spread functions and software supplied by the manufacturer. Fluorescent images are maximum intensity projections of deconvolved 3D stacks. The brightness and contrast of images used in the final figures were optimized for color prints. ImmunoGold Labeling 36107 RH parasites were first suspended in 5 ml PBS containing 5 mM calcium ionophore, A23187, and then spotted onto clean parafilm and overlayed with a carbon-film–coated nickel grid for 1 hour in a humid chamber. Parasites were then permeabilized with 10 mM DOC in PBS for 25 minutes and fixed with 3.7% formaldehyde in PBS, and washed 365 minutes with PBS, followed by 10 minutes of blocking in 5% BSA +0.1% (v/v) fish gelatin. Free aldehyde groups were blocked by incubation with 50 mM glycine in PBS for 15 minutes (pH 7.5) followed by 15 minute incubation with 0.1% (w/v) NaBH4 in PBS. Grids were washed twice with PBS, blocked again with 5% BSA +0.1% fish gelatin in PBS for 30 minutes, washed 265 minutes with incubation buffer (0.8% BSA, 0.1% fish gelatin in PBS plus 10 mM NaN3), incubated for ,2 hours with primary antibody (rat anti-TgICMAP1), diluted 1:100 in incubation buffer; washed 665 minutes in incubation buffer, inverted on 15 ml drops of secondary antibody (goat anti–rat IgG-conjugated with ultra-small gold (EMS Inc Cat#25181)), diluted 1:50 in incubation buffer; incubated ,19 hours at 4uC, then washed with incubation buffer as follows: 361 minute, 2610 minutes, 465 minutes, and then 561 minute with PBS. The samples were post-fixed for 5 minutes with 1% glutaraldehyde in PBS and washed in distilled water 365 minutes. Silver enhancement was carried out using the HQ silver enhancement kit (Nanoprobes) by floating grids on mixtures of the initiator, activator, and modulator for 6 minutes in a light-tight chamber, then washing briefly with distilled water once followed by 265 minutes washes in distilled water. Grids were negatively stained using 2% phosphotungstic acid (pH 7.0). Western blot 56106 extracellular parasites were lysed by incubating in 1x sample buffer (62.5 mM Tris pH 6.8, 2% (w/v) sodium deodeoyl sulfate, 10% (v/v) glycerol, ,0.5 mg of bromophenol blue) containing 50 mM DTT at 100uC for 10 minutes. Proteins were resolved on 4–12% gradient bis-tris gels (Cat#NP0332, Invitro- gen) at 200 V for 1 hour. Proteins were transferred to poly- vinylidene fluoride (PVDF) membrane using the XCellTM blot module (Cat#EI9051, Invitrogen) per manufacturers instructions. References 1. Morrissette NS, Murray JM, Roos DS (1997) Subpellicular microtubules associate with an intramembranous particle lattice in the protozoan parasite Toxoplasma gondii. J Cell Sci 110 (Pt 1): 35–42. 2. Shaw MK, Compton HL, Roos DS, Tilney LG (2000) Microtubules, but not actin filaments, drive daughter cell budding and cell division in Toxoplasma gondii. J Cell Sci 113 (Pt 7): 1241–1254. 3. Nichols BA, Chiappino ML (1987) Cytoskeleton of Toxoplasma gondii. J Protozool 34: 217–226. 4. Morrissette NS, Sibley LD (2002) Cytoskeleton of apicomplexan parasites. Microbiol Mol Biol Rev 66: 21–38. 5. Morrissette NS, Sibley LD (2002) Disruption of microtubules uncouples budding and nuclear division in Toxoplasma gondii. J Cell Sci 115: 1017–1025. 6. Hu K, Johnson J, Florens L, Fraunholz M, Suravajjala S, et al. (2006) Cytoskeletal Components of an Invasion Machine-The Apical Complex of Toxoplasma gondii. PLoS Pathog 2: e13. 7. Hu K, Roos DS, Murray JM (2002) A novel polymer of tubulin forms the conoid of Toxoplasma gondii. J Cell Biol 156: 1039–1050. 8. Hu K (2008) Organizational changes of the daughter basal complex during the parasite replication of Toxoplasma gondii. PLoS Pathog 4: e10. 9. Remington JS, McLeod R, Desmonts G (1995) Toxoplasmosis. In: Infectious diseases of the fetus and the newborn infant; J.S. R, J.O. K, eds. Philadelphia: W.B. Saunders Company. Characterization of TgICMAP1 Western blot was carried out using BM chemiluminescence Western blotting kit (Cat#1152709001, Roche) per the instruc- tions of the manufacturer. Washes were carried out in TBS (20 mM Tris base pH 7.4, 150 mM NaCl) containing 0.1% (v/v) Tween-20 (Sigma Cat#P7949). Rat anti-TgICMAP1 was diluted 1:250 and mouse-anti-tubulin B-5-1-2 was diluted 1:2,000 in TBS containing 0.5% (v/v) blocking buffer (Cat#1152709001, Roche). Goat anti-rat IgG HRP (Cat#NA935V, GE Healthcare, United Kingdom) was diluted 1:1000 and goat anti-mouse/rabbit HRP (Cat#1152709001, Roche) was diluted 1:20,000 in TBS contain- ing 0.5% blocking buffer. Supporting Information Table S1 Primer sequences used for the construction of plasmids used in this paper. Abbreviations: S; sense primer, AS; anti-sense primer. Found at: doi:10.1371/journal.pone.0007406.s001 (0.05 MB DOC) Figure S1 NcICMAP1 is homologous to TgICMAP1. ClustalW alignment of TgICMAP1 and NcICMAP1, which share 82% identity and 89% similarity. Sequence homologous to the coiled- coil SMC domain is highlighted in red. ‘‘*’’ indicates conserved amino acids; ‘‘:’’ indicates conserved substitution; ‘‘.’’ indicates semi-conserved substitution. EuPathDB accession numbers are TgICMAP1: TgME49_039300, NcICMAP1: Nc_LIV_ 070470. Found at: doi:10.1371/journal.pone.0007406.s002 (0.74 MB TIF) Acknowledgments We thank Dr. Claire Walczak (Indiana University, Bloomington) and the members of her lab for helpful discussions during the course of these experiments. We also thank Dr. Claire Walczak for providing GMPcPP, tubulin, and 6xHis-eGFP for in vitro MT binding assays, Dr Gary Ward (University of Vermont) for monoclonal anti-IMC1 antibody, Dr. John Murray (University of Pennsylvania) for YL1/2 tubulin antibody and pc22- eGFP-TubA1 plasmid, Dr. Wolfgang Mages (Universita¨t Regensburg, Germany) for pQE30-DIP13 plasmid and Dr. Pat Foster (Indiana University, Bloomington) for pBAD24 plasmid. Author Contributions Conceived and designed the experiments: ATH KH. Performed the experiments: ATH SEM KH. Analyzed the data: ATH KH. Contributed reagents/materials/analysis tools: ATH SEM KH. Wrote the paper: ATH KH. Revised the paper: SEM. 10. Luft BJ, Hafner R, Korzun AH, Leport C, Antoniskis D, et al. (1993) Toxoplasmic encephalitis in patients with the acquired immunodeficiency syndrome. Members of the ACTG 077p/ANRS 009 Study Team. N Engl J Med 329: 995–1000. 11. Levine ND (1988) Progress in taxonomy of the Apicomplexan protozoa. J Protozool 35: 518–520. 12. Montoya JG, Liesenfeld O (2004) Toxoplasmosis. Lancet 363: 1965–1976. 13. Montoya JG, Remington JS (1996) Toxoplasmic chorioretinitis in the setting of acute acquired toxoplasmosis. Clin Infect Dis 23: 277–282. 14. Holland GN (1999) Reconsidering the pathogenesis of ocular toxoplasmosis. Am J Ophthalmol 128: 502–505. 15. Cerede O, Dubremetz JF, Soete M, Deslee D, Vial H, et al. (2005) Synergistic role of micronemal proteins in Toxoplasma gondii virulence. J Exp Med 201: 453–463. 16. Huynh MH, Carruthers VB (2006) Toxoplasma MIC2 is a major determinant of invasion and virulence. PLoS Pathog 2: e84. 17. Mazumdar J, E HW, Masek K, C AH, Striepen B (2006) Apicoplast fatty acid synthesis is essential for organelle biogenesis and parasite survival in Toxoplasma gondii. Proc Natl Acad Sci U S A 103: 13192–13197. 18. Carruthers VB, Sibley LD (1997) Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur J Cell Biol 73: 114–123. PLoS ONE | www.plosone.org 11 October 2009 | Volume 4 | Issue 10 | e7406