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Abstract
ABSTRACTMicroscopy-based localisation of proteins during malaria parasite (Plasmodium) invasion of the erythrocyte is widely used for tentative assignment of protein function. To date, however, imaging has been limited by the rarity of invasion events and the poor resolution available, given the micron size of the parasite, which leads to a lack of quantitative measures for definitive localisation. Here, using computational image analysis we have attempted to assign relative protein localisation during invasion using wide-field deconvolution microscopy. By incorporating three-dimensional information we present a detailed assessment of known parasite effectors predicted to function during entry but as yet untested or for which data are equivocal. Our method, termed longitudinal intensity profiling, resolves confusion surrounding the localisation of apical membrane antigen 1 (AMA1) at the merozoite–erythrocyte junction and predicts that the merozoite thrombospondin-related anonymous protein (MTRAP) is unlikely to play a direct role in the mechanics of entry, an observation supported with additional biochemical evidence. This approach sets a benchmark for imaging of complex micron-scale events and cautions against simplistic interpretations of small numbers of representative images for the assignment of protein function or prioritisation of candidates as therapeutic targets.
Highlights
In the biological sciences, microscopy is generally used to translate information contained within a tissue or cell specimen into a more useful format, such as a processed digital image
Variations in the extent of invasion still prohibit wholesale quantification across multiple parasites, the realignment provided by rotation and skew correcting steps in this workflow should allow unprecedented reliability in evaluating large numbers of merozoite invasion events
Interacts (Lamarque et al, 2011; Tyler and Boothroyd, 2011). These results suggest that apical membrane antigen 1 (AMA1) is present at the tight junction of invading P. falciparum merozoites, with micronemal and surface populations regularly present, but that selection of the appropriate immune label is critical
Summary
Microscopy is generally used to translate information contained within a tissue or cell specimen into a more useful format, such as a processed digital image. The degradation of data due to imaging and image analysis is generally well understood and accepted (North, 2006), the loss of information resulting from image selection and presentation is frequently ignored. One of the key strengths of high-throughput automated and quantitative imaging studies is that they circumvent these issues and allow the presentation of large quantities of data without interference from direct bias (Zhan et al, 2015). Received 22 July 2015; Accepted 16 November 2015 imaging workflows, are not amenable to such approaches. Imaging of Plasmodium falciparum merozoites caught during invasion of the human erythrocyte (Boyle et al, 2010b; Riglar et al, 2011) is one such example
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