Biology subjects

Guizetti, J.

Publications and source records attributed to Guizetti, J..

9 recordsLinked to original sources

bioRxiv2024

Real-Time Imaging of Antimalarial Drug Effects on Plasmodium falciparum-Infected Red Blood Cells

Malaria remains a significant global health challenge, exacerbated by emerging drug resistance, which has been recurring ever since Chloroquine resistance was reported to spread in the 1960s. Cabamiquine (M5717), a promising antimalarial candidate, targets Plasmodium falciparum elongation factor 2 (PfeEF2) and has demonstrated high potency and multi-stage activity. However, differences in parasite-killing kinetics require further exploration. This proof-of-concept study employs time-lapse microscopy to assess the effects of Chloroquine and Cabamiquine on P. falciparum-infected red blood cells. Using a fluorogenic DNA dye, we monitored morphological changes and cell death at the single-cell level. Unlike the faster-killing Chloroquine, our findings confirm a lag phase of the effect of Cabamiquine and reveal a delay before host cell lysis. This delayed cytotoxic effect underscores the cytostatic phase of the drug prior to killing and highlights its potential in combination therapies. Our novel imaging approach provides a comprehensive fingerprinting method to quantify cytostatic, cytotoxic, and cytolytic activities of antimalarial drugs, offering valuable insights into their mechanisms of action at a cellular level therefore enabling to discriminate the steps prior to parasite clearance in hosts.

cell biology↗

bioRxiv2023

Malaria parasite proliferation implicates a resource-dependent counter mechanism

Malaria is caused by the rapid proliferation of Plasmodium parasites in patients and disease severity correlates with the number of infected red blood cells in circulation. Parasite multiplication within red blood cells is called schizogony and occurs through an atypical multinucleated cell division mode. The mechanisms regulating the number of daughter cells produced by a single progenitor are poorly understood. We investigated underlying regulatory principles by quantifying nuclear multiplication dynamics in Plasmodium falciparum and knowlesi using super-resolution time-lapse microscopy. This revealed that the number of daughter cells was statistically independent of the duration of the nuclear division phase, which confirms that a counter mechanism, rather than a timer, regulates multiplication. P. falciparum cell volume at the start of nuclear division correlated with the final number of daughter cells. As schizogony progressed, the nucleocytoplasmic volume ratio, which has been found to be constant in all eukaryotes characterized so far, increased significantly, possibly to accommodate the exponentially multiplying nuclei. Depleting nutrients by dilution of culture medium caused parasites to produce less merozoites and reduced proliferation but did not affect cell volume or total nuclear volume at the end of schizogony. Our findings suggest that the counter mechanism implicated in malaria parasite proliferation integrates extracellular resource status to modify progeny number during blood stage infection.

cell biology↗

bioRxiv2022

Plasmodium falciparum CRK4 links early mitotic events to the onset of S-phase during schizogony

Plasmodium falciparum proliferates through schizogony in the clinically relevant blood stage of infection. During schizogony, consecutive rounds of DNA replication and nuclear division give rise to multinucleated stages before cellularization occurs. Although these nuclei reside in a shared cytoplasm, DNA replication and nuclear division occur asynchronously. Here, by mapping the proteomic context of the S-phase-promoting kinase PfCRK4, we show that it has a dual role for nuclear-cycle progression: PfCRK4 orchestrates not only DNA replication, but also the rearrangement of intranuclear microtubules. Live-cell imaging of a reporter parasite showed that these microtubule rearrangements coincide with the onset of DNA replication. Together, our data render PfCRK4 the key factor for nuclear-cycle progression, linking entry into S-phase with the initiation of mitotic events. In part, such links may compensate for the absence of canonical cell cycle checkpoints in P. falciparum.

microbiology↗

bioRxiv2022

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 microtubule homeostasis caused polymerization of excess microtubules and aberrant mitotic spindles. Time-lapse microscopy revealed that this prevented or delayed spindle extension. Our study thereby identifies a novel extranuclear centriolar plaque factor and establishes a functional link to the intranuclear compartment of this divergent eukaryotic centrosome.

cell biology↗

bioRxiv2022

Ca2+-inducible phase separation of centrins in proliferating malaria parasites

Rapid proliferation of the malaria-causing parasite Plasmodium falciparum in the human blood depends on a particularly divergent and acentriolar centrosome, which incorporates several essential centrins. Centrins are small calcium-binding proteins that have a variety of roles and are universally associated with eukaryotic centrosomes. Their precise mode of action, however, remains unclear. In this study calcium-inducible liquid-liquid phase separation is revealed as an evolutionary conserved principle of assembly for Plasmodium and human centrins. Furthermore, the disordered N-terminus and calcium-binding motifs are defined as essential features for reversible biomolecular condensation and demonstrate that certain centrins can co-condensate. In vivo analysis using live-cell STED microscopy shows liquid-like dynamics of centrosomal centrin. Additionally, implementation of an inducible protein overexpression system reveals concentration-dependent formation of extra-centrosomal centrin assemblies with condensate-like properties. The timing of foci formation and dissolution suggests that centrin assembly is regulated. This study thereby provides a new model for centrin accumulation at eukaryotic centrosomes.

cell biology↗

bioRxiv2021

Asynchronous nuclear cycles in multinucleated Plasmodium falciparum enable rapid proliferation

Malaria-causing parasites proliferate within erythrocytes through schizogony, forming multinucleated stages before cellularization. Nuclear multiplication does not follow a strict geometric 2n progression and each proliferative cycle produces a heterogeneous number of progeny. Here, by tracking nuclei and DNA replication, we show that individual nuclei replicate their DNA at different times, despite residing in a shared cytoplasm. Extrapolating from experimental data using mathematical modeling, we demonstrate that a limiting factor must exist that slows down the nuclear multiplication rate. Indeed, our data show that temporally overlapping DNA replication events were significantly slower than partially or non-overlapping events. Our findings suggest an evolutionary pressure that selects for asynchronous DNA replication, balancing available resources with rapid pathogen proliferation.

microbiology↗

bioRxiv2021

An extended DNA-free intranuclear compartment organizes centrosomal microtubules in Plasmodium falciparum

Rapid proliferation of Plasmodium falciparum parasites in human red blood cells is the cause of malaria and is underpinned by an unconventional cell division mode, called schizogony. Contrary to model organisms, P. falciparum replicates by multiple rounds of closed and asynchronous nuclear divisions that are not interrupted by cytokinesis. Organization and dynamics of the critical nuclear division factors are, however, poorly understood. Centriolar plaques, the centrosomes of P. falciparum, are important regulators of division and serve as microtubule organizing centers. Early microscopy studies reveal an acentriolar, amorphous structure although its detailed organization remains elusive. Intranuclear microtubules mediate chromosome segregation, but the small size of parasite nuclei has precluded detailed analysis of their arrangement by classical fluorescence microscopy. We apply recently developed STED, expansion microscopy, and live cell imaging protocols to describe the reconfiguration of microtubules during schizogony. Analysis of centrin, nuclear pore, and microtubule positioning reveals a bipartite organization of the centriolar plaque. While centrin is extranuclear, we confirm by correlative light and electron tomography that microtubules are nucleated in a previously unknown and extended intranuclear compartment, which is devoid of chromatin. This study enables us to build a working model of the organization of an unconventional centrosome and better understand the diversity of eukaryotic cell division modes.

cell biology↗

bioRxiv2021

Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence

The pathology associated with malaria infection is largely due to the ability of infected human erythrocytes to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. Taken together, our data reveal a role for PFA66 in host cell modification, implicate human HSP70 as also being essential in this process, and uncover a KAHRP-independent mechanism for correct knob biogenesis. Our observations open up exciting new avenues for the development of new anti-malarials.

cell biology↗

bioRxiv2018

Improved immunofluorescence staining protocol for STED nanoscopy of Plasmodium-infected red blood cells

Immunofluorescence staining is the key technique for visualizing organization of endogenous cellular structures in single cells. Labeling and imaging of blood stage Plasmodium falciparum has always been challenging since it is a small intracellular parasite. The gold standard for parasite immunofluorescence is fixation in suspension with addition of minute amounts of glutaraldehyde to the paraformaldehyde-based solution. While this maintains red blood cell integrity, it has been postulated that antigenicity of the parasite proteins was, if at all, only slightly reduced. Here we show the deleterious effect that even these small quantities of glutaraldehyde can have on immunofluorescence staining quality and present an alternative cell seeding protocol that allows fixation with only paraformaldehyde. The highly improved signal intensity and staining efficiency enabled us to carry out RescueSTED nanoscopy on microtubules and nuclear pores and describe their organization in greater detail throughout the blood stage cycle.\n\nHighlights- Omitting glutaraldehyde from fixative improves immunofluorescence staining\n\n- STED nanoscopy is readily applicable to infected red blood cells\n\n- Intranuclear microtubules are nucleated from distinct sites

cell biology↗