This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.
Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1’s behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.
A. Cell Preparation
B. Microscope Setup and Image Acquisition
This protocol was developed using the Zeiss Cell Observer SD spinning disk confocal microscope equipped with an AxioObserver Z1 stand, a dual-channel Yokagawa CSU-X1A 5000 spinning disk unit, 2 Photometrics QuantEM 512SC emCCD cameras, an HXP 120C fiber-based illuminator, 4 lasers (a Lasos 100mW multi-line Argon [458, 488, 514 nm], 50 mW 405 nm diode, 40 mW 561 nm diode, and 30 mW 635 nm diode), AOTF, a Pecon XL multiS1 stage incubation system, Zeiss incubation modules (O2 Module S, CO2 Module S, TempModule S, Heating Unit XL S) and a Prior motorized XY stage with a NanoScanZ piezo Z insert. To minimize spherical aberrations while imaging live cells supported in an aqueous medium, a C-Apochromat 63x/1.20 Water/Corr objective lens and Zeiss Immersol W immersion fluid (with a refractive index n = 1.334) were used. For 2 channel confocal imaging, a RQFT 405/488/568/647 dichroic mirror and BP525/50 (green) and BP629/62 (red) emission filters were used. The system software used was Zeiss Axiovision (ver. 4.8.2.0) with AV4 Multi-channel/Z/T, Fast Imaging, Physiology, MosaiX, Mark & Find, Dual Camera, Inside 4D, Autofocus and 3-D Deconvolution modules.
C. Image Processing and Analysis
This protocol was developed using Volocity software (PerkinElmer). The software used to acquire this data (AxioVision) also has the ability to process and analyze images. Users are encouraged to utilize the software available to them, and consult appropriate literature regarding their application.
D. Representative Results
An example of 53BP1 foci formation in response to CPT is shown in Figure 1. Cells exposed to CPT form foci within 5-10 min, and maintain these foci throughout the duration of recording. As shown in Figure 2, 53BP1 dissociates from chromatin at the onset of mitosis, forming a thin haze around the condensing chromosomes. As telophase occurs and mitosis comes to an end, 53BP1 once again aggregates into distinct foci. While the HEK293 cells were not exposed to CPT, they nevertheless formed abundant, spontaneous 53BP1 repair foci generated by endogenous DNA damage. This observation allowed us to conclude that 53BP1 does not form foci during early mitosis, in line with a previous report showing a similar effect after the exposure of cells to ionizing radiation and radiomimetic drugs 5.
Figure 1. Camptothecin (10 μM) causes DSBs and 53BP1 foci formation in cycling fibroblasts within 30 min of adding the drug to the medium.
Figure 2. 53BP1 does not form foci during mitosis until telophase/G1 in HEK293 cells.
Maintenance of genomic integrity is crucial for cell survival. Failure to preserve the genome results in premature aging, carcinogenesis, or death 8. There is intense interest in discerning how the DDR functions, stemming from its importance to both basic and clinical research. Many techniques have been developed over the years to aid in the study of how cells detect and repair DNA damage. Traditional methods such as immunocytochemistry and western blotting have been mainstays of the field, though recent advances in technology have allowed increasingly sophisticated methods to evolve. Live cell imaging, as detailed in this protocol, allows us to study characteristics of the DDR overlooked by more traditional techniques.
Central to the use of live cell imaging is the creation of fluorescently labeled proteins. Here we describe the use of a mCherry-tagged protein involved in the DDR, 53BP1, as well as a histone H2B-GFP fusion product. Fluorescently labeled proteins are used extensively in molecular and cellular biology; however, they are not without their limitations. Attaching a novel structure to an endogenous protein creates the obvious risk of altering natural function. Additionally, certain constructs can and will be expressed at different levels in different cell lines, under various conditions. We have observed divergent levels of fluorescent intensity in all of the genetically engineered cells used in this study. The 53BP1 construct used in this protocol lacks most functional domains; however, it retains the ability to bind to sites of DNA damage which does not seem to adversely affect the cell 2.
Additionally, it is important to consider the method of gene delivery. In this protocol, we used two methods: lentiviral transduction and stable transfection. Our lentivirus-infected cells are stably transduced with the fluorescent construct; therefore, they will continue to express these proteins indefinitely. However, this method is somewhat more labor-intensive compared to transfection and is dictated by which kind of cells are being targeted; some cells are not amenable to efficient transfection. On the other hand, lentiviruses are able to enter most cells, including human. Investigators are encouraged to weigh the pros and cons to each method when it comes to their own experiments. As a possible means of circumventing the problems inherent in fluorescently labeled proteins, the use of cell-permeable fluorescently labeled probes were recently reported for live cell experiments 10. However, this technique has yet to be fully developed.
A primary advantage of live cell imaging is the ability to study the spatiotemporal relationships of the DDR and DSB repair. Indeed, Dimitrova et al. (2008) were able to observe fluorescently labeled live cells and reveal novel information regarding 53BP1 binding to telomeres and how it affects chromatin dynamics. This analysis would be significantly more challenging, though not impossible, with other methods. Having the ability to monitor the DDR in both space and time allows us to discern functions and relationships that we might otherwise overlook.
In summary, using live cell imaging enables researchers to monitor the kinetics of DSB formation and resolution in real time, and allows quantitative analysis at a single cell level. In addition to the technique used in this study, other applications of live cell imaging can be used, such as FRET and FRAP 3. The technology of observing cells in real-time will continue to be improved upon and used to study a variety of cellular processes.
The authors have nothing to disclose.
Supported in part by R01NS064593 and R21ES016636 (K.V.). Microscopy was performed at the VCU – Department of Neurobiology & Anatomy Microscopy Facility, supported, in part, with funding from NIH-NINDS Center core grant 5P30NS047463. The spinning disc confocal microscope was purchased with an NIH-NCRR award (1S10RR027957).
Product | Company |
CellStar culture dishes | Greiner Bio-one |
FluroDish glass bottom dishes | World Precision Instruments, Inc. |
MEM media | GIBCO |
Non-essential amino acids | GIBCO |
Amino acids | GIBCO |
Vitamins | GIBCO |
Sodium Pyruvate | Invitrogen |
Penicillin/Streptomycin | HyClone |
Fetal Bovine Serum | GIBCO |
N-Myc-53BP1 WT pLPC-Puro; plasmid 19836 |
Addgene |
pCLNR-H2BG; plasmid 17735 | Addgene |
SuperFect | Qiagen |
Zeiss Cell Observer SD Imaging system | Zeiss |
AxioVision (release 4.8.2) | Zeiss |
Zeiss Immersol W Oil | Zeiss |
Volocity software (version 6.0) | PerkinElmer |