Nucleoplasmic Lamin C Rapidly Accumulates at Sites of Nuclear Envelope Rupture with BAF and cGAS

In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that lamin C but not the other lamin isoforms rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The immunoglobulin-like fold domain and the NLS were required for the recruitment from the nucleoplasm to the rupture sites with the Barrier-to-autointegration factor (BAF). The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP (cGAMP) synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF and cGAS concertedly accumulate at sites of NE rupture for repair.

Application of Laser Microirradiation in the Investigations of Cellular Responses to DNA Damage

Since the laser has been invented it has been highly instrumental in ablating different parts of the cell to test their functionality. Through induction of damage in a defined sub-micron region in the cell nucleus, laser microirradiation technique is now established as a powerful real-time and high-resolution methodology to investigate mechanisms of DNA damage response and repair, the fundamental cellular processes for the maintenance of genomic integrity, in mammalian cells. However, irradiation conditions dictate the amounts, types and complexity of DNA damage, leading to different damage signaling responses. Thus, in order to properly interpret the results, it is important to understand the features of laser-induced DNA damage. In this review, we describe different types of DNA damage induced by the use of different laser systems and parameters, and discuss the mechanisms of DNA damage induction. We further summarize recent advances in the application of laser microirradiation to study spatiotemporal dynamics of cellular responses to DNA damage, including factor recruitment, chromatin modulation at damage sites as well as more global damage signaling. Finally, possible future application of laser microirradiation to gain further understanding of DNA damage response will be discussed.

Recruitment of MRE-11 to complex DNA damage is modulated by meiosis-specific chromosome organization

DNA double-strand breaks (DSBs) are one of the most dangerous assaults on the genome, and yet their natural and programmed production are inherent to life. When DSBs arise close together (clustered) they are particularly deleterious, and their repair may require an altered form of the DNA damage response. Our understanding of how clustered DSBs are repaired in the germline is unknown. Using UV laser microirradiation, we examine early events in the repair of clustered DSBs in germ cells within whole, live, Caenorhabditis elegans. We use precise temporal resolution to show how the recruitment of MRE-11 to complex damage is regulated, and that clustered DNA damage can recruit proteins from various repair pathways. Abrogation of non-homologous end joining or COM-1 attenuates the recruitment of MRE-11 through distinct mechanisms. The synaptonemal complex plays both positive and negative regulatory roles in these mutant contexts. These findings together indicate that MRE-11 is regulated by modifying its accessibility to chromosomes.

NAD+ consumption by PARP1 in response to DNA damage triggers metabolic shift critical for damaged cell survival

DNA damage signaling is critical for the maintenance of genome integrity and cell fate decision. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor rapidly activated in a damage dose- and complexity-dependent manner playing a critical role in the initial chromatin organization and DNA repair pathway choice at damage sites. However, our understanding of a cell-wide consequence of its activation in damaged cells is still limited. Using the phasor approach to fluorescence lifetime imaging microscopy and fluorescence-based biosensors in combination with laser microirradiation, we found a rapid cell-wide increase of the bound NADH fraction in response to nuclear DNA damage, which is triggered by PARP-dependent NAD+ depletion. This change is linked to the metabolic balance shift to oxidative phosphorylation (oxphos) over glycolysis. Inhibition of oxphos, but not glycolysis, resulted in parthanatos due to rapid PARP-dependent ATP deprivation, indicating that oxphos becomes critical for damaged cell survival. The results reveal the novel prosurvival response to PARP activation through a change in cellular metabolism and demonstrate how unique applications of advanced fluorescence imaging and laser microirradiation-induced DNA damage can be a powerful tool to interrogate damage-induced metabolic changes at high spatiotemporal resolution in a live cell.

NAD consumption by PARP1 in response to DNA damage triggers metabolic shift critical for damaged cell survival

DNA damage signaling is critical for the maintenance of genome integrity and cell fate decision. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor rapidly activated in a damage dose and complexity-dependent manner playing a critical role in the initial chromatin organization and DNA repair pathway choice at damage sites. However, the cell-wide consequence of its activation in damaged cells is not well delineated. Using the phasor approach to fluorescence lifetime imaging microscopy (FLIM) and fluorescence-based biosensors in combination with laser microirradiation, we found a rapid cell-wide increase of the bound/free NADH ratio in response to nuclear DNA damage, which is triggered by NAD+ depletion by PARP activation. This change is linked to the metabolic balance shift to oxidative phosphorylation (oxphos) over glycolysis. Inhibition of the respiratory chain resulted in rapid PARP-dependent ATP reduction and intracellular acidification, and eventually, PARP-dependent, AIF-independent, apoptosis indicating that oxphos becomes critical for damaged cell survival. The results reveal the novel pro-survival effect of PARP activation through a change in cellular metabolism, and demonstrate how unique applications of advanced fluorescence imaging technologies in combination with laser microirradiation can be a powerful tool to interrogate damage-induced metabolic changes at high spatiotemporal resolution in a live cell.

Q-FADD: A mechanistic approach for modeling the accumulation of proteins at sites of DNA damage by free diffusion

The repair of DNA damage requires the ordered recruitment of many different proteins that are responsible for signaling and subsequent repair. A powerful tool for studying the orchestrated accumulation of these proteins at damage sites is laser microirradiation in live cells, followed by monitoring of the accumulation of the fluorescently labeled protein in question. Despite the widespread use of this approach, there exists no rigorous method for characterizing this process quantitatively. Here we introduce a free diffusion model that explicitly accounts for the unique topology of individual nuclei and quantitatively describes the accumulation of two test proteins, poly-ADP-ribose polymerases 1 and 2. Application of our model to other proteins will yield novel insights into the timing and mechanism of DNA repair.