Figure 4. Mcm4 recovery results from... | Rockefeller University Press

Figure 4.

$Figure 4. Mcm4 recovery results from eviction during replication. (A) The IF was measured from a set of short FRAP experiments (each lasting <1 min) on a pool of cells at various stages of progression through S phase. The mean IF gradually decreased and was well fit by a line or single weak exponential decay (broken line), yielding a decay time of ∼13.5 h. The number of cells analyzed for each time interval is shown above the bar. (B) Long FRAP experiments (>150 min each) were performed on 10 cells in G1 phase that were all ∼100 min from entering S phase. The mean FRAP recovery after S phase entry was well fit with a line or single weak exponential (broken line), yielding a recovery time of ∼13.6 h. The consistency of this recovery time and the IF decay time suggests that Mcm4 undergoes little or no exchange throughout S phase. The gray bar indicates the appearance of PCNA foci. (C–F) Replication arrest prevents Mcm4 unloading. (C) Cells were cultured in roscovitine for 24 h, and a cell lacking PCNA foci but displaying heterogeneous Mcm4-mEm distribution (indicating G1 arrest) was subjected to FRAP and tracked for 403 min with no detectable IF recovery. (D) Cells were cultured in aphidicolin for 24 h, and a cell displaying PCNA foci characteristic of very early S phase as well as heterogeneous Mcm4-mEm distribution (Fig. 1 F) was subjected to FRAP and tracked for 296 min with no detectable IF recovery. (E) A cell cultured in aphidicolin for 24 h and displaying PCNA foci characteristic of early S phase and homogeneous Mcm distribution was subjected to FLIP/FRAP, as described in the text. The FLIP laser was directed in the broken yellow circle, and the FRAP bleach was directed along the solid yellow line. Similar results were obtained with roscovitine-arrested cells. Note that, although we observed a substantial reduction in the intensity of PCNA foci during aphidicolin arrest, as described previously (Görisch et al., 2008), characteristic focal patterns of PCNA were still discernable. Bars, 10 µm. (F) Quantification of the average bound fractions for groups of cells treated and analyzed as illustrated in C–E, and imaged for a minimum of 200 min. The number of cells analyzed and the standard deviation of the mean are shown above each bar (error bars). FRAP curves similar to those shown in Fig. 3 showed no recovery of the IF throughout the imaging period, so the bound fraction at the end of the entire imaging period is shown for simplicity. Note that for FLIP/FRAP of cells in early to mid S phase (ES/MS), the IF is actually considerably lower, but is measured after a significant fraction of the soluble molecules have been bleached.$

Mcm4 recovery results from eviction during replication. (A) The IF was measured from a set of short FRAP experiments (each lasting <1 min) on a pool of cells at various stages of progression through S phase. The mean IF gradually decreased and was well fit by a line or single weak exponential decay (broken line), yielding a decay time of ∼13.5 h. The number of cells analyzed for each time interval is shown above the bar. (B) Long FRAP experiments (>150 min each) were performed on 10 cells in G1 phase that were all ∼100 min from entering S phase. The mean FRAP recovery after S phase entry was well fit with a line or single weak exponential (broken line), yielding a recovery time of ∼13.6 h. The consistency of this recovery time and the IF decay time suggests that Mcm4 undergoes little or no exchange throughout S phase. The gray bar indicates the appearance of PCNA foci. (C–F) Replication arrest prevents Mcm4 unloading. (C) Cells were cultured in roscovitine for 24 h, and a cell lacking PCNA foci but displaying heterogeneous Mcm4-mEm distribution (indicating G1 arrest) was subjected to FRAP and tracked for 403 min with no detectable IF recovery. (D) Cells were cultured in aphidicolin for 24 h, and a cell displaying PCNA foci characteristic of very early S phase as well as heterogeneous Mcm4-mEm distribution (Fig. 1 F) was subjected to FRAP and tracked for 296 min with no detectable IF recovery. (E) A cell cultured in aphidicolin for 24 h and displaying PCNA foci characteristic of early S phase and homogeneous Mcm distribution was subjected to FLIP/FRAP, as described in the text. The FLIP laser was directed in the broken yellow circle, and the FRAP bleach was directed along the solid yellow line. Similar results were obtained with roscovitine-arrested cells. Note that, although we observed a substantial reduction in the intensity of PCNA foci during aphidicolin arrest, as described previously (Görisch et al., 2008), characteristic focal patterns of PCNA were still discernable. Bars, 10 µm. (F) Quantification of the average bound fractions for groups of cells treated and analyzed as illustrated in C–E, and imaged for a minimum of 200 min. The number of cells analyzed and the standard deviation of the mean are shown above each bar (error bars). FRAP curves similar to those shown in Fig. 3 showed no recovery of the IF throughout the imaging period, so the bound fraction at the end of the entire imaging period is shown for simplicity. Note that for FLIP/FRAP of cells in early to mid S phase (ES/MS), the IF is actually considerably lower, but is measured after a significant fraction of the soluble molecules have been bleached.