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First published online 20 November 2002
doi: 10.1242/jcs.00194

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Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites

¹ Division of Molecular Parasitology, Department of Microbiology & Immunology, Drexel University College of Medicine, Philadelphia, PA 19129, USA
² Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA
³ Infectious Diseases Section, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8022, USA
⁴ Michael Heidelberger Division, Department of Pathology, New York University School of Medicine, New York, NY 10016, USA

View larger version (19K): [in a new window]   Fig. 2. Localization of regions involved in MyoA and MTIP interaction. (A) The putative neck (amino acids 842-902) and tail (amino acids 903-917) domains of P. yoelii MyoA were cloned together or separately into the Gal4 DNA binding domain vector pAS2.1. The resulting plasmids were transformed into yeast strain PJ69-4a, containing the full length P. yoelii MTIP in the Gal4 activation domain vector (pAD-MTIP), and assayed for their ability to grow in the absence of histidine or adenine (growth is indicated as +; no growth as -). Only the tail domain of MyoA was necessary and sufficient for interaction with MTIP. (B) N- and C-terminal deletions of P. yoelii MTIP were constructed in the Gal4 activation domain vector, transformed into yeast strain PJ69-4a containing pAS2.1-MyoA (amino acids 842-917) and assayed as described above. The minimal interacting region located to amino acids 80-204. Further N-terminal deletion of 15 amino acids or C-terminal deletion of 15 amino acids abrogated interaction. (C) Site-directed mutagenesis of the basic motif within the tail of P. yoelii MyoA in the vector pAS2.1-MyoA. Plasmids were analyzed as described in A. Change of RKRAAA or RKRAAR abrogated interaction. *Values for ß-galactosidase activity are the mean of three independent cultures assayed in duplicate±s.d.

View larger version (50K): [in a new window]   Fig. 1. (A) Predicted tertiary structure of P. yoelii MyoA based on the crystal structure of chicken smooth muscle myosin. The ATP-binding pocket is shown with a bound ATP analog. The MyoA primary amino acid sequence corresponding to the neck domain is predicted to form an -helix. (B) Amino acid sequence alignment of P. yoelii (Py) MTIP, the MTIP ortholog from P. falciparum(Pf), obtained by BLAST analysis of the P. falciparum genome sequence information (Stanford_Chr12Contig01.010524, nucleotides 1931214-1031825) and the putative myosin light chain of T. gondii (Tg) (accession no. AY048862). The putative EF hand motif is indicated by asterisks. Identical amino acid residues are shown in white letters on black. Conserved amino acid changes are shown as white letters on gray. Radical amino acid changes are shown as black letters. The P. yoelii MTIP sequence is available from GenBank/EMBL/DDBJ under accession no. AF465245.

View larger version (70K): [in a new window]   Fig. 3. MTIP is expressed in invasive Plasmodium zoite stages. (A) Western blot analysis of P. yoelii sporozoite extracts (spz) and of extracts from a schizont-enriched fraction of P. yoelii blood stages (bs). The anti-MTIP antiserum specifically recognized a protein doublet in both zoite preparations that closely migrated at approximately 25 kDa. (B) Indirect immunofluorescence assay (IFA) with anti-MTIP antiserum showed that MTIP is concentrated around the periphery of merozoites during the late stages of blood stage schizogony (Bar, 2 µm). (C) IFA with anti-MTIP showed strong peripheral fluorescence in P. yoelii sporozoites that were isolated from mosquito salivary glands (Bar, 2 µm).

View larger version (133K): [in a new window]   Fig. 4. MTIP localizes to the inner membrane complex of Plasmodium sporozoites and co-localizes with MyoA. (A) The transmission electron micrograph of a longitudinal sporozoite section shows the architecture of the sporozoite cortex. The trilaminar pellicle consists of the plasma membrane (white arrow) and the IMC (black arrow) separated by the cortical cytoplasm. m, micronemes; mt, microtubules. (B) Immunoelectron microscopy of sporozoite sections labeled with anti-MTIP (15 nm gold particles). MTIP localized to the periphery of sporozoites and showed circumferential distribution. Almost no labeling was observed in the internal cytoplasm. The gold particles decorated an electron dense structure located 15 nm internal to the presumed plasma membrane. The position of gold particles is consistent with an IMC localization of MTIP. (C,D) Sporozoite sections double-labeled with anti-MyoA (5 nm gold particles) and anti-MTIP (15 nm gold particles) localized both proteins to the periphery of the sporozoite. MTIP was frequently clustered with MyoA. (E,F) Cryo-immunoelectron microscopy localized MTIP (10 nm gold particles) to the inner membrane complex and cortical cytoplasm of sporozoites. Note the absence of plasma membrane and the persistence of MTIP labeling in some positions. The white arrow indicates the plasma membrane; the black arrow indicates the IMC. The inset in F shows the apical prominence of a sporozoite. No gold particles label the prominence beyond the termination points of the IMC.

View larger version (51K): [in a new window]   Fig. 5. Extraction of MTIP and CS with Triton X-100 (TX-100). (A) Sporozoites were permeabilized with saponin and fixed with paraformaldehyde (PFA) (upper panel, —TX100) or treated with 1% TX-100 followed by PFA fixation (lower panel, +TX100). Each preparation was labeled with anti-CS (red) and anti-MTIP (green) antibodies. In untreated sporozoites MTIP and CS were localized to the periphery of sporozoites and showed a relatively homogenous distribution. TX-100-treated sporozoites showed no significant change in MTIP distribution or fluorescent intensity. However, CS fluorescence was lost from the periphery of sporozoites by TX-100 treatment, indicating a complete removal of the plasma membrane. The inset in each panel shows overview fluorescence micrographs for each sporozoite preparation at lower magnification. Left and right panels show the same sporozoites labeled with MTIP (left panels) and CS (right panels). Scale bars are 0.5 µm for individual sporozoite micrographs and 10 µm for overview micrographs. (B) Quantification of MTIP and CS fluorescence shown in A. Graphs are the mean of relative fluorescent intensities measured on 50 sporozoites for each the TX-100-treated and untreated populations with constant exposure times±s.d. (C) Immunoblot analysis of TX-100-treated sporozoites with anti-MTIP and anti-CS antibodies. Pellet (P) and supernatant (S) were separated by high speed centrifugation and analyzed by SDS-PAGE followed by blotting and probing with anti-MTIP (upper panel) or anti-CS (lower panel). Most CS was detected in the supernatant, indicating effective solubilization of the sporozoite plasma membrane and associated proteins. However, most MTIP was detected in the pellet, indicating its retention in the IMC. Total parasite extracts are shown for comparison (C). Note that CS was detected as 40/60 kDa species.

View larger version (83K): [in a new window]   Fig. 6. MTIP is progressively lost during development of hepatic stages, reflecting the disassembly of the inner membrane complex (IMC). (A) Early hepatic trophozoite stage showing a typical transformation bulb. Heat shock protein 70, which is not significantly expressed in sporozoites shows increase of expression. (B) A spherical hepatic trophozoite (24 hours after invasion) shows still complete circumferential MTIP staining. (C,D) Later stage hepatic stages (36-45 hours after invasion) show progressive loss of MTIP staining closely resembling the progressive loss of the IMC during these stages of development (Meis et al., 1985). DIC, differential interference contrast.

View larger version (21K): [in a new window]   Fig. 7. Two models of the molecular motility machinery in apicomplexan zoites. The diagrams show the periphery of a Plasmodium sporozoite and the possible arrangements of identified and hypothetical intracellular components of the motor complex. (A) In the currently prevailing model, actin filaments are tethered to the outer membrane of the IMC by a hypothetical protein. The cytoplasmic domain of TRAP directly or indirectly interacts with the tail of MyoA. The MyoA head domain interacts with actin filaments and moves towards the plus end of the filaments. This leads to displacement of the MyoA/TRAP complex from anterior to posterior and results in a forward movement of the zoite. (B) In the alternative model the N-terminal portion of MTIP anchors it to the outer membrane of the IMC by interaction with a hypothetical protein at the IMC. MTIP binds the tail domain of MyoA, immobilizing it, and this determines MyoA orientation with the head domain projecting outward. The head domain interacts with short actin filaments that are directly or indirectly linked to the cytoplasmic domain of TRAP. The MyoA head domain interacts with actin filaments and moves towards the plus end. Because MyoA is fixed to the IMC, the actin/TRAP complex is displaced from anterior to posterior resulting in a forward movement of the zoite. Note that in model A, the plus end of the actin filaments is oriented towards the posterior of the zoite. In model B the plus end of the actin filaments is oriented towards the anterior end of the zoite. IMC, inner membrane complex; MTIP, MyoA tail domain interacting protein; MyoA, myosin A; TRAP, thrombospondin-related anonymous protein.

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