Michael W. Berns

Astrocytes in the brain are rapidly recruited to sites of injury where they phagocytose damaged material and take up neurotransmitters and ions to avoid the spreading of damaging molecules. In this study we investigate the calcium (Ca²⁺) response in astrocytes to nearby cell death. To induce cell death in a nearby cell we utilized a laser nanosurgery system to photolyze a selected cell from an established astrocyte cell line (Ast1). Our results show that the lysis of a nearby cell is disruptive to surrounding cells' Ca²⁺ activity. Additionally, astrocytes exhibit a Ca²⁺ transient in response to cell death which differs from the spontaneous oscillations occurring in astrocytes prior to cell lysis. We show that the primary source of the Ca²⁺ transient is the endoplasmic reticulum.

Piezo1 belongs to mechano-activatable cation channels serving as biological force sensors. However, the molecular events downstream of Piezo1 activation remain unclear. In this study, we used biosensors based on fluorescence resonance energy transfer (FRET) to investigate the dynamic modes of Piezo1-mediated signaling and revealed a bimodal pattern of Piezo1-induced intracellular calcium signaling. Laser-induced shockwaves (LIS) and its associated shear stress can mechanically activate Piezo1 to induce transient intracellular calcium (Ca_[i] ) elevation, accompanied by an increase in FAK activity. Interestingly, multiple pulses of shockwave stimulation caused a more sustained calcium increase and a decrease in FAK activity. Similarly, tuning the degree of Piezo1 activation by titrating either the dosage of Piezo1 ligand Yoda1 or the expression level of Piezo1 produced a similar bimodal pattern of FAK responses. Further investigations revealed that SHP2 serves as an intermediate regulator mediating this bimodal pattern in Piezo1 sensing and signaling. These results suggest that the degrees of Piezo1 activation induced by both mechanical LIS and chemical ligand stimulation may determine downstream signaling characteristics.

Background: In recent years the functions of astrocytes have shifted from conventional supportive roles to also include active roles in altering synapses and engulfment of cellular debris. Recent studies have implicated astrocytes in both protective and pathogenic roles impacting Huntington’s disease (HD) progression. Objective: The goal of this study is to determine if phagocytosis of cellular debris is compromised in HD striatal astrocytes. Methods: Primary adult astrocytes were derived from two HD mouse models; the fast-progressing R6/2 and slower progressing Q175. With the use of laser nanosurgery, a single astrocyte was lysed within an astrocyte network. The phagocytic response of astrocytes was observed with phase contrast and by fluorescence microscopy for GFP-LC3 transiently transfected cells. Results: Astrocyte phagocytosis was significantly diminished in primary astrocytes, consistent with the progression of HD in R6/2 and Q175 mouse models. This was defined by the number of astrocytes responding via phagocytosis and by the average number of vesicles formed per cell. GFP-LC3 was found to increasingly localize to phagocytic vesicles over a 20-min imaging period, but not in HD mice, suggesting the involvement of LC3 in astrocyte phagocytosis. Conclusion: We demonstrate a progressive decrease in LC3-associated phagocytosis in HD mouse striatal astrocytes.

Editorial: Optical Trapping (Laser Tweezers) and Nanosurgery (Laser Scissors)

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.

In this paper, we propose a new system for studying cellular injury. The system is a biophotonic work station that can generate Laser-Induced Shockwave (LIS) in the cell culture medium combined with a Quantitative Phase Microscope (QPM), enabling the real-time measurement of intracellular dynamics and quantitative changes in cellular thickness during the damage and recovery processes. In addition, the system is capable of Phase Contrast (PhC) and Differential Interference Contrast (DIC) microscopy. Our studies showed that QPM allows us to discern changes that otherwise would be unnoticeable or difficult to detect using phase or DIC imaging. As one application, this system enables the study of traumatic brain injury in vitro. Astrocytes are the most numerous cells in the central nervous system (CNS) and have been shown to play a role in the repair of damaged neuronal tissue. In this study, we use LIS to create a precise mechanical force in the culture medium at a controlled distance from astrocytes and measure the quantitative changes, in order of nanometers, in cell thickness. Experiments were performed in different cell culture media in order to evaluate the reproducibility of the experimental method.

Starting in 1969 laser scissors have been used to study and manipulate chromosomes in mitotic animal cells. Key studies demonstrated that using the “hot spot” in the center of a focused Gaussian laser beam it was possible to delete the ribosomal genes (secondary constriction), and this deficiency was maintained in clonal daughter cells. It wasn’t until 2020 that it was demonstrated that cells with focal-point damaged chromosomes could replicate due to the cell’s DNA damage repair molecular machinery. A series of studies leading up to this conclusion involved using cells expressing different GFP DNA damage recognition and repair molecules. With the advent of optical tweezers in 1987, laser tweezers have been used to study the behavior and forces on chromosomes in mitotic and meiotic cells. The combination of laser scissors and tweezers were employed since 1991 to study various aspects of chromosome behavior during cell division. These studies involved holding chromosomes in an optical while gradually reducing the laser power until the chromosome recovered their movement toward the cell pole. It was determined in collaborative studies with Prof. Arthur Forer from York University, Toronto, Canada, cells from diverse group vertebrate and invertebrates, that forces necessary to move chromosomes to cell poles during cell division were between 2 and 17pN, orders of magnitude below the 700 pN generally found in the literature.

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 DNA-damaged 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.

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 DNA-damaged 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.

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.