Final Report Summary - MALGENEXPRESSION (The role of a nuclear expression site in the regulation of virulence genes in malaria parasites)
Project context and objectives
As a first step towards understanding nuclear structure and dynamics in malaria parasites, we were interested in identifying and characterising the organisation of Plasmodium falciparum nuclear pores during the intra-erythrocytic development cycle (IDC). To our knowledge, the nuclear pore complexes (NPCs) of blood stage parasites have previously only been superficially described. Transmission electron microscopy (TEM) of early schizonts prepared by conventional chemical fixation revealed the classic NPC structure as an electron-dense disk perforating the double membrane of the nuclear envelope (NE). Encouraged by these observations of the NPC in thin sections, we were interested in having a broader view of their organisation at the NE. Red blood cell (RBC) samples infected with late-stage parasites were vitrified by high pressure freezing and subjected to freeze-fracture, then imaged by cryogenic scanning electron microscopy (cryo-SEM). This method affords a surface view of the fracture plane, which in numerous instances coincided with the parasite NE. In some fractures we were able to view the cytoplasmic face of the nuclear pores, while in others the chromatin was removed, revealing the nucleoplasmic face and the double membrane in section. The size of the pores is slightly smaller than what have been described in yeast (around 80 nm). Interestingly, the pores seemed not to be evenly distributed around the NE.
In budding yeast, the nuclear pores are distributed around the NE and create islands of loose euchromatin that may allow transcription within the condensed heterochromatic region of the nuclear periphery. Therefore we were interested to determine how the uneven distribution of nuclear pores observed by freeze-fracture may be correlated to the chromatin organisation within the nucleus. We prepared thin sections of parasites by high pressure freezing and freeze substitution. This method provides superior ultrastructural preservation of chromatin due to cryogenic immoblilisation and gentle dehydration of the sample. There are distinct differences in chromatin organisation between parasites at different stages of the IDC. In late schizonts, one can clearly distinguish between the densely stained heterochromatic regions and the lighter euchromatic regions in the nuclei. In sharp contrast, the organisation of chromatin in the earlier trophozoite phase shows patches of electron-dense genetic matter scattered throughout the nucleoplasm. In one telling example, a single RBC was infected by two parasites at different stages, a trophozoite and a late schizont, side by side. We also noted that while numerous nuclear pores could be seen in trophozoites, very few could be detected in thin sections through schizonts. In both stages, we noted that the nuclear pores are located near regions of loose euchromatin and not adjacent to the electron-dense heterochromatin.
A full verification of this suggestive association is not possible using isolated thin sections, but requires a full 3D view of multiple nuclei. To that end, we applied the emerging technique of ion abrasion by focused ion beam (FIB) milling within a scanning electron microscope (SEM). Known as 'slice and view', this method provides automated acquisition of serial section images as thin as 10 nm. Thus a single nucleus is spanned typically by around 150 sections. A three-dimensional (3D) model is generated directly, without the need for tomographic reconstruction and without the limitations imposed by sample tilt geometry. We applied this method to investigate nuclear dynamics in trophozoite, and through schizogony from early schizont to late schizont stage parasites. Analysis of these 3D models of whole nuclei (in fact, whole parasites) shows distinctly different patterns of sub-nuclear organisation among the different stages. In trophozoites there are numerous nuclear pores spread around the NE, similar to what is observed in other eukaryotic organisms. In addition, at that stage the heterochromatic regions are distributed as patches throughout the nucleoplasm. At early schizogony, the nuclear architecture is similar to that of trophozoites, albeit with an apparent decrease in the number of NPCs. However, as schizogony progresses and the number of nuclei increases, the nuclear organization changes dramatically. The heterochromatin takes on a very distinctive compact pattern, cuts through which are entirely consistent with previous observations in TEM of thin sections. Late schizonts contain still fewer NPCs per nucleus than mid schizonts. The nuclear pores now cluster together at the nuclear envelope at a location corresponding, within the nucleus, to a groove like structure of loose matter surrounded by condensed heterochromatin. The 3D information demonstrates unambiguously that nuclear pores are found adjacent to euchromatin regions only.
As a first step towards understanding nuclear structure and dynamics in malaria parasites, we were interested in identifying and characterising the organisation of Plasmodium falciparum nuclear pores during the intra-erythrocytic development cycle (IDC). To our knowledge, the nuclear pore complexes (NPCs) of blood stage parasites have previously only been superficially described. Transmission electron microscopy (TEM) of early schizonts prepared by conventional chemical fixation revealed the classic NPC structure as an electron-dense disk perforating the double membrane of the nuclear envelope (NE). Encouraged by these observations of the NPC in thin sections, we were interested in having a broader view of their organisation at the NE. Red blood cell (RBC) samples infected with late-stage parasites were vitrified by high pressure freezing and subjected to freeze-fracture, then imaged by cryogenic scanning electron microscopy (cryo-SEM). This method affords a surface view of the fracture plane, which in numerous instances coincided with the parasite NE. In some fractures we were able to view the cytoplasmic face of the nuclear pores, while in others the chromatin was removed, revealing the nucleoplasmic face and the double membrane in section. The size of the pores is slightly smaller than what have been described in yeast (around 80 nm). Interestingly, the pores seemed not to be evenly distributed around the NE.
In budding yeast, the nuclear pores are distributed around the NE and create islands of loose euchromatin that may allow transcription within the condensed heterochromatic region of the nuclear periphery. Therefore we were interested to determine how the uneven distribution of nuclear pores observed by freeze-fracture may be correlated to the chromatin organisation within the nucleus. We prepared thin sections of parasites by high pressure freezing and freeze substitution. This method provides superior ultrastructural preservation of chromatin due to cryogenic immoblilisation and gentle dehydration of the sample. There are distinct differences in chromatin organisation between parasites at different stages of the IDC. In late schizonts, one can clearly distinguish between the densely stained heterochromatic regions and the lighter euchromatic regions in the nuclei. In sharp contrast, the organisation of chromatin in the earlier trophozoite phase shows patches of electron-dense genetic matter scattered throughout the nucleoplasm. In one telling example, a single RBC was infected by two parasites at different stages, a trophozoite and a late schizont, side by side. We also noted that while numerous nuclear pores could be seen in trophozoites, very few could be detected in thin sections through schizonts. In both stages, we noted that the nuclear pores are located near regions of loose euchromatin and not adjacent to the electron-dense heterochromatin.
A full verification of this suggestive association is not possible using isolated thin sections, but requires a full 3D view of multiple nuclei. To that end, we applied the emerging technique of ion abrasion by focused ion beam (FIB) milling within a scanning electron microscope (SEM). Known as 'slice and view', this method provides automated acquisition of serial section images as thin as 10 nm. Thus a single nucleus is spanned typically by around 150 sections. A three-dimensional (3D) model is generated directly, without the need for tomographic reconstruction and without the limitations imposed by sample tilt geometry. We applied this method to investigate nuclear dynamics in trophozoite, and through schizogony from early schizont to late schizont stage parasites. Analysis of these 3D models of whole nuclei (in fact, whole parasites) shows distinctly different patterns of sub-nuclear organisation among the different stages. In trophozoites there are numerous nuclear pores spread around the NE, similar to what is observed in other eukaryotic organisms. In addition, at that stage the heterochromatic regions are distributed as patches throughout the nucleoplasm. At early schizogony, the nuclear architecture is similar to that of trophozoites, albeit with an apparent decrease in the number of NPCs. However, as schizogony progresses and the number of nuclei increases, the nuclear organization changes dramatically. The heterochromatin takes on a very distinctive compact pattern, cuts through which are entirely consistent with previous observations in TEM of thin sections. Late schizonts contain still fewer NPCs per nucleus than mid schizonts. The nuclear pores now cluster together at the nuclear envelope at a location corresponding, within the nucleus, to a groove like structure of loose matter surrounded by condensed heterochromatin. The 3D information demonstrates unambiguously that nuclear pores are found adjacent to euchromatin regions only.