Systematic analysis of DNA damage induction and DNA repair pathway activation by continuous wave visible light laser micro-irradiation.

Laser micro-irradiation can be used to induce DNA damage with high spatial and temporal resolution, representing a powerful tool to analyze DNA repair in the context of chromatin. However, most lasers induce a mixture of DNA damage leading to the activation of multiple DNA repair pathways and making it impossible to study individual repair processes. Hence, we aimed to establish and validate micro-irradiation conditions together with inhibition of several key proteins to discriminate different types of DNA damage and repair pathways using lasers commonly available in confocal microscopes.

Discrimination of Kinetic Models by a Combination of Microirradiation and Fluorescence Photobleaching.

Fluorescence recovery after photobleaching (FRAP) is an excellent tool to measure the chemical rate constants of fluorescently labeled proteins in living cells. Usually FRAP experiments are conducted with the protein concentrations being in a steady state, i.e.

Altered spatio-temporal dynamics of RNase H2 complex assembly at replication and repair sites in Aicardi-Goutières syndrome.

Ribonuclease H2 plays an essential role for genome stability as it removes ribonucleotides misincorporated into genomic DNA by replicative polymerases and resolves RNA/DNA hybrids. Biallelic mutations in the genes encoding the three RNase H2 subunits cause Aicardi-Goutières syndrome (AGS), an early-onset inflammatory encephalopathy that phenotypically overlaps with the autoimmune disorder systemic lupus erythematosus. Here we studied the intracellular dynamics of RNase H2 in living cells during DNA replication and in response to DNA damage using confocal time-lapse imaging and fluorescence cross-correlation spectroscopy.

Autophagy proteins LC3B, ATG5 and ATG12 participate in quality control after mitochondrial damage and influence lifespan.

Mitochondrial health is maintained by the quality control mechanisms of mitochondrial dynamics (fission and fusion) and mitophagy. Decline of these processes is thought to contribute to aging and neurodegenerative diseases. To investigate the role of mitochondrial quality control in aging on the cellular level, human umbilical vein endothelial cells (HUVEC) were subjected to mitochondria-targeted damage by combining staining of mitochondria and irradiation.

Respiratory chain complexes in dynamic mitochondria display a patchy distribution in life cells.

Background: Mitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism.

Do UCP2 and mild uncoupling improve longevity?

Mild uncoupling of mitochondrial respiration is considered to prolong life span of organisms by reducing the production of reactive oxygen species (ROS). Experimental evidence against this hypothesis has been brought forward by premature senescence in cell cultures treated with uncouplers. Exposing HUVEC to a mixture of nutritionally important fatty acids (oil extract of chicken yolk) mild uncoupling with "naturally acting substances" was performed.

Aging of different avian cultured cells: lack of ROS-induced damage and quality control mechanisms.

Elevated reactive oxygen species (ROS) levels have been observed in mammals during aging, implying an important role of ROS in the aging process. Most bird species are known to live longer and to contain lower ROS levels than mammals of the same body weight. The influence of ROS on the aging process of birds has been investigated using pigeon embryonic fibroblasts (PEF) and chicken embryonic fibroblasts (CEF).