TY  - GEN
CY  - Heidelberg
AV  - public
Y1  - 2023///
TI  - The role of phase separation in centrin interactions during Plasmodium falciparum schizogony
ID  - heidok34219
A1  - Voß, Yannik
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/34219/
N2  - Extensive proliferation of the malaria-causing parasite Plasmodium falciparum inside red blood cells is essential for its pathogenesis and survival strategy. Here Plasmodium spp. proliferate asexually via schizogony, an atypical form of cell division, wherein the parasite undergoes multiple rounds of asynchronous nuclear division in a shared cytoplasm. The Plasmodium centrosome, the centriolar plaque, is a critical regulator in this process that nucleates mitotic spindle microtubules and limits proliferation as it needs to duplicate before each nuclear division. However, little is known about the molecular and physiochemical composition of this protein-dense and amorphous organelle.

Within this thesis, I investigated the localization, dynamics, and biochemical interactions of the four Plasmodium centrins, PfCen1-4, a family of highly conserved centrosomal proteins. I demonstrated that all four localize to the centriolar plaque at the onset schizogony and defined a coordinated disassembly event during its final stages. To study their localization in more detail, I adapted live-cell Stimulated Emission Depletion Microscopy (STED) to Plasmodium for the first time, which revealed a highly dynamic rearrangement of PfCen1-Halo within the centriolar plaque. Investigating the underlying interactions of the Ca2+-binding centrins via microscopy-assisted in vitro assays revealed that specifically PfCen1 and PfCen3 can undergo Ca2+-dependent liquid-liquid phase separation, individually or as joint condensates. By testing a phylogenetically diverse panel of eukaryotic centrins, including human centrin 2, I further demonstrated that phase separation capability is an evolutionarily conserved feature. In vitro mutagenesis revealed a critical dependence on the N-terminus, wherein the presence of an intrinsically disordered region is predictive of phase separation capability. As the protein density and absence of structural features within the centriolar plaque could be well explained by it being a proteinaceous liquid, I investigated whether centrin dynamics and localization might be driven by phase separation in vivo. As one of the defining features of phase separation is its dependency on a critical concentration, I developed pFIO, a new DiCre/loxP based inducible overexpression system for P. falciparum. This allowed me to exert better control over intracellular PfCen1 levels, where increasing concentration correlated with formation of artificial non-centrosomal assemblies and premature recruitment to the centriolar plaque. Loss of Ca2+-binding capability also abolished both in vitro phase separation and in vivo assembly. However, machine-learning assisted analysis of PfCen1 distribution in a large dataset of live cells revealed an inconsistent cellular concentration during assembly, arguing against a concentration threshold as a key trigger for localization. Centrosomal and artificial PfCen1-GFP assemblies also showed little turnover, suggesting a more solid state, but could still be dissolved and had no affinity to aggregate stains. This might point towards a gradual hardening process. 

This work represents the first study of liquid-liquid phase separation in Plasmodium and pioneered useful toolsets for its subsequent research. It further provides important context for future studies on centrins across the eukaryotic spectrum and has potential implications for the material state and assembly mechanism of the centriolar plaque.
ER  -