Yannik Voß (Heidelberg / DE), Christoph Wenz (Heidelberg / DE), Caroline Simon (Heidelberg / DE), Vanessa Stürmer (Heidelberg / DE), Tatiany Patricia Romao (Heidelberg / DE), Dr. Severina Klaus (Heidelberg / DE), Marta Machado (Heidelberg / DE), Anja Klemmer (Heidelberg / DE), Dr. Markus Ganter (Heidelberg / DE), Mathieu Brochet (Geneva / CH), Dr. Julien Guizetti (Heidelberg / DE)
Abstract text
Throughout its life cycle Plasmodium falciparum undergoes several stages of extensive proliferation that are integral to survival and transmission of this malaria-causing parasite. Rapid parasite multiplication in the human red blood cell occurs via an atypical cell division mode, called schizogony, and relies on a particularly divergent centrosome. This so called centriolar plaque, functions as the main microtubule organizing center (MTOC) for the mitotic spindle and lacks conventional structures, such as centrioles. Our previous investigation using a combination of super-resolution, live cell, and correlative microscopy have revealed that it rather consists of two amorphous protein dense regions spanning across the nuclear membrane (see Figure). Composition and assembly of this atypical centrosome remain largely unknown.
To better understand the centriolar plaque we investigated centrins, which are small calcium-binding proteins and among the very few universally conserved MTOC components. Contrary to previous hypotheses we found liquid-liquid phase-separation (LLPS) as a driver behind the calcium-inducible self-assembly of some centrins in vitro. LLPS has emerged as biophysical model explaining the accumulation of concentrated proteins in droplet-like structures, which can contribute to the formation of membraneless compartments, such as centrosomes. To verify LLPS of PfCentrin1 in parasites we employed live cell STED and designed a novel inducible overexpression system.
Co-immunoprecipitation of PfCentrin1 further revealed a Sfi1-like protein (PfSlp) as novel centriolar plaque component. Conditional knock down of PfSlp caused a growth delay in blood stages and a reduced number of daughter cells. Surprisingly, intranuclear tubulin abundance was significantly increased, which raises the hypothesis that the centriolar plaque might be implicated in regulating tubulin homeostasis. Time-lapse microscopy revealed that this misregulation prevented or delayed spindle extension.
Taken together our data indicates that the centrin/Slp protein pair is a core component of the centriolar plaque and might implicate phase separation in its assembly.