Cells were washed three times with PBS and incubated with Alexa-Fluor 555-conjugated anti-mouse IgG. antibody (mAbB17). This antibody was obtained from NZB/NZW mice, which spontaneously develop a systemic autoimmune disease that closely resembles human systemic lupus. Due to unknown reasons, this antibody is not able to identify kinetoplast DNA. Immunofluorescence assays using anti-TcOrc1/Cdc6 or anti-TcPCNA showed that TcOrc1/Cdc6 SPRY4 and TcPCNA labels just the nucleus. It is expected once the replication origins, as well as replication machineries working in kinetoplast DNA replication are quite different from that working on nuclear DNA replication. TcOrc1/Cdc6 and TcPCNA present two patterns of nuclear distribution in an exponentially growing culture. Analysis of different Z-sections, obtained by confocal microscopy, illustrate the following two main patterns: a peripheral pattern, in which molecules are constrained close to JAK/HDAC-IN-1 nuclear periphery (Fig. 2A and C), and a dispersed pattern, in which TcOrc1/Cdc6 or TcPCNA is usually dispersed throughout the nuclear space (Fig. 2B and D). The TcOrc1/Cdc6 and TcPCNA labeled area of dispersed and peripheral patterns were measured (as showed in the bottom of Fig. 2F). We found that in fact the labeled area of peripheral pattern is usually smaller than the dispersed pattern (Fig. 2F). Comparing the anti-TcOrc1/Cdc6 labeling with anti-DNA labeling we found that when TcOrc1/Cdc6 is usually constrained close to nuclear periphery, DNA is also constrained at this region (Fig. 2A). However, when TcOrc1/Cdc6 is usually dispersed through the nuclear space, DNA is also dispersed (Fig. 2B). These data JAK/HDAC-IN-1 are not unexpected once we have shown that TcOrc1/Cdc6 binds DNA.10 But it is interesting to note that the entire DNA (and not only the replication origins) can also be found constrained close to nuclear periphery (Fig. 2A). Also, even when DNA is usually close to nuclear periphery, TcOrc1/Cdc6 is usually outsider (Fig. 2A), strongly suggesting that chromatin structures in loops putting replication origin closer to nuclear periphery. Open in a separate window Physique 2 You will find two patterns of TcOrc1/Cdc6 and TcPCNA distribution in the nuclei JAK/HDAC-IN-1 of epimastigote cells. Epimastigote cells were fixed with 2% paraformaldehyde, permeabilized with Triton X-100 and incubated with (A and B) anti-TcOrc1/Cdc6 (reddish) or (C and D) anti-TcPCNA antibodies (reddish). All cells were labeled with mAbB17 (green), a monoclonal anti-DNA antibody. Images shown for each pattern were acquired at different Z-sections by a confocal microscope. The white figures indicate the distance between the section and the top limit of each nucleus. N indicates nuclei, k indicates kinetoplasts and bars represent 1 m. (E) Graph shows average standard deviation of three impartial experiments (n = 100), indicating the proportion of cells in an exponentially growing culture presenting TcOrc/Cdc6 or TcPCNA constrained at the nuclear periphery (gray box) or dispersed throughout the entire nucleus JAK/HDAC-IN-1 (white box). (F) Graphs show labeled area by anti-TcOrc1/Cdc6 or anti-TcPCNA in dispersed (black) or peripheral (white) patterns (n = 50). To perform this analysis the labeled areas were circled as exemplified in the bottom of (F) and these areas were quantified using the Image J program. **p 0.01. (G) Graph shows the central non-labeled area by anti-TcOrc1/Cdc6 (dark gray) or anti-TcPCNA (light gray) antibodies. To perform this analysis the central non-labeled areas were circled as exemplified in the bottom of (G) and these areas were quantified by Image J. *p 0.05. Images suggest that the peripheral pattern of TcOrc1/Cdc6 is usually more constrained close to nuclear periphery than the TcPCNA peripheral pattern. To confirm that, we measured the central non-labeled area (as represented in the bottom of Fig. 2G) of nuclei presenting peripheral patterns. We found that central nonlabeled area from nuclei labeled with anti-TcPCNA is usually smaller than the central non-labeled area from nuclei labeled with anti-TcOrc1/Cdc6 (Fig. 2G). Quantitative analyses of the distribution of these patterns in exponentially growing cells (n = 100 in three experiments) showed that TcOrc1/Cdc6 is usually constrained at the nuclear periphery in 54% of cells, whereas TcPCNA is usually localized at the nuclear periphery in 24% of cells (Fig. 2E). To further explore the nuclear localization of these molecules, ultrathin sections were labeled with each antibody and sections observed by transmission electron microscope. While in some cells TcOrc1/Cdc6 and TcPCNA are constrained in a more peripheral nuclear region (between nuclear membrane and the eletrodense chromatin), these molecules are not juxtaposed with nuclear membrane in 90% of the cells (Fig. 3A and left parts), suggesting that this physical contact between.
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