UNRAVELING THE NUCLEAR ARCHITECTURE OF PLASMODIUM FALCIPARUM USING CRYO-SCANNING TRANSMISSION ELECTRON TOMOGRAPHY (CSTET)
2Department of Chemical and Structural Biology, Weizmann Institute of Science, Rehovot, Israel
3Department of Microbiology & Molecular Genetics, Hebrew University of Jerusalem-Hadassah Medical School, Jerusalem, Israel
4Laboratory for Bio- and Nano-Instrumentation, École polytechnique fédérale de Lausanne (EPFL), Laussane, Switzerland
5Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel
6Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel
Malaria is caused by the eukaryotic protozoan parasite-Plasmodium falciparum. The parasites have a complex life-cycle alternating between the female Anopheles mosquito and the human host. Following an initial asymptomatic phase of development within the human hepatocytes, the parasites infect red blood cells (RBCs), where they undergo cyclic asexual multiplication thereby enabling disease progression. The life-cycle of the parasite within the infected RBC (or iRBC) is marked by the presence of morphologically distinct parasitic forms (rings, trophozoites, and schizonts). These forms are characterized by concomitant changes in transcription, translation, and also DNA synthesis1. The controlled modulation of gene expression is dependent on the three-dimensional (3D) organization of the chromatin and its regulatory elements within the nucleus2. Emerging views suggest that regions of apparent compaction are not always hallmarks of gene silencing and that the chromatin domains are dynamic. These aspects of gene regulation in conjunction with the nuclear architecture largely remain unexplored at the ultrastructural levels.
Classic electron microscopy (EM) methods that have been employed to study genome organization have traditionally relied on the use of harsh chemical fixation that can change native cellular structures. To reliably understand the ultrastructure of the parasite and the nuclear architecture in situ, new cryogenic methods that rely on ultra-rapid vitrification to preserve biological ultrastructure in the near-native state are preferred. Cryo-scanning transmission electron tomography (CSTET) is a new addition to the 3D microscopy tool chest3,4. In CSTET, a focused electron probe raster scans the sample and a series of projection images are obtained by tilting the sample, which can be used to create a 3D map of the cellular structures.
The data obtained from CSTET supports our initial hypothesis that in the early stages of the asexual blood-stage of the life-cycle in the iRBCs (viz rings and trophozoites), the chromatin appears decondensed and diffused, followed by compaction of the chromatin in the nuclei of the later stages (late trophozoites and schizonts)5. Combined with cryo-fluorescence light microscopy, we are able to correlate nuclear domains across different stages of development (CLEM). Additionally, we have used a parasite line expressing mCherry-heterochromatin protein -1 (HP1)- a protein that naturally associates with heterochromatin, to understand the presence of chromatin domains within the nucleus6. Currently, we are integrating innovative image analysis tools as a newer approach to further the understanding of chromatin domains within the parasite7.
References:
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2) Batugedara, G. and Le Roch, K. G. (2019) doi: 10.1016/j.semcdb.2018.07.015.
3) Dahan-Pasternak, N. et al. (2013) doi: 10.1242/jcs.122119.
4) Waugh, B. et al. (2020) doi: 10.1073/PNAS.2000700117/-/DCSUPPLEMENTAL.
5) Weiner, A. et al. (2011) doi: 10.1111/j.1462-5822.2011.01592.x.
6) Wolf, S. G. et al. (2017) doi: 10.7554/eLife.29929.
7) Wolf, S. G., Houben, L. and Elbaum, M. (2014) doi: 10.1038/nmeth.2842.