DAPI staining and Tc38 signal are indicated. Left panel shows the pooled ISIS software (MetaSystems GmbH) captured image.

For the merge image, Tc38-Alexa 488 signal is shown in green and DAPI nucleic acid staining in blue. Bars = 10 μm. Tc38 intramitochondrial distribution changes during the cell cycle Since Tc38 was found to predominantly co-localize with the kDNA and to recognize single stranded mini and maxicircle replication related sequences, we focused on the intramitochondrial localization during the cell cycle. For this purpose, we first analyzed asynchronic cultures. We based the identification of each cell cycle stage on morphological markers including both the number of nuclei and kinetoplasts determined by DAPI staining together with the number and appearance of flagella assessed by phase contrast microscopy [25]. Figure 5 shows the sequential changes in Tc38 localization www.selleckchem.com/products/PD-0325901.html during the cell cycle. It shows that

G1/S cells usually exhibit a homogeneous signal over the kDNA (Figure 5A) even though in some cases Tc38 condenses in two small antipodal sites. Cells at G2 (see arrow showing the second flagellum in phase contrast image) exhibit a diffuse signal connecting what now has become two clearly defined spots (Figure 5B). The two Tc38 spot signals do not seem to exactly co-localize LBH589 with the DAPI staining. As the cell cycle progresses the defined spots of Tc38 disappear and the diffuse dotted signal spreads out, covering a region far beyond the kinetoplast and without an evident association with it (Figure 5C and 5D). Finally in late cytokinesis the signal of Tc38 tends to regain the homogenous distribution over the kDNA (Figure 5E). Figure 5 Progesterone Tc38 patterns in T. cruzi epimastigotes during the cell cycle. Phase contrast, DAPI staining and Tc38 signal are indicated. For the merge images, Tc38-Alexa 488 signal is shown in green and DAPI nucleic acid staining in blue. Selected parasites that show the most frequent patterns seen in the cell cycle phases are presented. A corresponds to G1/S, B to G2 and C to E show images from mitosis to cytokinesis. Each one of the Tc38 labeling patterns were found

in the majority of examined cells (n ≥ 20). The arrow indicates the position of the second flagellum, indicative of G2. Black bars = 5 μm. The dotted lines in the phase contrast indicate the position enlarged in the fluorescent images. White bars = 2 μm. We also studied Tc38 localization in cultures synchronized with hydroxyurea (HU). HU inhibits the enzyme ribonucleotide reductase and the resulting depletion of deoxyribonucleotides arrests DNA replication in late G1/early S phase [26]. Previous reports on the effects of HU treatment on the T. cruzi cell cycle phases considered S phase to occur between 3–6 h and G2 at 9 h after HU removal [27, 28]. Progression of the cell cycle was followed using the same time schedule.