Figure 1.

Real-time visualization of a single Cyclin D1 gene locus in human cells. (a) Schematic representation of a stably inserted construct (ANCH3-CCND1-MS2) comprising the Cyclin D1 (CCND1) gene under its endogenous promoter, adjacent to a unique ANCH3 sequence, 24 × MS2 repeats within the 3′UTR, and a hygromycin selection gene (HYG). The construct is flanked by FRT sites for integration into MCF-7 FRT cells. Transient transfection with OR3 and MCP-tagged fluorescent proteins results in their accumulation at the ANCH3 and MS2 sequences (after estradiol (E2) stimulation), respectively (raw 3D images in Movie S1). (b) Fluorescent spots are easily detectable in transfected cells. A representative cell with an OR3-EGFP spot is shown. Region imaged during fluorescence recovery after photobleaching (FRAP) is indicated in orange. At time t = 0 s, a circular region enclosing the ANCHOR spot was bleached and fluorescence recovery of the spot was followed over time. Relative fluorescence intensity (RFI) was calculated as described in the Materials and Methods and Fig. S1 (right panel; solid line: mean, shadowed region: lower and upper quartile; n = 44 cells, four experiments with n ≥ 6 cells per experiment). Data were fitted to a single exponential. The 95% confidence interval is indicated in brackets. Scale bars, 2 μm. (c) Representative images of transiently transfected ANCH3-CCND1-MS2 cells expressing OR3-Santaka and MCP-EGFP (raw images in Movies S1 and S2). CCND1 DNA (red spot) colocalizes with transcribed mRNA (green spot) as MCP-EGFP associates with MS2 stem loops 45 min after adding 100 nM estradiol (E2). The same cell is shown before and after addition of E2. Scale bars, 5 and 2 μm (for cropped images). (d) Example of two-dimensional trajectories and area explored over 50 s (250 ms acquisition, 200 steps) of the OR3-Santaka-labeled CCND1 locus recorded before (−E2) and after (+E2) transcription activation. To see this figure in color, go online.