Molecular and Functional Characterization of a Trypanosoma cruzi Nuclear Adenylate Kinase Isoform
Figure 3
TcADKn nuclear exportation and nutrient availability regulation.
A) T. cruzi Epimastigotes in exponential growth phase were treated with leptomycin B, 0.1 µg.mL−1 for 5 h (LMB), results were analyzed by immunofluorescence and fluorescence was quantified for 40 treated and untreated parasites. In each parasite the fluorescence from the green channel (GFP) was quantified in an area selected according to blue signal (DAPI) fluorescence (nucleus) using the RGB plugin in ImageJ (http://rsb.info.nih.gov/ij). Cytoplasmic fluorescence was quantified in the same way selecting the brightest perinuclear areas in the green channel (GFP). Selection criterion was the same for all transfected parasites. Bars represent mean ± S.D. Statistically significant difference was calculated by t-student test (p<0,05). B) T. cruzi epimastigotes in stationary growing phase in BHT medium, were supplemented with glucose (Glu) or proline (Pro) 2% for 12 h. Epimastigotes from day 2 of culture were maintained in starvation conditions (PBS) for 24 h. Data were analyzed by indirect immunofluorescence using anti-TcADKn antibodies. C) Epimastigotes from day 2 of culture were maintained in starvation conditions (PBS) for 24 followed by indirect immunofluorescence for colocalization with p-bodies (TcDhh1) and stress granules markers (TcPABP1).