Fig. 4: Knock sideways of FRM2 leads to an arrest in gametocyte development and disrupted actin and microtubule networks.
From: Plasmodium falciparum formins are essential for invasion and sexual stage development
a Live-cell microscopy of stage IV gametocytes treated with ethanol (Control) or Rapalog (Rapa). Mislocalisation of FRM2-GFP to the nucleus using the NLS mislocaliser resulted in irregular morphologies including abnormal (middle panel) and rounded morphologies (bottom panel). The nucleus is delineated by the native fluorescence from the mCherry reporter incorporated into the NLS mislocaliser (red). FRM2-GFP (green) is evident at the periphery of the nucleus. Scale bars = 5 μm. b Images of Giemsa-stained cells following treatment with and without Rapa. Representative examples of gametocytes from day 7 of the assay when control cells exhibited mostly stage IV morphology. Scale bars = 5 μm. c Graphs showing the gametocyte stage distribution for day 4, 6, 8 and 10. Giemsa counts were performed from four separate experiments. Gametocytes were separated based on morphology (stage III–V). Irregular parasites include those that are abnormal or rounded. The means and standard error of the mean are shown from three independent experiments. d, e 3D-Structured Illumination Microscopy of stage IV control (ethanol) and Rapalog (Rapa) treated gametocytes. d Gametocytes were labelled with anti-GFP (green) and anti-actin (red). White arrows point to short, stabilised actin filaments. Yellow arrows highlight long, stabilised actin filaments. Blue arrows depict FRM2-GFP puncta positioned between actin filaments. e Gametocytes labelled with anti-GFP (green) and anti-β-tubulin (red). White arrows highlight the preferential bundling of microtubules to one side of the gametocyte. Blue arrows point to a lone bundle of microtubules present in Rapa-treated gametocytes with an abnormal morphology (middle panel). Yellow arrows depict FRM2-GFP puncta positioned where microtubules terminate. Scale bars = 5 μm. Zoomed images are displayed below each panel. Zoom scale bars = 1 μm. Source data for this figure is provided in the Source Data files.
