Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNAdamaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.
Laser induced shockwave (LIS) can be utilized to subject neuronal cells to conditions similar to those occurring during a blast induced traumatic brain injury. We utilized a 532nm Coherent Flare laser to induce a shockwave near cells which had been transfected with a FRET calcium biosensor (D3CPV) so that we could monitor the immediate cellular responses. Our shockwave system was characterized with a high-speed camera to monitor cavitation bubble dynamics and calculate the shear forces cells were subjected to. We found that we could induce forces which have been previously shown to induce injury. Using both phase and fluorescence microscopy we monitored the effects of shear on our cells. We found that at distances up to 120 microns from the laser focal point cells experienced shears greater than 10kPa. At those distances cell fragmentation was observed. Cells that survived and expressed the FRET biosensor demonstrated an immediate calcium elevation irrespective of extracellular or cytoplasmic calcium concentration. Cells recovered to pre-shockwave calcium levels within ~30s. In conclusion, LIS can be utilized to simultaneously monitor the neuronal response to shear stress and nearby cell death or injury.
Laser-induced shockwaves (LIS) can be utilized as a method to subject cells to conditions similar to those occurring during a blast-induced traumatic brain injury. The pairing of LIS with genetically encoded biosensors allows researchers to monitor the immediate molecular events resulting from such an injury. In this study, we utilized the genetically encoded Ca2+ FRET biosensor D3CPV to study the immediate Ca2+ response to laser-induced shockwave in cortical neurons and Schwann cells. Our results show that both cell types exhibit a transient Ca2+ increase irrespective of extracellular Ca2+ conditions. LIS allows for the simultaneous monitoring of the effects of shear stress on cells, as well as nearby cell damage and death.
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