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. 2011 Apr;10(4):556-64.
doi: 10.1128/EC.00244-10. Epub 2011 Jan 14.

Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum

Jeffrey A Yeoman  1 , Eric Hanssen, Alexander G Maier, Nectarios Klonis, Bohumil Maco, Jake Baum, Lynne Turnbull, Cynthia B Whitchurch, Matthew W A Dixon, Leann Tilley
Affiliations

Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum

Jeffrey A Yeoman et al. Eukaryot Cell. 2011 Apr.

Abstract

The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.

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Figures

Fig. 1.
Fig. 1.
Schematic diagram of the PfGAP50 chimera construct and Western analysis of PfGAP50-GFP transfectants. (A) The four domains of PfGAP50 are as follows: a predicted signal sequence (SS), an acid phosphatase homology domain, a predicted transmembrane domain (TM), and a short cytoplasmic tail. (B) Late-stage 3D7 parent and full-length PfGAP50-GFP transfectants were enriched, treated with streptolysin O, and then lysed by freeze-thaw cycling. Supernatant (S) and pellet (P) fractions were subjected to Western blotting and probed with antibodies recognizing PfGAP50 or GFP. The blots were reprobed with antibodies recognizing the soluble parasite protein PfGAPDH.
Fig. 2.
Fig. 2.
PfGAP50-GFP interacts with PfGAP45 and PfMTIP. Proteins were detergent solubilized from enriched, mature-stage PfGAP50-GFP transfectants. The extract was immunoprecipitated (IP) using rabbit preimmune serum (Pre), anti-PfGAP45, anti- PfMTIP, or anti-GFP. Western blots (WB) of the pellet fractions were probed with mouse anti-GFP, anti-PfGAP45, or anti-PfMTIP. The antibodies used for IP are shown at the top of the blot, and those for WB are shown at the bottom.
Fig. 3.
Fig. 3.
Dynamics of PfGAP50-GFP-labeled compartments in live transfectants. Shown is live confocal fluorescence microscopy of highly synchronized PfGAP50-GFP transfectants. Single section scans (collected at 25 μs/pixel) from different cells at 2-h intervals in the schizont stage are shown (2 cells are represented per time point). At 40 to 42 h after invasion, reticular structures with looped extensions and focal concentrations of PfGAP50-GFP are apparent, coalescing into punctate structures by 44 h. The PfGAP50-GFP-containing structures then appear to expand around each of the daughter merozoites. A more extended time series is shown in Fig. S1 in the supplemental material. Bars = 1 μm.
Fig. 4.
Fig. 4.
PfGAP50 is located in the ER prior to recruitment to the IMC. (A) Parasites expressing PfGAP50-GFP (green) were incubated with ER-Tracker (depicted in red). The GFP and ER-Tracker fluorescence signals are colocated in trophozoite stage parasites. Upon relocation of GFP fluorescence to the apical ends of nascent merozoites during the early schizont stage, the reticular ER-Tracker labeling persists. PfGAP50-GFP is concentrated at one pole of released merozoites, while ER-Tracker labels internal structures. (B) Fixed-cell immunofluorescence microscopy of the 3D7 parent strain labeled with antibodies raised against PfGAP50 (green) and PfERC (red). (C) Immunofluorescence microscopy of PfGAP50-GFP transfectants at the mature- and ruptured-schizont stages labeled with anti-GFP (green), anti-PfGAP45 (red), and DAPI (blue). Bars = 1 μm.
Fig. 5.
Fig. 5.
Photobleach analysis of PfGAP50-GFP organization. The panels are prebleach images with and without DIC overlay and postbleach images after application of a laser pulse at the positions indicated by arrows. (A) A region of the ER in an early-trophozoite stage parasite shows complete loss of fluorescence at the point of bleaching, partial loss from a connected compartment, and little loss from an adjacent compartment. There was no recovery after 3 min. (B) Application of a laser pulse to PfGAP50-GFP in an apical cap in an early schizont ablates fluorescence with no recovery from adjacent structures. (C) Spot bleaching of a region of PfGAP50-GFP in a region of a mature merozoite results in complete loss of fluorescence at the point of bleaching, with little loss from other regions of the same merozoite or from adjacent merozoites. Bars = 1 μm.
Fig. 6.
Fig. 6.
The IMC forms around the apical pore of developing merozoites. 3D-SIM was performed on parasites expressing PfGAP50-GFP (green) labeled with DAPI nuclear stain (depicted in red). (A and C) Schizont with eight nuclei, each with two associated PfGAP50-GFP-containing ellipsoids. Each ellipsoid has two unlabeled “pores” (arrow). (B and D) A schizont with eight nuclei that are undergoing division showing the PfGAP50-GFP-containing structures separating into claw-like structures (arrow). (E and F) Mature schizonts in which the PfGAP50-GFP fluorescence is more evenly distributed around the periphery of the parasite. (G and H) Transmission EM (TEM) images of early schizonts with electron-dense apical prominences with overlying membrane caps (arrowheads) forming in pairs on either side of a nucleus (n). The apical caps are connected to the underlying electron-dense rhoptries (r) and form close to a mitotic spindle (m). (I) TEM image of a maturing schizont showing invagination of the parasite plasma membrane around the daughter cells and separation of the apical caps (arrowheads). (J) TEM image of a mature schizont showing electron-dense structures at the apical ends of closely apposed daughter cells (arrowheads). (K) Selected virtual section (22 nm) through a tomogram showing a merozoite developing within a schizont. The electron-dense polar rings are indicated with black arrows, and the IMC with a white arrow. A microneme (mc) is visible. (L) Rendered tomographic reconstructions of merozoites, generated from serial sections through the schizont shown in panel K, showing the nucleus in yellow, rhoptries in burnt orange, dense granules in brown, the polar rings in blue, the mitochondrion in green, and a cytostomal ring as a yellow circle. Rotations of the 3D projection in panels A, B, E, and F are presented in movies S1 to S4 in the supplemental material. Bars = 1 μm (A to F, G, and J) and 500 nm (H, I, K, and L).
Fig. 7.
Fig. 7.
The C terminus of PfGAP50 is required for correct trafficking. (A) A truncation mutant of PfGAP50 lacking the C-terminal domains (PfGAP501-369-GFP) and a mutant in which the acid phosphatase homology domain is replaced with GFP (PfGAP50ΔAP-GFP) were generated. (B and C) Location and solubility of episomally expressed PfGAP501-369-GFP. (B) Live confocal fluorescence images of trophozoite and schizont stage parasites (top 2 panels) show mistrafficking of PfGAP501-369-GFP to the PV outside the region labeled with ER-Tracker (bottom). Bar = 1 μm. (C) Enriched PfGAP501-369-GFP transfectants were treated with streptolysin O, followed by freeze-thaw cycling. Supernatant (S) and pellet (P) fractions were subjected to Western blotting, probed with antibodies recognizing GFP, and then reprobed with anti-PfGAPDH. (D and E) Location and solubility of episomally expressed PfGAP50ΔAP-GFP. (D) Live-cell images show a fluorescence pattern similar to that of the full-length PfGAP50-GFP. Bar = 1 μm. (E) Late-stage parasites expressing PfGAP50ΔAP-GFP were harvested and lysed, and the Western blot of the pellet and supernatant fractions was probed with mouse anti-GFP.
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