Figure 1.
TcSR62 behaviour under several stress conditions.
(A) Transcription inhibition was induced incubating epimastigotes with ActD for 24 h. Genotoxic stress was induced with phleomycin (phleo), and protein synthesis inhibition with cycloheximide (CHX) for 24 h, respectively. Oxidative stress was induced subjecting the cells to sodium arsenite (ARS) for 2 h. Acid pH stress was induced incubating cells in BHT pH 5.5 for 24 h. Heat shock was induced after incubating cells at 37°C for 24 h. (B) Localization detail of TcSR62 in parasites subjected or not to transcription inhibition. TcSR62 (green) was detected by immunofluorescence. Nuclei were counterstained with DAPI (blue). N: nucleus, K: kinetoplast, Nu: nucleolus. Size bars represent 2 µm. Representative nuclei are shown.
Figure 2.
TcSR62 is relocalized to the nucleolus after ActD treatment.
(A) Immunofluorescence images of double staining for TcSR62 (green) and the nucleolar marker L1C6 (red) in ActD-treated and untreated epimastigotes (top and middle panels) or in ActD-Chx-treated epimastigotes (bottom panels). The fourth column on the right is an overlap of the TcSR62, L1C6 and DNA stain, showing nucleolar relocalization of TcSR62 upon transcription inhibition. Green and red pixels overlapped in the digital images yielded yellow signals. (B) Immunofluorescence images of double staining for TcSR62 (green) and DAPI in untreated (top panels), sinefungin-treated epimastigotes (middle panels) or in chloroquine-treated parasites (bottom panels). The third column on the right is an overlap of the TcSR62 and DNA stain. Nuclei were counterstained with DAPI (blue). N: nucleus, K: kinetoplast, Nu: nucleolus. Size bars represent 2 µm. Representative nuclei are shown. (C) Immunoblot showing an extract of 3×107 epimastigotes under normal growth conditions or exposed to transcription inhibition. The membrane was sequentially stained with TcSR62 and TcGDH antibodies, respectively.
Figure 3.
Effects of ActD treatment on the localization of several RNA binding proteins and TcHSP70.
(A) Immunofluorescence images of TcPTB2, TcPABP1 in ActD-treated and untreated epimastigotes. Each protein (in green) was colocalized with the nucleolar marker L1C6 (red). Nuclei were counterstained with DAPI (blue). The fourth column on the right is an overlap of each protein, L1C6 and DNA staining. Green and red pixels overlapped in the digital images yielded yellow signals. (B) Immunofluorescence images for TcRNP38, TcLA and TcHSP70. Each protein is shown in green. Nuclei were counterstained with DAPI (blue). The third column on the right is an overlap of each protein and DNA stain. Size bars represent 2 µm. Representative nuclei are shown. The graphic in panel (C) shows the percentage of cells showing nucleolar localization for TcSR62, TcPTB2, TcPABP1 and L1C6 in control (blue bars) and Act-D-treated cells (red bars). (D) Quantitative analysis of TcLA behaviour under transcription inhibition treatment. The results are expressed as mean +/− SD from at least three independent experiments.
Figure 4.
Nucleolar accumulation of RBPs upon ActD treatment depends on an active transport mechanism.
(A) Epimastigotes were incubated with both sodium Azide (Az) and 2-Deoxy-Glucose (2De) and ActD at 28°C for 24 h. (B) Epimastigotes were incubated with ActD at 4°C for 24 h. Then, immunofluorescence studies were performed using antibodies against TcSR62 (green) and TcPTB2 (red). Recovery of both treatments was allowed either by washing up the cultures and then incubating with fresh medium (−Az−2De+ActD) or reincubating at 28°C (↑28°+ActD) for 24 h. Each inhibitor treatment alone or cell culture at 4°C, are shown as controls. Nuclei were counterstained with DAPI (blue). The fourth column on the right is an overlap of the TcSR62 and TcPTB2 and DNA stain. Green and red pixels overlapped in the digital images yielded yellow signals. Size bars represent 2 µm. Representative nuclei are shown. The graphic in panel (C) is a quantification of the experiments shown in panels (A) and (B). Results are expressed as mean +/− SD from at least three independent experiments.
Figure 5.
Stress-induced nucleolar localization of RBPs is inhibited by blocking phosphorylation but not dephosphorylation.
Epimastigotes were incubated either with both okadaic acid (Oka) and ActD for 24 h or first preincubated with staurosporine (Stau) for 16 h and then with ActD for 24 h. Panel (A) shows TcSR62 (green), L1C6 (red) and DAPI. The fourth column on the right is an overlap of the TcSR62 and L1C6 and DNA stain. Green and red pixels overlapped in the digital images yielded yellow signals. (B) TcPTB2 (green), (C) TcPABP1 (green), each counterstained with DAPI. The third column on the right is an overlap of each protein and DNA stain. Each inhibitor treatment alone is shown as control. Nuclei were counterstained with DAPI (blue). Size bars represent 2 µm. Representative nuclei are shown. The graphic in panel (D) is a quantitative analysis of the experiments shown in panels (A) and (B). The graphic in panel (E) is a quantitative analysis of TcPABP1 behaviour. Results are expressed as mean +/− SD from at least three independent experiments.
Figure 6.
Nucleolar determinants of TcSR62 are located in the COOH terminal region.
(A) Cartoon showing TcSR62 main domains and mutants expressed as eGFP fusion proteins in T. cruzi. (B) SR62-eGFP (C) NH2-eGFP (D) COOH-eGFP parasites were untreated or incubated with ActD for 24 h. Fusion proteins are shown in green and L1C6 in red. The fourth column on the right is an overlap of each fusion protein, L1C6 and DNA stain. Green and red pixels overlapped in the digital images yielded yellow signals. Nuclei were counterstained with DAPI (blue). N: nucleus, K: kinetoplast, Nu: nucleolus. Size bars represent 2 µm. Representative nuclei are shown. The graphic in panel (E) is a quantification of the experiments showed in panels (B), (C) and (D). Results are expressed as mean +/− SD from three independent experiments.
Figure 7.
Poly(A)+ RNA is accumulated into the nucleolus in response to ActD treatment.
(A) Poly(A)+ was detected by FISH using a Cy3-labelled oligo(dT)30 probe in parasites untreated or incubated with ActD for 24 h. In addition, cells were pre-treated with RNAse A before performing FISH (bottom panels). (B) RNA FISH using a Cy3-labelled oligo(dA)30 probe in parasites untreated or incubated with ActD for 24 h. (C) Immunofluorescence against TcSR62 (green) coupled to FISH using a Cy3-labelled oligo(dT)30 probe in parasites untreated or incubated with ActD for 24 h. Nuclei were counterstained with DAPI (blue). Representative nuclei are shown. Nu: nucleolus. Size bars represent 2 µm. The graphic in panel (D) is a quantitative analysis of poly(A)+ behaviour. Results are expressed as mean +/− SD from at least three independent experiments.
Figure 8.
Severe heat shock could reversible promote nucleolar localization of both TcSR62 and TcPTB2 but not TcPABP1.
(A) Immunofluorescence images of double labelling for nucleolar marker L1C6 (green) and TcPTB2 (red), under normal conditions, 2 h at 40°C or 6 h of recovery at 28°C. (B) TcPTB2 (green) and TcSR62 (red) in untreated cells or cells incubated at 40°C for 2 h. Nuclei were counterstained with DAPI (blue). The graphic in panel (C) shows the percentage of cells showing nucleolar localization for TcSR62 and TcPTB2 in control (blue bars), at 40°C 2 h (red bars) and recovered at 28°C for 6 h (green). The results are expressed as mean +/− SD from three independent experiments. (D) TcPABP1 (green) and TcPTB2 (red) in untreated cells and cells incubated at 40°C for 2 h. The fourth column on the right is an overlap of TcPTB2, DAPI and each protein evaluated. N: nucleus Nu: nucleolus. K: kinetoplast. Size bars represent 2 µm. Representative nuclei are shown.