Imaging malaria parasites across scales and time.

The idea that disease is caused at the cellular level is so fundamental to us that we might forget the critical role microscopy played in generating and developing this insight. Visually identifying diseased or infected cells lays the foundation for any effort to curb human pathology. Since the discovery of the Plasmodium-infected red blood cells, which cause malaria, microscopy has undergone an impressive development now literally resolving individual molecules.

Malaria parasite centrins can assemble by Ca2+-inducible condensation.

Centrins are small calcium-binding proteins that have a variety of roles and are universally associated with eukaryotic centrosomes. 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. Their precise mode of action, however, remains unclear.

Progeny counter mechanism in malaria parasites is linked to extracellular resources.

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.

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

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.

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.

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.

Visualization of Infected Red Blood Cell Surface Antigens by Fluorescence Microscopy.

Immunofluorescence labeling enables the detection and characterization of various parasite proteins presented on the surface of the infected red blood cell. Several approaches for immunofluorescence detection of red blood cell surface-presented proteins of Plasmodium spp. have been successfully established and published over the years.

Asynchronous nuclear cycles in multinucleated facilitate rapid proliferation.

Malaria-causing parasites proliferate within erythrocytes through schizogony, forming multinucleated stages before cellularization. Nuclear multiplication does not follow a strict geometric 2 progression, and each proliferative cycle produces a variable 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.

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 RBCs 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.

An extended DNA-free intranuclear compartment organizes centrosome microtubules in malaria parasites.

Proliferation of in red blood cells is the cause of malaria and is underpinned by an unconventional cell division mode, called schizogony. Contrary to model organisms, replicates by multiple rounds of nuclear divisions that are not interrupted by cytokinesis. Organization and dynamics of critical nuclear division factors remain poorly understood.

How Many Is Enough? - Challenges of Multinucleated Cell Division in Malaria Parasites.

Regulating the number of progeny generated by replicative cell cycles is critical for any organism to best adapt to its environment. Classically, the decision whether to divide further is made after cell division is completed by cytokinesis and can be triggered by intrinsic or extrinsic factors. Contrarily, cell cycles of some species, such as the malaria-causing parasites, go through multinucleated cell stages.

Structural analysis of the SRP Alu domain from Plasmodium falciparum reveals a non-canonical open conformation.

The eukaryotic signal recognition particle (SRP) contains an Alu domain, which docks into the factor binding site of translating ribosomes and confers translation retardation. The canonical Alu domain consists of the SRP9/14 protein heterodimer and a tRNA-like folded Alu RNA that adopts a strictly 'closed' conformation involving a loop-loop pseudoknot. Here, we study the structure of the Alu domain from Plasmodium falciparum (PfAlu), a divergent apicomplexan protozoan that causes human malaria.

Apicomplexans: A conoid ring unites them all.

Apicomplexan parasites are defined by complex apical structures, which are necessary for interaction with incredibly diverse host cells. Two studies now amend a long-standing paradigm by showing conservation of an essential ring structure in the entire phylum.

CRISPR Interference of a Clonally Variant GC-Rich Noncoding RNA Family Leads to General Repression of Genes in Plasmodium falciparum.

The human malaria parasite uses mutually exclusive expression of the PfEMP1-encoding gene family to evade the host immune system. Despite progress in the molecular understanding of the default silencing mechanism, the activation mechanism of the uniquely expressed member remains elusive. A GC-rich noncoding RNA (ncRNA) gene family has coevolved with species that express genes.

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. A widely-used standard for parasite immunofluorescence is fixation in suspension with addition of minute amounts of glutaraldehyde to the paraformaldehyde-based solution.

Genome organization and DNA accessibility control antigenic variation in trypanosomes.

Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time.

CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for Gene Activation or Silencing in .

relies on monoallelic expression of 1 of 60 virulence genes for antigenic variation and host immune evasion. Each gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs.

Trans-acting GC-rich non-coding RNA at var expression site modulates gene counting in malaria parasite.

Monoallelic expression of the var multigene family enables immune evasion of the malaria parasite Plasmodium falciparum in its human host. At a given time only a single member of the 60-member var gene family is expressed at a discrete perinuclear region called the 'var expression site'. However, the mechanism of var gene counting remains ill-defined.

Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria.

Antigenic variation of the Plasmodium falciparum multicopy var gene family enables parasite evasion of immune destruction by host antibodies. Expression of a particular var subgroup, termed upsA, is linked to the obstruction of blood vessels in the brain and to the pathogenesis of human cerebral malaria. The mechanism determining upsA activation remains unknown.

Nuclear pores and perinuclear expression sites of var and ribosomal DNA genes correspond to physically distinct regions in Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum modifies the erythrocyte it infects by exporting variant proteins to the host cell surface. The var gene family that codes for a large, variant adhesive surface protein called P. falciparum erythrocyte membrane protein 1 (PfEMP1) plays a particular role in this process, which is linked to pathogenesis and immune evasion.

Silence, activate, poise and switch! Mechanisms of antigenic variation in Plasmodium falciparum.

Phenotypic variation in genetically identical malaria parasites is an emerging topic. Although antigenic variation is only part of a more global parasite strategy to create adaptation through epigenetically controlled transcriptional variability, it is the central mechanism enabling immune evasion and promoting pathogenesis. The var gene family is the best-studied example in a wide range of clonally variant gene families in Plasmodium falciparum.

ESCRT-III polymers in membrane neck constriction.

The endosomal sorting complex required for transport (ESCRT)-III machinery contributes to membrane deformation and scission in cytokinesis, intraluminal vesicle formation, autophagy and virus budding. Recombinant ESCRT-III subunits polymerize in vitro into filaments, tubes, sheets or rings, and ESCRT-III-dependent filaments have been observed in cells at virus bud necks and at the cytokinetic abscission site. These observations have inspired speculation about how ESCRT-III could mediate constriction and fission of membrane necks.

Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments.

After partitioning of cytoplasmic contents by cleavage furrow ingression, animal cells remain connected by an intercellular bridge, which subsequently splits by abscission. Here, we examined intermediate stages of abscission in human cells by using live imaging, three-dimensional structured illumination microscopy, and electron tomography. We identified helices of 17-nanometer-diameter filaments, which narrowed the cortex of the intercellular bridge to a single stalk.

Correlative time-lapse imaging and electron microscopy to study abscission in HeLa cells.

HeLa cells are widely used as a model system to study cell division. The last step of cell division, abscission, occurs at an about 1 μm wide intercellular bridge that connects the post-mitotic sister cells. Abscission often occurs long after ingression of the cleavage furrow, and no efficient methods to synchronize cells to this stage are available.