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【Cell】科学家第一次观测并且记录到疟原虫感染
http://www.cell.com/cell-host-microbe/abstract/S1931-3128(10)00413-0
Cell Host & Microbe, Volume 9, Issue 1, 9-20, 20 January 2011
Summary
Erythrocyte invasion by the merozoite is an obligatory stage in Plasmodium parasite infection and essential to malaria disease progression. Attempts to study this process have been hindered by the poor invasion synchrony of merozoites from the only in vitro culture-adapted human malaria parasite, Plasmodium falciparum. Using fluorescence, three-dimensional structured illumination, and immunoelectron microscopy of filtered merozoites, we analyze cellular and molecular events underlying each discrete step of invasion. Monitoring the dynamics of these events revealed that commitment to the process is mediated through merozoite attachment to the erythrocyte, triggering all subsequent invasion events, which then proceed without obvious checkpoints. Instead, coordination of the invasion process involves formation of the merozoite-erythrocyte tight junction, which acts as a nexus for rhoptry secretion, surface-protein shedding, and actomyosin motor activation. The ability to break down each molecular step allows us to propose a comprehensive model for the molecular basis of parasite invasion.
Figure 1 Electron and Immunofluorescence Microscopy of P. falciparum Merozoite Invasion
Time courses of invasion by transmission electron microscopy (TEM) (A) and wide-field immunofluoresence assay (IFA) microscopy ( with deconvolution (single slice or three-dimensional reconstruction) labeled with PfRON4. For TEM numbering, see the main text. The IFA scale bar represents 2.0 μm. In 3D images, gamma settings were altered, and the grid represents 0.5 μm intervals. DG, dense granules; Mi, micronemes; N, nucleus; PV, parasitophorous vacuole; R, rhoptries; TJ, tight junction.
Figure 2 PfAMA1 and PfRON4 Define Core Components of the Merozoite-Erythrocyte Tight Junction
(A) Three-dimensional reconstruction of IFA z stacks showing steps of invasion from attachment through to <10 min after invasion labeled with PfRON4 (green) and PfAMA1 (red). The inset shows a bright-field and single slice with deconvolution. The scale bar represents 2.0 μm.
( Three-dimensional structured illumination microscopy (3D SIM) of PfAMA1 and PfRON4 double-labeled merozoites during invasion. The scale bar represents 0.3 μm.
(C) Representative z slices through tight junctions used to measure fluorescence intensity profile across the junction to explore PfAMA1 localization with respect to PfRON4 compared to PfRON4/RON4 double-labeled preparations.
(D) The chart shows the mean distances in arbitrary units between peak intensities across the tight junction (see schematic). Values ±standard deviation comparing PfAMA1 and PfRON4 compared to PfRON4/RON4 double-labeled preparations (p < 0.0001, unpaired t test).
(E) Quantification of gold particles either side of the erythrocyte membrane in merozoites with tight junctions immunogold labeled with anti-PfRON4. The chart shows mean and 95% spread.
(F) Representative transmission electron microscopy with immunogold labeling (white arrows) of PfRON4 (the scale bar represents 0.2 μm).
Figure 3 Stepwise Secretion of Apical-Organelle Resident Proteins from Rhoptries and Dense Granules, during Invasion
(A and Three-dimensional reconstruction of IFA z stacks showing steps of invasion from attachment (early), through mid and late invasion, to <10 min after invasion labeled with resident proteins of the rhoptry bulb (RAP1) (A) and dense granules (RESA) (. Insets show bright-field and single slice deconvolution. The scale bar represents 2.0 μm. In 3D images, gamma settings were altered, and the grid represents 0.5 μm intervals.
(C) 3D SIM time course of invasion labeled with anti-RAP1.
(D) Electron microscopy with immunogold labeling of RAP1 (white arrows). The scale bar represents 0.2 μm.
(E) Electron microscopy of merozoite after invasion showing intact dense granules (white arrows and insets). The scale bar represents 0.2 μm.
(F) Serial sections of electron microscopy with immunogold labeling of RAP1 10 min after invasion (white arrows). The scale bar represents 0.2 μm.
Figure 4 Coordination of Surface Protein Shedding and Actomyosin Motor Engagement during Invasion
Three-dimensional reconstruction of IFA z stacks showing steps of invasion from attachment (early), through mid and late invasion labeled with markers for the merozoite surface (MSP1) (A), the MSP1 sheddase (PfSUB2—tagged with an endogenous HA epitope) (, and parasite actin (C). Insets show bright-field and single slice deconvolution. The scale bar represents 2.0 μm. In 3D images, gamma settings were altered, and the grid represents 0.5 μm intervals.
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作者:admin@医学,生命科学 2011-01-26 00:17
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