The role of phase separation in centrin interactions during Plasmodium falciparum schizogony

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

Plasmodium schizogony, a chronology of the parasite's cell cycle in the blood stage.

Malaria remains a significant threat to global health, and despite concerted efforts to curb the disease, malaria-related morbidity and mortality increased in recent years. Malaria is caused by unicellular eukaryotes of the genus Plasmodium, and all clinical manifestations occur during asexual proliferation of the parasite inside host erythrocytes. In the blood stage, Plasmodium proliferates through an unusual cell cycle mode called schizogony. Contrary to most studied eukaryotes, which divide by binary fission, the parasite undergoes several rounds of DNA replication and nuclear division that are not directly followed by cytokinesis, resulting in multinucleated cells. Moreover, despite sharing a common cytoplasm, these nuclei multiply asynchronously. Schizogony challenges our current models of cell cycle regulation and, at the same time, offers targets for therapeutic interventions. Over the recent years, the adaptation of advanced molecular and cell biological techniques have given us deeper insight how DNA replication, nuclear division, and cytokinesis are coordinated. Here, we review our current understanding of the chronological events that characterize the unusual cell division cycle of P. falciparum in the clinically relevant blood stage of infection

An Sfi1-like centrin-interacting centriolar plaque protein affects nuclear microtubule homeostasis.

Malaria-causing parasites achieve rapid proliferation in human blood through multiple rounds of asynchronous nuclear division followed by daughter cell formation. Nuclear divisions critically depend on the centriolar plaque, which organizes intranuclear spindle microtubules. The centriolar plaque consists of an extranuclear compartment, which is connected via a nuclear pore-like structure to a chromatin-free intranuclear compartment. Composition and function of this non-canonical centrosome remain largely elusive. Centrins, which reside in the extranuclear part, are among the very few centrosomal proteins conserved in Plasmodium falciparum. Here we identify a novel centrin-interacting centriolar plaque protein. Conditional knock down of this Sfi1-like protein (PfSlp) caused a growth delay in blood stages, which correlated with 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 levels. Disruption of tubulin homeostasis caused excess microtubules and aberrant mitotic spindles. Time-lapse microscopy revealed that this prevented or delayed mitotic spindle extension but did not significantly interfere with DNA replication. Our study thereby identifies a novel extranuclear centriolar plaque factor and establishes a functional link to the intranuclear compartment of this divergent eukaryotic centrosome

Measurement of the top quark pole mass using tt‾ \textrm{t}\overline{\textrm{t}} +jet events in the dilepton final state in proton-proton collisions at s \sqrt{s} = 13 TeV

A measurement of the top quark pole mass ${{m_{\mathrm{t}}} ^{\text{pole}}}$ in events where a top quark-antiquark pair ($\mathrm{t\bar{t}}$) is produced in association with at least one additional jet ($\mathrm{t\bar{t}}$+jet) is presented. This analysis is performed using proton-proton collision data at $\sqrt{s} =$ 13 TeV collected by the CMS experiment at the CERN LHC, corresponding to a total integrated luminosity of 36.3 fb$^{-1}$. Events with two opposite-sign leptons in the final state (e$^{+}$e$^{-}$, $\mu^{+}\mu^{-}$, e$^{\pm}\mu^{\mp}$) are analyzed. The reconstruction of the main observable and the event classification are optimized using multivariate analysis techniques based on machine learning. The production cross section is measured as a function of the inverse of the invariant mass of the $\mathrm{t\bar{t}}$+jet system at the parton level using a maximum likelihood unfolding. Given a reference parton distribution function (PDF), the top quark pole mass is extracted using the theoretical predictions at next-to-leading order. For the ABMP16NLO PDF, this results in ${{m_{\mathrm{t}}} ^{\text{pole}}} =$ 172.94 $\pm$ 1.37 GeV.A measurement of the top quark pole mass ${m}_{\textrm{t}}^{\textrm{pole}}$ in events where a top quark-antiquark pair ($\textrm{t}\overline{\textrm{t}}$) is produced in association with at least one additional jet ($\textrm{t}\overline{\textrm{t}}$+jet) is presented. This analysis is performed using proton-proton collision data at $\sqrt{s}$ = 13 TeV collected by the CMS experiment at the CERN LHC, corresponding to a total integrated luminosity of 36.3 fb$^{−1}$. Events with two opposite-sign leptons in the final state (e$^{+}$e$^{−}$, μ$^{+}$μ$^{−}$, e$^{±}$μ$^{∓}$) are analyzed. The reconstruction of the main observable and the event classification are optimized using multivariate analysis techniques based on machine learning. The production cross section is measured as a function of the inverse of the invariant mass of the $\textrm{t}\overline{\textrm{t}}$+jet system at the parton level using a maximum likelihood unfolding. Given a reference parton distribution function (PDF), the top quark pole mass is extracted using the theoretical predictions at next-to-leading order. For the ABMP16NLO PDF, this results in ${m}_{\textrm{t}}^{\textrm{pole}}$ = 172.93 ± 1.36 GeV.[graphic not available: see fulltext]A measurement of the top quark pole mass $m_\mathrm{t}^\text{pole}$ in events where a top quark-antiquark pair ($\mathrm{t\bar{t}}$) is produced in association with at least one additional jet ($\mathrm{t\bar{t}}$+jet) is presented. This analysis is performed using proton-proton collision data at $\sqrt{s}$ = 13 TeV collected by the CMS experiment at the CERN LHC, corresponding to a total integrated luminosity of 36.3 fb$^{-1}$. Events with two opposite-sign leptons in the final state (e$^+$e$^-$, $\mu^+\mu^-$, e$^\pm\mu^\mp$) are analyzed. The reconstruction of the main observable and the event classification are optimized using multivariate analysis techniques based on machine learning. The production cross section is measured as a function of the inverse of the invariant mass of the $\mathrm{t\bar{t}}$+jet system at the parton level using a maximum likelihood unfolding. Given a reference parton distribution function (PDF), the top quark pole mass is extracted using the theoretical predictions at next-to-leading order. For the ABMP16NLO PDF, this results in $m_\mathrm{t}^\text{pole}$ = 172.93 $\pm$ 1.36 GeV