Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Summary

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Abstract

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Introduction

DNA damage leads to the appearance of DNA lesions consisting of cyclobutane pyrimidine dimers (CPDs), 8-oxo-7,8-dihydro-2'-deoxyguanosine, and single-strand or double-strand breaks^1,2. γ-rays are the form of ionizing radiation with the highest energy and high penetrance, thus this source of radiation is widely used in radiotherapy³. On the other hand, experimentally induced DNA damage caused by UV lasers mimics natural exposure to UV light. UVA microirradiation, as a microscopic method, represents an experimental tool for studying DNA damage in individual living cells. Microirr....

Protocol

1. Cultivation of Cell Lines

1. HeLa-derived cell lines
   NOTE: Use HeLa cervical carcinoma cells: either HeLa cells stably expressing histone H2B tagged with GFP or HeLa-Fucci cells expressing RFP-Cdt1 in the G1 and early S phases and GFP-geminin in the S/G2-M phases (Figure 1).
   1. For cultivation of all HeLa-derived cell lines, use Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and appropriate antibiotics at 37 °C in a humidified atmosphere containing 5% CO₂. Replace the medium 2 - 3 times per week.
   2. Remove culture medium, and r....

Results

Using advanced confocal microscopy, we observed an accumulation of mCherry-tagged 53BP1 and mCherry-PCNA proteins at DNA lesions. Analyses were performed by local microirradiation of living cells. To recognize the nuclear distribution patterns of DNA-repair-related proteins in individual cell cycle phases, we used the Fucci cellular system, by which it is possible to determine the G1, early S, and G2 phases of the cell cycle (Figure 1). The biological applica.......

Discussion

Microscopy techniques represent basic tools in research laboratories. Here, a brief description of the methods used for the study of protein recruitment and kinetics at DNA lesions is presented. We especially noted our experimental experience in the field of local microirradiation of living cells, and we discuss the study of protein kinetics by FRAP and protein-protein interaction at DNA lesions by acceptor-bleaching FRET²⁸ and its advanced modification FRET-FLIM (Figure 4A

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell cultivation
HeLa	ATCC	CCL-2TM
ES-D3 [D3]	ATCC	CRL-11632TM
HeLa -Fucci cells			http://ruo.mbl.co.jp/bio/e/product/flprotein/fucci.html
DMEM	PAN-Biotech	P03-0710
DMEM high glucose	Sigma-Aldrich	D6429-500ML
Fetal bovine serum (FBS)	HyClone	SV30180.03
ES Cell FBS	Gibco	16141-079
Non-Essential Amino Acids (NEAA)	Gibco	11140-035
mLIF (mouse Leukemia Inhibitor Factor)	Merck Millipore	ESG1107
MTG (1-Thioglycerol)	Sigma-Aldrich	M6145-25ML
Penicillin-Streptomycin Solution	Biosera	XC-A4122/100
Trypsin - EDTA	Biosera	XC-T1717/100	dilute with 1 × PBS in ratio 1:6
Nunclon cell culture dishes	Sigma-Aldrich	P7866	cultivation of mESCs D3 cells
µ-Dish 35m+A15:I35m Grid-500	Ibidi GmbH	81166	microscopic dish
0.2% Gelatine	Sigma-Aldrich	G1890-100G	dilute in destille water and autoclaved
Name	Company	Catalog Number	Comments
Cell transfection
GFP-p53 plasmid	Addgene	12091
mCherry-PCNA plasmid			generous gift from Cristina Cardoso, Technische Universität Darmstadt
mCherry-53BP1 plasmid	Addgene	19835
Metafecetene	Biontex Laboratories GmbH	T020–2.0
10 × PBS	Thermo Fisher Scientific	AM9625	for transfection use 1 × PBS diluted in nuclease-free water
5-bromo-2’-deoxy-uridine	Sigma-Aldrich	11296736001
Name	Company	Catalog Number	Comments
Confocal microscopy
Microscope Leica TCS SP5	Leica Microsystems
Microscope Leica TCS SP8	Leica Microsystems
White-light laser	Leica Microsystems
355-nm laser	Coherent Inc.		laser power 80 mW
405-nm laser	Leica Microsystems		laser power 50 mW
Name	Company	Catalog Number	Comments
Immunofluorescence staining
Coverslip	VWR International Ltd	631-1580
4% paraformaldehyde	Affymetrix	19943 1 LT
Triton X100	MP Biomedicals	2194854
Saponin from quillaja bark	Sigma-Aldrich	S4521
BSA	Sigma-Aldrich	A2153
53BP1	Abcam	ab21083	primary antibody
AlexaFluore 647	Thermo Fisher Scientific	A27040	secondary antibody
Vectashield	Vector Laboratories Ltd	H-1000	mounting medium