown are representative sections at the periphery in the apical region (a and b) and through the center (c and d). Rods are indicated by asterisks in panel (c). Scale bar (a and b) 2 m, (c and d) 5 m. The protocol for staining of actin in ookinetes has been described previously [14]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
slightly different in each ookinete, rods were consistently seen. In order to assess in which compartment the actin rods were localized we performed double stainings with an antibody against the Pb70 protein of the inner membrane complex (IMC) [18] (Fig. 1C). The results indicate that the rods are localized in close proximity with the Pb70 protein, at the cell periphery. This staining pattern is consistent with the glideosome model [19,20], where putative actin filaments were proposed to be localized in the narrow space between the IMC and the plasmamembrane. We next measured the lengths of the rods (n = 18 from 3 different ookinetes). In contrast to what has been measured in vitro [7,9] the rods were considerably longer being ∼ 600 nm in length ±117 nm (S.D.). This discrepancy may be due to differences in the properties of the filament in the cell where actin-binding proteins (ABF) will influence the length and stability of the polymer. The detected rods may also consist of bundled or networked F-actin, which could be formed in the
presence of ABFs. At present we cannot distinguish between these possibilities. To further corroborate our findings and investigate whether this staining pattern indeed reflects F-actin, we next treated ookinetes with cytochalasin D, a potent inhibitor of F-actin formation and jasplakinolide, an F-actin stabilizing drug. We hypothesized that in the presence of cytochalasin D the rod-like staining should disappear, whereas in ookinetes treated with jasplakinolide the filaments should be elongated and more prominent than in untreated ookinetes. Our results (Fig. 2A) are fully consistent with this prediction and support the notion that the Ab-actinI-I specifically detects actin filaments. In cytochalasin D-treated parasites (Fig. 2A, center) a diffuse cytoplasmic staining was observed, while in the jasplakinolide-treated ookinetes (Fig. 2A, bottom rows) filaments were detected. These filaments are much longer than the rods detected in the untreated parasites (Fig. 2A, top rows),
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I. Siden-Kiamos et al. / Molecular & Biochemical Parasitology 181 (2012) 186–189
Fig. 2. Changes in actin rods after treatment with the F-actin inhibitors cytochalasin D and jasplakinolide (A) Ab-actinI-I staining of control ookinetes (top) and ookinetes after treatment with 100 nM cytochalasin D, which disrupts actin filaments (center), and 25 nM jasplakinolide, an actin stabilizing drug (bottom). Actin staining is shown in red (top row in each section), and double staining with anti-Pb70 (green) below. Individual Z-sections from confocal analysis (center to periphery of the cell) are shown. (B) Western blot analysis of extracts from control ookinetes (left) and ookinetes treated with jasplakinolide (right). P1 = low speed pellet (3000 × g for 10 min), P2 = pellet at 10,000 × g (10 min), P3 = ultra-high speed pellet at 100,000 × g (1 h), S = supernatant after ultra centrifugation. Blots were probed with Ab-actinI-I (top) and mAb 224–236–1 (bottom). Both antibodies recognized one major band at the predicted molecular weight for actin I (42 kD). Molecular weight standards are shown on the right. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
and they are found also in the cytoplasm, i.e. the center of the ookinete. To further verify that the antibody preferentially recognizes actin in the filamentous state, we used sedimentation analysis of endogenous Plasmodium F-actin (Fig. 2B). Before ultracentrifugation, freshly prepared ookinetes were lysed in F-actin buffer (5 mM Tris, pH 8.0; 50 mM KCl; 2 mM MgCl2 ; 0.2 mM CaCl2 ; 1 mM ATP; 0.5 mM DTT, and 1% Triton X-100) on ice for 30 min. The parasite extract was cleared with two low-speed centrifugations (500 × g and 10,000 × g for 10 min each). F-actin was then sedimented at 100,000 × g for 1 h and analyzed by Western blot analysis. As a control, ookinetes, which had been pre-treated with jasplakinolide, were included. A large proportion of the Ab-actinI-I
signal was detected with F-actin present in the 100,000 × g pellet (Fig. 2B, lanes 3). Interestingly, the Ab-actinI-I antibody detected only actin in the pellet and not in the supernatant after ultracentrifugation (Fig. 2B, lanes 4), irrespective of whether the ookinetes had been pre-treated with jasplakinolide or not. This indicates that this antiserum recognizes actin only in a filamentous state, perhaps in a specific conformation or in a modified form. We also probed the blots with mAb 224–236–1. In this case, actin was recognized both in the ultra-high speed pellet as well as in the supernatant, suggesting that this antibody is conformation independent. In the jasplakinolide-treated ookinetes this antibody reacted only with the ultra-high speed pellet, confirming that actin had been effectively polymerized into filaments.
I. Siden-Kiamos et al. / Molecular & Biochemical Parasitology 181 (2012) 186–189
In conclusion we describe a novel tool to probe for Plasmodium Factin in whole parasites and provide cell biological and biochemical evidence for the presence of rod-like filaments in gliding ookinetes. In vivo, the actin-positive filamentous structures are substantially longer than anticipated from in vitro studies; the significance of this remains to be elucidated. The presence of similar rod-like F-actin in other intra- and extracellular life cycle stages of the malaria parasite can now be tested. Acknowledgements This work was partly funded by Intermal, a Marie Curie Initial Training Consortium (Grant no. PITN-GA-2008-215281 to I S-K) and the EVIMalaR network (Grant agreement 242095) of which K.L. and K.M. are members. References [1] Frenal K, Soldati-Favre D. Role of the parasite and host cytoskeleton in apicomplexa parasitism. Cell Host Microbe 2009;5:602–11. [2] Sibley LD. How apicomplexan parasites move in and out of cells. Curr Opin Biotechnol 2010;21:592–8. [3] Pollard TD, Cooper JA. Actin, a central player in cell shape and movement. Science 2009;326:1208–12. [4] Baum J, Papenfuss AT, Baum B, Speed TP, Cowman AF. Regulation of apicomplexan actin-based motility. Nat Rev Microbiol 2006;4:621–8. [5] Schuler H, Matuschewski K. Regulation of apicomplexan microfilament dynamics by a minimal set of actin-binding proteins. Traffic 2006;7:1433–9. [6] Sahoo N, Beatty W, Heuser J, Sept D, Sibley LD. Unusual kinetic and structural properties control rapid assembly and turnover of actin in the parasite Toxoplasma gondii. Mol Biol Cell 2006;17:895–906.
189
[7] Schmitz S, Grainger M, Howell S, Calder LJ, Gaeb M, Pinder JC, et al. Malaria parasite actin filaments are very short. J Mol Biol 2005;349:113–25. [8] Schmitz S, Schaap IA, Kleinjung J, Harder S, Grainger M, Calder L, et al. Malaria parasite actin polymerization and filament structure. J Biol Chem 2010;285:36577–85. [9] Schuler H, Mueller AK, Matuschewski K. Unusual properties of Plasmodium falciparum actin: new insights into microfilament dynamics of apicomplexan parasites. FEBS Lett 2005;579:655–60. [10] Skillman KM, Diraviyam K, Khan A, Tang K, Sept D, Sibley LD. Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites. PLoS Pathog 2011;7:e1002280. [11] Kudryashev M, Lepper S, Baumeister W, Cyrklaff M, Frischknecht F. Geometric constrains for detecting short actin filaments by cryogenic electron tomography. PMC Biophys 2010;3:6. [12] Schatten H, Sibley LD, Ris H. Structural evidence for actin-like filaments in Toxoplasma gondii using high-resolution low-voltage field emission scanning electron microscopy. Microsc Microanal 2003;9:330–5. [13] Mehta S, Sibley LD. Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii. Mol Biol Cell 2011;22:1290–9. [14] Siden-Kiamos I, Pinder JC, Louis C. Involvement of actin and myosins in Plasmodium berghei ookinete motility. Mol Biochem Parasitol 2006;150:308–17. [15] Westphal M, Jungbluth A, Heidecker M, Muhlbauer B, Heizer C, Schwartz JM, et al. Microfilament dynamics during cell movement and chemotaxis monitored using a GFP-actin fusion protein. Curr Biol 1997;7:176–83. [16] Deibler M, Spatz JP, Kemkemer R. Actin fusion proteins alter the dynamics of mechanically induced cytoskeleton rearrangement. PLoS One 2011;6:e22941. [17] Deligianni E, Morgan RN, Bertuccini L, Kooij TW, Laforge A, Nahar C, et al. Critical role for a stage-specific actin in male exflagellation of the malaria parasite. Cell Microbiol 2011;13:1714–30. [18] Siden-Kiamos I, Vlachou D, Margos G, Beetsma A, Waters AP, Sinden RE, et al. Distinct roles for pbs21 and pbs25 in the in vitro ookinete to oocyst transformation of Plasmodium berghei. J Cell Sci 2000;19(113 Pt):3419–26. [19] Keeley A, Soldati D. The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa. Trends Cell Biol 2004;14:528–32. [20] Sibley LD. Intracellular parasite invasion strategies. Science 2004;304: 248–53.
Contents lists available at SciVerse ScienceDirect
Molecular & Biochemical Parasitology
Short communication
Evidence for filamentous actin in ookinetes of a malarial parasite Inga Siden-Kiamos a,∗ , Christos Louis a,b , Kai Matuschewski c a
Institute of Molecular Biology and Biotechnology, FORTH, N. Plastira 100, Vassilika Vouton, Heraklion 700 13, Crete, Greece Department of Biology, University of Crete, Heraklion, Greece c Parasitology Unit, Max Planck Institute for Infection Biology, Berlin, Germany b
a r t i c l e
i n f o
Article history: Received 21 September 2011 Received in revised form 13 October 2011 Accepted 2 November 2011 Available online 11 November 2011 Keywords: Malaria Ookinete Actin Motility Cytoskeleton
a b s t r a c t Extracellular stages of apicomplexan parasites utilize their own actin myosin motor machinery for gliding locomotion, penetration of cell barriers, and host cell invasion. Thus far, filamentous actin could not be visualized by standard microscopic techniques in vivo. Here, we describe the generation of a novel peptide antibody against the divergent amino-terminal portion of the major Plasmodium isoform, actin I. We show that our antiserum, termed Ab-actinI-I, is conformation-specific. In motile ookinetes it recognizes actin in rod-like structures, which are sensitive to inhibitors interfering with actin polymerization. The average size of the rods is 600 nm, which is considerably longer than what has been detected in in vitro studies of actin filaments. © 2011 Elsevier B.V. All rights reserved.
Apicomplexan parasites use an actin–myosin based motor machinery for their motility [1,2]. Although they share many features with actin–myosin based motility in higher organisms there are also substantial differences. Here, we concentrate on the ability of the major isoform of actin in Plasmodium parasites to form filaments. In most well-studied eukaryotic organisms formation of filamentous actin (F-actin) is regulated by around 150 different proteins [3], while in Apicomplexa, there are substantially fewer regulators identified [4,5]. Importantly, in vitro studies have revealed that actin from Plasmodium or Toxoplasma forms considerably shorter and more fragile filaments than actin from yeast or vertebrates [6–10]. This is also reflected by the fact that, so far, actin filaments have not been readily detected in these parasites [11,12]. Analysis of a Toxoplasma gondii mutant, in which ADF (actin depolymerizing factor) was conditionally down-regulated, revealed actin staining in a pattern consistent with filaments [13] and this pattern was also detected in jasplakinolide treated T. gondii tachyzoites expressing mutated forms of actins [10]. We are interested in understanding the role of actin in parasite motility, and for this we systematically tested antibodies recognizing actin, in order to identify one that would specifically recognize Plasmodium berghei actin I, the major actin isoform (PBANKA 145930; gi: 74991781), but not actin from animals or
Abbreviations: ADF, actin depolymerizing factor; G-actin, globular actin; F-actin, filamentous actin; IMC, inner membrane complex; ABF, actin-binding proteins. ∗ Corresponding author. Tel.: +30 2810 391118; fax: +30 2810 391104. E-mail address: [email protected] (I. Siden-Kiamos). 0166-6851/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2011.11.002
yeast. Both the antiserum (anti-PfACT1) against a specific peptide of Plasmodium falciparum actin I (peptide DEEMKTSEQSSDI, residues 224–236 in subdomain 4, conserved in Plasmodium berghei) [14] and the monoclonal antibody mAb 224–236–1, originally developed against Dictyostelium actin [15], detected the presence of actin in a well-defined region in the apical tip of the parasite, as previously described [14]. Furthermore, a similar localization was seen in transgenic parasites, expressing as a second copy YFP-tagged actin I fusion protein under the control of the circumsporozoite and TRAP-related protein (CTRP) promoter, which is active in ookinetes but not in other stages (manuscript in preparation) (Fig. 1A). Fluorescently tagged actin may interfere with the normal function of endogenous actin; although not well understood, this effect may be dependent on the ratio of non-modified to modified form (Deligianni and Siden-Kiamos, unpublished [16]). We, therefore, hypothesize that the observed accumulation of YFPactin I might reflect the fusion protein in the globular (G−) form. Clearly, we did not detect YFP-actin in a pattern consistent with F− actin. In an attempt to further characterize actin I in P. berghei we developed an antiserum against a synthetic peptide from actin I, corresponding to amino acids 16–30 in subdomain 1, named AbactinI-I; this peptide is sufficiently divergent from the Plasmodium isoform actin II [17] (Fig. 1B). The antibody specifically recognized recombinant P. berghei actin I expressed in Escherichia coli on Western blots (data not shown). Surprisingly, when we used this antibody to detect actin in ookinetes of P. berghei, we detected the protein in short rods, while staining at the apical tip was absent or less pronounced (Fig. 1C). Although the pattern of staining was
I. Siden-Kiamos et al. / Molecular & Biochemical Parasitology 181 (2012) 186–189
187
Fig. 1. Rod-like structures in the cell periphery are detected in ookinetes using the Ab-actinI-I antibody. (A) YFP-actin is detected only at the apical tip. (a) Live imaging of transgenic ookinetes expressing an YFP-actin I fusion protein (b) Same ookinete surface labeled with the 13.1 antibody recognizing the Pbs21 antigen. Scale bar 5 m. (B) Sequence alignment of the amino-terminal region of P. berghei actin I in comparison to other actin proteins. Strictly conserved amino acid residues are boxed in grey. The polypeptide sequence used for immunization is shown in bold and marked by a bar. P. berghei actin I, gi: 74991781; P. berghei actin II, gi: 74989223; T. gondii actin, gi: 606857; D. melanogaster actin 5, gi: 61677879; G. gallus actin A, gi: 71894831; and S. cerevisiae actin 1, gi: 170986. (C) Ookinete immunofluorescence staining with Ab-actinI-I (red) and anti-Pb70 mAb (green) labeling the IMC. Shown are representative sections at the periphery in the apical region (a and b) and through the center (c and d). Rods are indicated by asterisks in panel (c). Scale bar (a and b) 2 m, (c and d) 5 m. The protocol for staining of actin in ookinetes has been described previously [14]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
slightly different in each ookinete, rods were consistently seen. In order to assess in which compartment the actin rods were localized we performed double stainings with an antibody against the Pb70 protein of the inner membrane complex (IMC) [18] (Fig. 1C). The results indicate that the rods are localized in close proximity with the Pb70 protein, at the cell periphery. This staining pattern is consistent with the glideosome model [19,20], where putative actin filaments were proposed to be localized in the narrow space between the IMC and the plasmamembrane. We next measured the lengths of the rods (n = 18 from 3 different ookinetes). In contrast to what has been measured in vitro [7,9] the rods were considerably longer being ∼ 600 nm in length ±117 nm (S.D.). This discrepancy may be due to differences in the properties of the filament in the cell where actin-binding proteins (ABF) will influence the length and stability of the polymer. The detected rods may also consist of bundled or networked F-actin, which could be formed in the
presence of ABFs. At present we cannot distinguish between these possibilities. To further corroborate our findings and investigate whether this staining pattern indeed reflects F-actin, we next treated ookinetes with cytochalasin D, a potent inhibitor of F-actin formation and jasplakinolide, an F-actin stabilizing drug. We hypothesized that in the presence of cytochalasin D the rod-like staining should disappear, whereas in ookinetes treated with jasplakinolide the filaments should be elongated and more prominent than in untreated ookinetes. Our results (Fig. 2A) are fully consistent with this prediction and support the notion that the Ab-actinI-I specifically detects actin filaments. In cytochalasin D-treated parasites (Fig. 2A, center) a diffuse cytoplasmic staining was observed, while in the jasplakinolide-treated ookinetes (Fig. 2A, bottom rows) filaments were detected. These filaments are much longer than the rods detected in the untreated parasites (Fig. 2A, top rows),
188
I. Siden-Kiamos et al. / Molecular & Biochemical Parasitology 181 (2012) 186–189
Fig. 2. Changes in actin rods after treatment with the F-actin inhibitors cytochalasin D and jasplakinolide (A) Ab-actinI-I staining of control ookinetes (top) and ookinetes after treatment with 100 nM cytochalasin D, which disrupts actin filaments (center), and 25 nM jasplakinolide, an actin stabilizing drug (bottom). Actin staining is shown in red (top row in each section), and double staining with anti-Pb70 (green) below. Individual Z-sections from confocal analysis (center to periphery of the cell) are shown. (B) Western blot analysis of extracts from control ookinetes (left) and ookinetes treated with jasplakinolide (right). P1 = low speed pellet (3000 × g for 10 min), P2 = pellet at 10,000 × g (10 min), P3 = ultra-high speed pellet at 100,000 × g (1 h), S = supernatant after ultra centrifugation. Blots were probed with Ab-actinI-I (top) and mAb 224–236–1 (bottom). Both antibodies recognized one major band at the predicted molecular weight for actin I (42 kD). Molecular weight standards are shown on the right. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
and they are found also in the cytoplasm, i.e. the center of the ookinete. To further verify that the antibody preferentially recognizes actin in the filamentous state, we used sedimentation analysis of endogenous Plasmodium F-actin (Fig. 2B). Before ultracentrifugation, freshly prepared ookinetes were lysed in F-actin buffer (5 mM Tris, pH 8.0; 50 mM KCl; 2 mM MgCl2 ; 0.2 mM CaCl2 ; 1 mM ATP; 0.5 mM DTT, and 1% Triton X-100) on ice for 30 min. The parasite extract was cleared with two low-speed centrifugations (500 × g and 10,000 × g for 10 min each). F-actin was then sedimented at 100,000 × g for 1 h and analyzed by Western blot analysis. As a control, ookinetes, which had been pre-treated with jasplakinolide, were included. A large proportion of the Ab-actinI-I
signal was detected with F-actin present in the 100,000 × g pellet (Fig. 2B, lanes 3). Interestingly, the Ab-actinI-I antibody detected only actin in the pellet and not in the supernatant after ultracentrifugation (Fig. 2B, lanes 4), irrespective of whether the ookinetes had been pre-treated with jasplakinolide or not. This indicates that this antiserum recognizes actin only in a filamentous state, perhaps in a specific conformation or in a modified form. We also probed the blots with mAb 224–236–1. In this case, actin was recognized both in the ultra-high speed pellet as well as in the supernatant, suggesting that this antibody is conformation independent. In the jasplakinolide-treated ookinetes this antibody reacted only with the ultra-high speed pellet, confirming that actin had been effectively polymerized into filaments.
I. Siden-Kiamos et al. / Molecular & Biochemical Parasitology 181 (2012) 186–189
In conclusion we describe a novel tool to probe for Plasmodium Factin in whole parasites and provide cell biological and biochemical evidence for the presence of rod-like filaments in gliding ookinetes. In vivo, the actin-positive filamentous structures are substantially longer than anticipated from in vitro studies; the significance of this remains to be elucidated. The presence of similar rod-like F-actin in other intra- and extracellular life cycle stages of the malaria parasite can now be tested. Acknowledgements This work was partly funded by Intermal, a Marie Curie Initial Training Consortium (Grant no. PITN-GA-2008-215281 to I S-K) and the EVIMalaR network (Grant agreement 242095) of which K.L. and K.M. are members. References [1] Frenal K, Soldati-Favre D. Role of the parasite and host cytoskeleton in apicomplexa parasitism. Cell Host Microbe 2009;5:602–11. [2] Sibley LD. How apicomplexan parasites move in and out of cells. Curr Opin Biotechnol 2010;21:592–8. [3] Pollard TD, Cooper JA. Actin, a central player in cell shape and movement. Science 2009;326:1208–12. [4] Baum J, Papenfuss AT, Baum B, Speed TP, Cowman AF. Regulation of apicomplexan actin-based motility. Nat Rev Microbiol 2006;4:621–8. [5] Schuler H, Matuschewski K. Regulation of apicomplexan microfilament dynamics by a minimal set of actin-binding proteins. Traffic 2006;7:1433–9. [6] Sahoo N, Beatty W, Heuser J, Sept D, Sibley LD. Unusual kinetic and structural properties control rapid assembly and turnover of actin in the parasite Toxoplasma gondii. Mol Biol Cell 2006;17:895–906.
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[7] Schmitz S, Grainger M, Howell S, Calder LJ, Gaeb M, Pinder JC, et al. Malaria parasite actin filaments are very short. J Mol Biol 2005;349:113–25. [8] Schmitz S, Schaap IA, Kleinjung J, Harder S, Grainger M, Calder L, et al. Malaria parasite actin polymerization and filament structure. J Biol Chem 2010;285:36577–85. [9] Schuler H, Mueller AK, Matuschewski K. Unusual properties of Plasmodium falciparum actin: new insights into microfilament dynamics of apicomplexan parasites. FEBS Lett 2005;579:655–60. [10] Skillman KM, Diraviyam K, Khan A, Tang K, Sept D, Sibley LD. Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites. PLoS Pathog 2011;7:e1002280. [11] Kudryashev M, Lepper S, Baumeister W, Cyrklaff M, Frischknecht F. Geometric constrains for detecting short actin filaments by cryogenic electron tomography. PMC Biophys 2010;3:6. [12] Schatten H, Sibley LD, Ris H. Structural evidence for actin-like filaments in Toxoplasma gondii using high-resolution low-voltage field emission scanning electron microscopy. Microsc Microanal 2003;9:330–5. [13] Mehta S, Sibley LD. Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii. Mol Biol Cell 2011;22:1290–9. [14] Siden-Kiamos I, Pinder JC, Louis C. Involvement of actin and myosins in Plasmodium berghei ookinete motility. Mol Biochem Parasitol 2006;150:308–17. [15] Westphal M, Jungbluth A, Heidecker M, Muhlbauer B, Heizer C, Schwartz JM, et al. Microfilament dynamics during cell movement and chemotaxis monitored using a GFP-actin fusion protein. Curr Biol 1997;7:176–83. [16] Deibler M, Spatz JP, Kemkemer R. Actin fusion proteins alter the dynamics of mechanically induced cytoskeleton rearrangement. PLoS One 2011;6:e22941. [17] Deligianni E, Morgan RN, Bertuccini L, Kooij TW, Laforge A, Nahar C, et al. Critical role for a stage-specific actin in male exflagellation of the malaria parasite. Cell Microbiol 2011;13:1714–30. [18] Siden-Kiamos I, Vlachou D, Margos G, Beetsma A, Waters AP, Sinden RE, et al. Distinct roles for pbs21 and pbs25 in the in vitro ookinete to oocyst transformation of Plasmodium berghei. J Cell Sci 2000;19(113 Pt):3419–26. [19] Keeley A, Soldati D. The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa. Trends Cell Biol 2004;14:528–32. [20] Sibley LD. Intracellular parasite invasion strategies. Science 2004;304: 248–53.