Passage
Quantitative fluorescence recovery after photobleaching (FRAP) is used to study the movement of molecules in live cells. During FRAP, the fluorescence of a green fluorescent protein (GFP) -tagged molecule is first measured and then photodestroyed in targeted cell regions. Researchers then assess the time course for fluorescence recovery, which is an indicator of molecular mobility of the GFP-tagged protein into the photodestroyed region. The average time required to recover 80% of the pre-bleach fluorescence of protein histone 1 (H1) fused to GFP (H1-GFP) in the photodestroyed region is given as t80.FRAP was used to analyze the mobility of the architectural H1 alone and in the presence of high-mobility group (HMG) proteins. HMG proteins are dynamic modifiers that have been shown to have opposite effects but similar binding sites on chromatin structure.
Figure 1 Unique chromatin binding motifs of HMG proteinsDuring analysis, HMGA1 and HMGB1 were microinjected into the cytoplasm of mouse embryonic fibroblast cells expressing H1-GFP. FRAP was performed on euchromatin and heterochromatin. Euchromatin and heterochromatin domains were identified as regions weakly and strongly stained by H1-GFP, respectively. The relative intensity of H1-GFP fluorescence in euchromatin and heterochromatin of uninjected and injected cells was assessed, and t80 results are shown in Table 1.Table 1 Effect of HMGB1 and HMGA1 on H1-GFP Mobility
Adapted from Catez F, Yang H, Tracey KJ, Reeves R, Misteli T, Bustin M. Network of dynamic interactions between histone H1 and high-mobility-group proteins in chromatin. Mol Cell Biol. 2004;24(10) :4321-8.
-If HMGA1 were injected into cells expressing HMGB1-GFP instead of H1-GFP, which of the following FRAP analysis graphs would show conclusively that HMGA1 binds the same sites as HMGB1 on chromatin? [The t80 value for HMGB1-GFP is indicated by the arrows].

Question

Passage
Quantitative fluorescence recovery after photobleaching (FRAP) is used to study the movement of molecules in live cells. During FRAP, the fluorescence of a green fluorescent protein (GFP) -tagged molecule is first measured and then photodestroyed in targeted cell regions. Researchers then assess the time course for fluorescence recovery, which is an indicator of molecular mobility of the GFP-tagged protein into the photodestroyed region. The average time required to recover 80% of the pre-bleach fluorescence of protein histone 1 (H1) fused to GFP (H1-GFP) in the photodestroyed region is given as t80.FRAP was used to analyze the mobility of the architectural H1 alone and in the presence of high-mobility group (HMG) proteins. HMG proteins are dynamic modifiers that have been shown to have opposite effects but similar binding sites on chromatin structure.

Figure 1 Unique chromatin binding motifs of HMG proteinsDuring analysis, HMGA1 and HMGB1 were microinjected into the cytoplasm of mouse embryonic fibroblast cells expressing H1-GFP. FRAP was performed on euchromatin and heterochromatin. Euchromatin and heterochromatin domains were identified as regions weakly and strongly stained by H1-GFP, respectively. The relative intensity of H1-GFP fluorescence in euchromatin and heterochromatin of uninjected and injected cells was assessed, and t80 results are shown in Table 1.Table 1 Effect of HMGB1 and HMGA1 on H1-GFP Mobility

Adapted from Catez F, Yang H, Tracey KJ, Reeves R, Misteli T, Bustin M. Network of dynamic interactions between histone H1 and high-mobility-group proteins in chromatin. Mol Cell Biol. 2004;24(10) :4321-8.
-If HMGA1 were injected into cells expressing HMGB1-GFP instead of H1-GFP, which of the following FRAP analysis graphs would show conclusively that HMGA1 binds the same sites as HMGB1 on chromatin? [The t80 value for HMGB1-GFP is indicated by the arrows].

Quizplus · Accepted Answer

11ecba2d_0f57_5992_a3bf_ed4032cfd48a_MD0008_00 GFP is frequently used to track the mobility of proteins in a cell. This scenario states that HMGA1 would be injected into cells already expressing HMGB1-GFP. For the result to reveal that HMGA1 competes with HMGB1, HMGA1 would have to decrease HMGB1 mobility (tracked by GFP) into the photodestroyed region by competing for similar binding sites on chromatin. The passage defines t80 as the average amount of time required to recover 80% of the pre-bleach fluorescence (ie, the time it takes for HMGB1-GFP to move back and restore 80% of the original fluorescence to the photobleached region). The graph would have to show that HMGB1-GFP + HMGA1 has a greater t₈₀ value (ie, takes longer) than HMGB1-GFP alone. Accordingly, the fourth graph (Choice D) shows that in the presence of HMGA1, it takes longer for HMGB1 to move back into the photobleached region and bind chromatin because HMGA1 is already binding to the same sites. (Choice A) This graph shows almost no difference in the t80 values of both states. The results show that HMGB1 and HMGA1 do not compete with each other, indicating that each protein may bind different sites on chromatin. (Choice B) This graph shows that HMGB1 never recovered fluorescence; this data may have been due to an experimental error as initial fluorescence before photobleaching was recorded. (Choice C) This graph shows the opposite result of the hypothetical scenario. In this figure, HMGB1-GFP + HMGA1 has a clearly lesser t80 value than that of HMGB1-GFP alone. Educational objective: Green fluorescent protein (GFP) is frequently used to track the mobility of proteins in a cell. The protein of interest is tagged with GFP, and its movement, localization, and expression can be studied in cells based on the fluorescence emitted.

Passage Quantitative Fluorescence Recovery After Photobleaching (FRAP) Is Used to Study