In situ fluorescence visualization of transcription sites and genomic Loci in blood stages of Plasmodium falciparum

Abstract

Fluorescence-based techniques have been used extensively in the malaria field to study the functional role of nuclear organization and gene positioning in blood stages of the human malaria parasite, Plasmodium falciparum. In this chapter, we present optimized protocols for bromouridine (BrUTP) incorporation into nascent RNA in live parasites and fluorescent in situ hybridization (FISH) in fixed parasites. Methodology to perform various combinations of the FISH assay, as well as a basic approach for quantitative analysis of nuclear position, is also described.

Fig. 1

General outline of the BrUTP transcription and FISH assays. (a) The BrUTP incorporation and immunodetection by microscopy that has been adapted to P. falciparum parasites is a 2-day experiment. The parasites are first released from erythrocytes, lightly permeabilized, and incubated for 10–20 min in a transcriptional buffer containing BrUTP. After washing off the non-incorporated nucleotides, parasites are fixed and subject to immunofluorescence to detect BrRNA. The anti-BrdU antibodies are incubated overnight and the fluorophore-conjugated secondary antibodies for 1 h. This protocol preserves transcriptional activity and nuclear structure; it is performed entirely with parasites in suspension and is suited for simultaneous detection of proteins or nuclear substructures. (b) The traditional FISH is a 2-day experiment: on day 1 parasites are fixed, permeabilized, and hybridized overnight with labeled oligonucleotide probes, which bind to their complementary sequences. On day 2, the unbound probes are washed off and the slides are mounted and examined (dashed lines are optional or method-specific steps). The FISH probes are usually prepared in advance and can be directly coupled to biotin, digoxigenin, or fluorochromes. Nonfluorescent probes require a second step of detection (dashed box) using fluorophore-conjugated antibodies, which offers the advantage that signal can be amplified by using additional antibodies. The FISH protocol can be combined with immunofluorescence (immuno-FISH) allowing colocalization of protein, specific genes, and either genomic DNA or nuclear substructures. After immunofluorescence, parasites are fixed again and then subjected to FISH.

Fig. 2

Representative images of pre-replicative blood-stage parasites in various combinations of BrUTP labeling and FISH techniques. (a–d) Br-UTP labeling of nascent RNA with and without α-amanitin, an inhibitor of Pol II and III. (c, d) Colocalization of BrRNA (green) with the nucleolus (red, PfPol I). (e) DNA-FISH analysis using the exon2 probe that cross-hybridizes to group B of the var multigene family. (f) RNA-FISH analysis of var transcripts from group B. Note that the same probe is used in (e, f) (only the hybridization and washing conditions were different for the DNA- and RNA-FISH). (g) RNA/DNA-FISH of var transcripts (green) and telomeric clusters (rep20, red). (h) Immunofluorescence using antibodies against the nucleolar protein PfNop1 (red) combined with DNA-FISH for an individual var gene (var2CSA, green). (i) Two-color DNA-FISH of telomeric clusters (rep20, green) and 28S rRNA (red). In all cases, parasites are in the ring stage and nuclear DNA stained with DAPI (blue). Representative examples are shown as merged images. Scale bar: 1 μm. (j) Current model of nuclear organization in ring-stage parasites. The nucleolus is represented in a half-moon shape at the nuclear periphery, opposing the telomeric clusters. These two DNA-based subnuclear compartments can be used as reference for colocalization studies. Other nuclear landmarks are the transcription sites or factories distributed in the nucleolus and nucleoplasm. It is speculated that the few transcription sites observed in the nucleoplasm do not correspond to single genes, thus implying nuclear clustering of multiple actively transcribing genes at each factory (6, 8).

Fig. 3

Microscope slide setup for FISH analysis. The Teflon-coated surface delimitates and reduces the region to apply the parasite suspension and solutions or antibodies. The rubber in situ frame when covered with a coverslip (also in plastic) creates a small hybridization chamber that allows a reduction on the amount of probe required for each assay. This system also prevents against evaporation during the overnight hybridization. The frames are adhesive on both sides, and can be applied and removed easily from the microscope slide using forceps. We usually use microscope slides with three wells (and three frames), thus allowing for testing three different conditions per slide.

Fig. 4

Subnuclear localization relative to the nuclear periphery. (a) DNA-FISH analysis of a single locus gene (PFB0540w, green) in ring-stage parasites. Nuclear DNA is visualized with DAPI (blue). Scale bar: 1 μm. (b) Schematic representation of the measurements of nuclear diameter (d, yellow line) and the distance of the green fluorescent spot to the nearest periphery (x, white line). The extremity of DAPI staining (defined by the low-intensity staining, see Note 26) represents the perimeter of the nucleus (dashed line). (c) Illustration of the three concentric zones of equal surface (zones 1, 2, and 3). As described in (15), the most peripheral zone (zone 1) corresponds to a ring of width 0.184 × nuclear radius (r). Zone 2 lies between 0.184r and 0.442r from the periphery, and zone 3 is a central core of radius 0.578r. In the example shown in a, p = 0.53, which falls in zone 3. Analysis of position is generally performed on 100–200 parasite nuclei, and the percentage of measured distribution is compared to a random distribution (33.3% in the 3 zones) or to another condition, using a chi-square test.