Department of Genetics, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem
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Nissim-Rafinia, M., Meshorer, E. Photobleaching Assays (FRAP & FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells. J. Vis. Exp. (52), e2696, doi:10.3791/2696 (2011).
Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.
1. Plating the ES Cells
T = 0 hrs
T = 6 hrs
ES cell plating
2. Transfecting the ES Cells
T = 24 hrs
3. Performing FRAP and FLIP
T = 48-72 hrs
4. FRAP and FLIP Data Analysis
5. Representative Results:
Figure 1. A and B show representative FRAP curves of HP1 (left), H1o (middle) and H1e (right) in R1 ES cells. For simplicity and clarity Figure 1A shows the raw data of a single cell before any normalization and calculation. The yellow curve corresponds to the bleached region, the purple curve corresponds to the non-bleached nuclear area (when the bleached region is negligible the entire nucleus can be selected for normalization purposes), and the red line corresponds to the background fluorescence, which is minimal in this case. Vertical arrow represents the bleach time. Normalized and averaged data is shown in Figure 1B. Note the slower recovery of H1 (blue) compared with HP1 (red). Also the H1e variant (dark blue) is slower than the H1o variant (light blue). Mobile and immobile fractions and Bleach Depth are indicated for HP1.
Figure 2. A and B show representative FRAP curves comparing euchromatin (left) with heterochromatin (right) of HP1 in R1 ES cells. Similarly to Figure 1, Figure 2A shows raw data of a single cell, yellow curve corresponds to the bleached region, purple curve corresponds to the non-bleached nuclear area, and the red line corresponds to the background fluorescence. Vertical arrow represents the bleach time. Normalized and averaged data is shown in Figure 2B. Note the slower recovery of heterochromatin (dark red) compared with euchromatin (light red). Mobile and immobile fractions and Bleach Depth are indicated for euchromatin.
Figure 3.. A typical FLIP experiment of H1o R1 ES cells is shown in Figure 3A (raw, un-normalized data) and B (normalized and averaged data). In this experiment the purple curve corresponds to the non-bleached nuclear area, the green line corresponds to a neighboring cell nucleus and the red line corresponds to the background fluorescence.
Unlike most available techniques, which involve purified chromatin from cell populations or fixed cells, FRAP experiments follow changes in chromatin protein dynamics in living cells. We found chromatin protein dynamics to be a good indicator for chromatin plasticity. However, because it requires fusing the gene of interest with GFP, the addition of the fluorescent tag can interfere with the protein's function. Thus, prior to proceeding with FRAP, the fusion protein must be rigorously tested to ensure it has the same properties and function as its native counterpart. The gold standard would be to complement the endogenous protein's function with the GFP-fusion in a knockout cell line. However, knockout cell lines are not always available and in many cases the protein's absence does not have a clear phenotype. Nevertheless, one can test the fusion protein's subcellular distribution, its expression level, its binding partners, all compared with the endogenous protein to ensure proper partial replacement.
Once verified, the GFP fusion protein must be transfected into ES cells. We found that TransIT works well with ES cells and achieves transfection efficiencies of over 50%. Transient transfection is convenient as it allows proceeding with the experiment directly, but stable transfection is often superior, ensuring long-term survival of the cells in the presence of the fusion protein of interest, and resulting in lower and a homogenous expression level. Additional methods of labeling proteins with GFP tags include GFP gene tagging in BACs using combineering10 or GFP-trapping endogenous genes directly with GFP/YFP exons11,12. These methods are preferable, as the fusion protein is driven by an endogenous promoter, but not always available. Transfection of BACs into ES cells is possible using standard transfection methods. Finally, ES cells from transgenic mice expressing the tagged chromatin protein can also be used. Although more cumbersome, this method allows the usage of early passage cells, unlike stably transfected cells, which require long term culturing to achieve pure selection of stable integration.
Once the protein of interest has been selected, fused with GFP, verified and transfected into ES cells, there are several essential conditions that must be fulfilled for a successful FRAP experiment: first, the fluorescent signal to be bleached must be clearly detectable over any background signal; second, the photobleaching must be fast relative to the period of recovery to provide sufficient temporal resolution for analysis of the recovery curve and to allow measurement of the half-time of recovery. Therefore, the laser used for bleaching should be powerful enough to allow this; third, the monitoring beam must be of low intensity to minimize photobleaching. A spinning disk confocal microscope, equipped with photobleaching capabilities (such as Andor Revolution system), is therefore ideal for this purpose. Finally, an environmental chamber keeping the cells at the proper growth conditions must be installed on the microscope to ensure proper cell homeostasis.
A major limitation when analyzing core histones is that they are tightly bound to DNA, and therefore, in FRAP experiments they appear almost immobile. To reach complete recovery of core histones, the FRAP curves should reach several hours. Since ES cells are highly mobile in culture, it is essentially technically impossible to perform hours-long FRAP experiments. To avoid this, one can perform up to 10 min FRAP experiments and extrapolate the kinetic behavior from minutes to hours. In contrast to core histones, most DNA binding proteins rapidly associate and dissociate from chromatin, resulting in short half lives in the order of seconds to several minutes at most1. In this paper we studied two DNA binding proteins, H1 and HP1, both dynamic in ES cells, yet HP1 is more dynamic than H1 (as shown in Figure 1). Of note, both these chromatin proteins are less dynamic in heterochromatin than euchromatin, therefore the fluorescence recovery after photobleaching of heterochromatin is slower (as seen in Figure 2). The slower recovery in heterochromatin likely reflects a higher concentration of binding sites for H1 and HP1 as well as molecular crowding.
To sum, photobleaching experiments provide means to study chromatin protein dynamics in living cells, reflecting chromatin plasticity, which is exaggerated in pluripotent cells.
No conflicts of interest declared.
We thank members of the Meshorer lab, especially Shai Melcer, Adi Alajem, Edupuganti Raghu Ram, Badi Sri Sailaja, Anna Mattout and Alva Biran, for critical comments and for trouble-shooting photobleaching experiments on a daily basis. EM is a Joseph H. and Belle R. Braun Senior Lecturer in Life Sciences and is supported by the Israel Science Foundation (ISF 943/09), the Israel Ministry of Health (6007) the European Union (IRG-206872 and 238176), the Israel Cancer Research Foundation, the Internal Applicative Medical Grants of the Hebrew University and the Israel Psychobiology Institute.
|Gelatin||Merck & Co., Inc.||1.04078|
|Opti-MEM||GIBCO, by Life Technologies||31985|
|TransIT-LT1||Mirus Bio LLC||MIR2300|