Figure 1. Characterization of cell... | Rockefeller University Press

Figure 1.

$Figure 1. Characterization of cell lines. (A) Structure of Mcm-fluorescent protein (Mcm-mEm) fusion proteins. Each Mcm-mEm contains a full-length Mcm2-7 cDNA (Kimura et al., 1996), followed by a flexible linker, the fluorescent protein, a second flexible linker, and an optimized peptide substrate (BLT) for the E. coli biotin ligase enzyme (BirA). (B) Homogenous expression of Mcm4-mEm. After transfection of the expression cassette shown in A, cells were selected in the presence of dox to establish cell lines in the absence of Mcm-mEm expression. Dox was then removed from aliquots of each cell line for 48 h to induce tagged protein. Shown is Mcm4-mEm fluorescence merged with a phase contrast image. Bar, 10 µm. (C) Cell cycle regulation of soluble and insoluble fractions of endogenous and tagged Mcm4-mEm. Cells were synchronized in mitosis by shake-off and detergent-extracted at the indicated times after replating. Aliquots of cells were plated into aphidicolin for 16 h to arrest cells at the G1/S phase border (G1/S Aph), and a portion of those cells were released into S phase for 6 h (G1/S+6). The soluble and insoluble fractions (Fig. S4 A) were subjected to immunoblotting with the indicated antibodies. Both tagged and endogenous Mcm4 are completely soluble and exhibit a molecular weight shift during mitosis, as expected (Pereverzeva et al., 2000; Okuno et al., 2001). During G1, both tagged and endogenous Mcm4 present as a doublet band when sufficient care is taken to inhibit phosphatases, as expected (Komamura-Kohno et al., 2006). Insoluble PCNA tracks S phase; note that aphidicolin arrest results in increased detergent extractability of PCNA. β-Tubulin and LaminB are used as loading controls for the soluble and insoluble fractions, respectively. Both tagged and endogenous Mcm4 are reduced in the insoluble fraction 6 h after release from aphidicolin, as expected (Okuno et al., 2001). (D) Autogenous regulation of Mcm4-mEm. Mcm4-mEm–expressing cells were grown in the indicated concentrations of dox, and whole cell extracts were subjected to immunoblotting with an anti-Mcm4 antibody. Anti–β-tubulin was used as a loading control. (E) Coprecipitation of Mcm4-mEm with endogenous Mcm subunits in chromatin from late G1 phase cells. Cells either expressing (+) or not expressing (−) Mcm4-mEm were synchronized in mitosis and collected 8 h after release into G1 phase, and Mcm4-mEm (indicated with a black arrow) was precipitated from the solubilized chromatin fragments (Chromatin-Bound; Fig. S4 A). Whole cell extracts (WCE) from the same cells are also shown. Mcm4-mEm–expressing cells were grown in 0.5 ng/ml dox for 24 h before harvesting to eliminate all cytoplasmic Mcm4-mEm expression. Note that endogenous Mcm4 did not pull down with the tagged Mcm4, which indicates that double hexamers containing both tagged and untagged subunits are rare under these conditions, but this does not imply that they do not exist. Their abundance may depend on the ratio of tagged and untagged proteins bound to chromatin (e.g., see Fig. S4 C). (F) RFP-PCNA displays cell cycle–dependent punctate patterns in Mcm-mEm cell lines. Shown are cells in very early (initial appearance of PCNA foci), early, mid, or late S phase displaying the characteristic spatial patterns of PCNA (red) at sites of ongoing DNA synthesis. Note that Mcm4-mEm (green) is distributed heterogeneously in very early S phase but more homogeneously as S phase proceeds. PCNA is distributed uniformly in both G1 and G2 phase (Fig. 2, B and C). Bars, 10 µm.$

Characterization of cell lines. (A) Structure of Mcm-fluorescent protein (Mcm-mEm) fusion proteins. Each Mcm-mEm contains a full-length Mcm2-7 cDNA (Kimura et al., 1996), followed by a flexible linker, the fluorescent protein, a second flexible linker, and an optimized peptide substrate (BLT) for the E. coli biotin ligase enzyme (BirA). (B) Homogenous expression of Mcm4-mEm. After transfection of the expression cassette shown in A, cells were selected in the presence of dox to establish cell lines in the absence of Mcm-mEm expression. Dox was then removed from aliquots of each cell line for 48 h to induce tagged protein. Shown is Mcm4-mEm fluorescence merged with a phase contrast image. Bar, 10 µm. (C) Cell cycle regulation of soluble and insoluble fractions of endogenous and tagged Mcm4-mEm. Cells were synchronized in mitosis by shake-off and detergent-extracted at the indicated times after replating. Aliquots of cells were plated into aphidicolin for 16 h to arrest cells at the G1/S phase border (G1/S Aph), and a portion of those cells were released into S phase for 6 h (G1/S+6). The soluble and insoluble fractions (Fig. S4 A) were subjected to immunoblotting with the indicated antibodies. Both tagged and endogenous Mcm4 are completely soluble and exhibit a molecular weight shift during mitosis, as expected (Pereverzeva et al., 2000; Okuno et al., 2001). During G1, both tagged and endogenous Mcm4 present as a doublet band when sufficient care is taken to inhibit phosphatases, as expected (Komamura-Kohno et al., 2006). Insoluble PCNA tracks S phase; note that aphidicolin arrest results in increased detergent extractability of PCNA. β-Tubulin and LaminB are used as loading controls for the soluble and insoluble fractions, respectively. Both tagged and endogenous Mcm4 are reduced in the insoluble fraction 6 h after release from aphidicolin, as expected (Okuno et al., 2001). (D) Autogenous regulation of Mcm4-mEm. Mcm4-mEm–expressing cells were grown in the indicated concentrations of dox, and whole cell extracts were subjected to immunoblotting with an anti-Mcm4 antibody. Anti–β-tubulin was used as a loading control. (E) Coprecipitation of Mcm4-mEm with endogenous Mcm subunits in chromatin from late G1 phase cells. Cells either expressing (+) or not expressing (−) Mcm4-mEm were synchronized in mitosis and collected 8 h after release into G1 phase, and Mcm4-mEm (indicated with a black arrow) was precipitated from the solubilized chromatin fragments (Chromatin-Bound; Fig. S4 A). Whole cell extracts (WCE) from the same cells are also shown. Mcm4-mEm–expressing cells were grown in 0.5 ng/ml dox for 24 h before harvesting to eliminate all cytoplasmic Mcm4-mEm expression. Note that endogenous Mcm4 did not pull down with the tagged Mcm4, which indicates that double hexamers containing both tagged and untagged subunits are rare under these conditions, but this does not imply that they do not exist. Their abundance may depend on the ratio of tagged and untagged proteins bound to chromatin (e.g., see Fig. S4 C). (F) RFP-PCNA displays cell cycle–dependent punctate patterns in Mcm-mEm cell lines. Shown are cells in very early (initial appearance of PCNA foci), early, mid, or late S phase displaying the characteristic spatial patterns of PCNA (red) at sites of ongoing DNA synthesis. Note that Mcm4-mEm (green) is distributed heterogeneously in very early S phase but more homogeneously as S phase proceeds. PCNA is distributed uniformly in both G1 and G2 phase (Fig. 2, B and C). Bars, 10 µm.