The Absalon Lab

Coupling nuclear division cycles with cell growth

During the asexual stage of the life cycle, Plasmodium falciparum replicates via schizogony, a division mode that can be divided into a growth phase and a budding phase.

During the growth phase, the parasite undergoes multiple asynchronous rounds of mitosis with segregation of uncondensed chromosomes followed by nuclear division with an intact nuclear envelope. The budding phase occurs when the multi-nucleated syncytium is subjected to an asynchronous round of karyokinesis, coinciding with the segmentation of dozens of daughter cells known as merozoites (see figure).

  • How does each nucleus cycle independently?

  • How do nuclei maintain independent cell cycle stages within a common cytoplasm?

  • What evolutionary advantage does asynchronous mitosis bring to Plasmodium spp asexual replication?

Immunofluorescence assay using anti-Inner Membrane Complex protein (green) and anti-parasite plasma membrane protein (Red) antibodies with DAPI-stained DNA (Blue) highlight the complete segmentation of newly formed daughter wild type cells upon egress. 

Left panel: PMT image / Right panel: Merge of fluorescent channels

Airyscan LSM880 microscopy, Scale is 1 um. Unpublished data from Absalon Lab

The molecular replicative machinery utilized by the Plasmodium parasite, as well as whether the parasite uses local and global checkpoints to control the timing of DNA replication and nuclear division, remain a major gap of knowledge in the biology of the parasite.

Nuclear envelope formation & shaping

The eukaryotic genome is housed by a double lipid bilayer called the nuclear envelope (NE). During schizogony, the NE remains intact before chromosome segregation. This form of division is termed closed mitosis and it is considered to be the most ancient mechanism of eukaryotic cell division. Plasmodium falciparum begins mitosis with an intact NE and assembles an intranuclear mitotic spindle. The formation of mitotic spindles causes the most dramatic rearrangements of nuclear shape when the spherical nucleus adopts to an elongated dumbbell shape.

To understand the dynamics of the NE during closed mitosis our laboratory studies multiple inducible knockdown parasite lines with a defect in NE, including one mutant with the loss of its NE during replication (see figure). In addition, we are mapping the proteome of the outer and inner nuclear membrane utilizing the method of proximal-dependent labeling of proteins.

An understanding of the molecular mechanism of cell division in malaria parasites could reveal vulnerable targets for antimalarials. In addition, a mutation in NE proteins results in a broad spectrum of human diseases including cancer, therefore studying the divergent mitotic mechanism of Plasmodium spp might provide new insights on common properties of NE biology.​

Transmission electron microscopy of early schizont-stage parasites from a wild-type parasite (upper panel) and a transgenic parasite (lower panel).

Unpublished data from Absalon Lab

Expansion microscopy of malaria parasite

Each schizont-stage parasite produces dozen of daughter cells, each of which is around 1.5 µm in length and 1 µm in diameter. To study the dynamic of the nuclear envelope during the nuclear division of the malaria blood-stage parasite, we need to monitor cellular events at the microscopic scale.

To tackle this challenge, we will deploy a recently-developed technique named ExM for Expansion Microscopy that enables to physically expand preserved cells isotropically via a chemical process (see figure).

For instance, the combination of ExM and lattice light-sheet microscopy has allowed mapping 40 million synapses of a fly brain in 3D (see lower panel of the figure). Despite the promises of the method to study microorganisms, ExM has not yet been applied to malaria parasites or any protozoan.

Expansion microscopy (ExM) workflow: 

A biological specimen is fixed, labeled and tethered to the hydrogel polymer chains. A hydrogel made of closely spaced highly charged monomers is then polymerized evenly throughout the cells or tissue, intercalating between and around the labels. Then the embedded specimen goes through a mechanical homogenization step. The specimen–hydrogel composite is now ready for physical expansion by dialysis in low-salt buffer or water. Biomolecules or labels of interest remain bound to the expanded polymer network, which has pulled them apart. Adapted from Tillberg PW et al. 2016 Nature Biotechnology.

After expanding the fruit fly brain to four times its usual size, scientists used lattice light-sheet microscopy to image all of the dopaminergic neurons (green). Image: Gao et al./ Science 2019.

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