Supplementary MaterialsSupplementary File
May 12, 2021
Supplementary MaterialsSupplementary File. monitored the endogenous protein expression level of the five remaining enzymes. A shown in Fig. 2and Fig. S4, the protein expression levels of the members enzymes remained constant over the time course studied. We performed single-cell analysis to evaluate the total fluorescence intensity in cells with and Ribocil B without purinosomes under a purine-depleted condition. No difference in the average fluorescence intensity per cell was observed between cells classified as purinosome-negative and those classified as purinosome-positive (Fig. S5). This result suggests that highly fluorescent cells (correlated with high protein expression of FGAMS-GFP) do not show a higher propensity to form purinosomes. Therefore, the formation of purinosome in the cells is not governed by protein expression level. Purinosome Characterization in Cell Models. We used an LND fibroblast model to evaluate the influence of the parallel salvage pathway in HeLa cells on purinosome appearance and levels in the phase of the cell cycle. These LND cells are HGPRT-deficient and rely primarily on the de novo purine biosynthetic pathway to meet purine demand. To properly classify purinosome-containing cells in the two cell models used for this study, Ribocil B we performed the basic morphological characterization of purinosomes in both HeLa Ribocil B and LND cells. We chose the average size and number of purinosomes in a given cell as the physical criteria to distinguish purinosomes from other cellular bodies. Purinosome diameter varied between 0.2 and 0.9 m, with an average of 0.56 0.16 m in HeLa cells (Fig. 3). The median number of purinosomes inside purinosome-positive HeLa cells was 278 (Fig. 3). We found no correlations between fluorescence intensity in a purinosome-positive cells and the average size and number of purinosomes in that cell (Fig. S6). For added measure, we evaluated the spatial organization of purinosomes in cells using superresolution stochastic optical reconstruction microscopy (STORM) (29). The size distribution in HeLa cells detected using STORM was consistent with previous observations (Fig. 3 and Fig. S7). Open in a separate window Fig. 3. Purinosome characterization in cell models. Shown are the general size and number distribution of purinosomes in HeLa cells and LND cells after single-cell analysis (= 200 for HeLa cells; = 50 for LND cells). Finally, we subjected nontransfected fixed LND cells to immunofluorescence imaging of the enzymes ASL and FGAMS, which demonstrated their clustering into purinosome punctates (Fig. S8). In LND cells, the average diameter of purinosomes was 0.41 0.11 m, and the median number of purinosomes inside LND purinosome-positive cells was 235. The results show Mouse monoclonal to LPA that purinosomes formed in LND cells are of similar size and Ribocil B number distribution as those formed in HeLa cells (Fig. 3). Therefore, the results are in accordance with the observation of the same cellular body, the purinosomes, in both cell types. Cell Cycle Dependency of HGPRT-Deficient Cells. LND fibroblast cells were transfected with FGAMS-GFP, and representative images of purinosome-positive cells in different phases of the cell cycle were acquired (Fig. 4and ?and4= 3). (= 3). In addition, we investigated the average size and quantity of purinosomes per cell in different cell cycle phases in both HeLa and LND fibroblast cells. Fig. S9 and illustrates the distribution of the average size of purinosomes in the three phases of the cell cycle, and Fig. S9 and shows the number of purinosomes per cell. No correlation between the average size and quantity of purinosomes per cell was observed across the different phases of the cell cycle (Fig. S9). Conversation Previous findings possess shown that de novo purine biosynthesis is definitely closely related to the cell cycle (19, 20, 25, 30C33). Studies of additional enzyme complexes have suggested the assembly or disassembly of an enzyme cluster may be correlated with cellular events, such as developmental cues or metabolic claims of the cell (33); for example, the replitase, a six-enzyme complex involved in DNA replication, offers been shown to exist only during S phase (34). In the present study, we aimed to understand purinosome formation like a function of the cell cycle phases. Through the use of time-lapse fluorescence microscopy, we.