A genetically encoded marker for imaging membranes of Plasmodium

Fluorescence microscopy is a powerful tool to analyze subcellular architecture, and long-term live-cell imaging permits the analysis of the dynamics of intracellular structures. Applying this approach to the membranes of Plasmodium spp., the causative agent of malaria, yielded important insight. Fluorescent labelling of Plasmodium ’s plasma membrane via membrane-resident proteins has been described, but proteins, which broadly mark internal membranes are currently elusive. Alternatively, general membrane dyes can be employed to study membrane dynamics, however, we find that the membrane dye BODIPY TR Ceramide has adverse effects on cell viability during live-cell imaging. To overcome this limitation, we present FLUMMI (Fixed-and-Live-cell Universal Membrane Marker for Imaging), a 32-amino acid long peptide derived from P. falciparum PCNA1, which targets (fluorescent) protein tags to internal membranes for detection in live or fixed samples. Importantly, FLUMMI enables non-invasive, long-term live-cell imaging of internal membranes without affecting P. falciparum viability and is functional in blood stages of both, the human malaria parasite P. falciparum and the rodent-infecting parasite P. berghei , as well as in P. berghei mosquito stages. FLUMMI is also compatible with advanced techniques such as expansion microscopy. Together, our results establish FLUMMI as a versatile and non-toxic tool for membrane imaging in Plasmodium .