A Genetically Encoded Marker for Imaging of Internal Plasmodium Membranes

Fluorescence microscopy is a powerful tool to analyze the subcellular architecture, and long-term live-cell imaging permits the analysis of the dynamics of intracellular structures, such as membranes. Fluorescent labeling of the plasma membrane of Plasmodium, the causative agent of malaria, via membrane-resident proteins has been described, but proteins that 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 IMMP (Internal Membrane Marker for Plasmodium), 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, IMMP 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. IMMP is also compatible with advanced techniques such as expansion microscopy. Together, our results establish IMMP as a tool for internal membrane imaging in Plasmodium.