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北京大学工学院
生物医学工程系学术报告 |
题目：USING LOCAL PHOTODAMAGE TO STUDY CELLULAR REPAIR MECHANISMS BY OPTICAL MICROSCOPY
Jurek W.Dobrucki, Ph.D.
Professor of Biophysics, Head of Division of Cell Biophysics
Faculty of Biochemistry, Biophysics and Biotechnology
Jagiellonian University
Kraków
Poland
时 间： 2010年3月26日（星期五）上午11：00—11：30
主持人 李长辉 博士
地 点：廖凯原楼2-401
Abstract: Photodamage can be inflicted by a focused laser beam in a selected small area of a cell. If the damage is sublethal, the cell activates repair mechanisms, therefore this approach has been used successfully to study repair of DNA or plasma membranes [1,2]. We inflicted local DNA damage by exposing cells to ethidium anion and focused green light, in order to study the repair processes. We noticed that, in addition to known repair factors, heterochromatin protein 1 (HP1) was also recruited to the damaged area [1,2,3], indicating that HP1 was involved in DNA damage response (DDR).
HP1 is a constitutive component of heterochromatin, and plays an important role in transcriptional repression and regulation of euchromatic genes, however it was not known to be required for repair of oxidative damage. The finding of HP1 recruitment is particularly puzzling, since in another study HP1 was shown to dissociate from chromatin as a result of DNA damage (Ayoub et al. 2008). Technical aspects of live cell imaging that may explain these contradictory results will be discussed.
I will demonstrate that this experimental approach to study repair mechanism (i.e. local photodamage inflicted by a focused laser beam), although elegant and useful in studies of DNA repair mechanisms, can also lead to a false negative. If the concentration of a photosensitiser or the intensity of exciting light is sufficiently high (more than approx. 1 mW) [5] the expected recruitment of HP1 to damage is abolished. Thus, the experimental conditions of inflicting local chromatin lesions must be carefully optimised in order to permit the recruitment of a repair factor to damage.
References:
[1]. M.Zarebski, Methods of inflicting local photooxidative damage in live cells and application to studies of repair of plasma membranes and chromatin. PhD Thesis, Jagiellonian University, Kraków, Poland, 2008
[2]. E. Wiernasz, Recruitment of heterochromatic proteins 1 to sites of photooxidative chromatin damage. MSc Thesis, Jagiellonian University, Kraków, Poland, 2004.
[3]. M.Zarebski et al. Recruitment of Heterochromatin Protein 1 to DNA Repair Sites, Cytometry Part A, 2009; 75A:619-625
[4]. N.Ayoub et al. HP1-beta mobilization promotes chromatin changes that initiate the DNA damage response.Nature 2008; 453:682-686.
[5]. J.W.Dobrucki et al. Scattering of exciting light by live cells in fluorescence confocal imaging: Phototoxic effects and relevance for FRAP studies. Biophys J 2007;93:1778-1786
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Biography
Jurek Dobrucki received M.Sc. degree from Jagiellonian Univerity in Krakow in 1980. Subsequently, he worked in Cambridge University, UK, on response of mammalian cells to radiation and hyperthermia, and in University of Illinois at Urbana-Champaign, USA, on applications of electron spin resonance microscopy to studies of cellular response to radiation and drugs. In 1994 he created a Laboratory of Confocal Microscopy and Image Analysis at Jagiellonian University in Krakow, and, in 2005, a Division of Cell Biophysics. His research uses mainly optical microscopy analytical approaches and focuses on studies of DNA repair and the role of heterochromatin protein 1 in DNA damage response (DDR), and interaction of drugs with DNA and chromatin in vivo. He also studies extracellular matrix, using a new fluorescent probe Col-F (patent pending). His group is interested in technical aspects of imaging, including a role of scattered exciting light in creating cell damage, and in methods of inflicting local, sublethal damage to cell components to study various cellular repair mechanisms. He is the author of over 40 peer-reviewed experimental papers. Currently, he serves as Vide-dean for Science and International Cooperation at Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
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