Shining Light in Mechanobiology: Optical Tweezers, Scissors, and Beyond.

Mechanobiology helps us to decipher cell and tissue functions by looking at changes in their mechanical properties that contribute to development, cell differentiation, physiology, and disease. Mechanobiology sits at the interface of biology, physics and engineering. One of the key technologies that enables characterization of properties of cells and tissue is microscopy. Combining microscopy with other quantitative measurement techniques such as optical tweezers and scissors, gives a very powerful tool for unraveling the intricacies of mechanobiology enabling measurement of forces, torques and displacements at play. We review the field of some light based studies of mechanobiology and optical detection of signal transduction ranging from optical micromanipulation-optical tweezers and scissors, advanced fluorescence techniques and optogenentics. In the current perspective paper, we concentrate our efforts on elucidating interesting measurements of forces, torques, positions, viscoelastic properties, and optogenetics inside and outside a cell attained when using structured light in combination with optical tweezers and scissors. We give perspective on the field concentrating on the use of structured light in imaging in combination with tweezers and scissors pointing out how novel developments in quantum imaging in combination with tweezers and scissors can bring to this fast growing field.

A single cell death is disruptive to spontaneous Ca2+ activity in astrocytes.

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.

Mechanosensor Piezo1 mediates bimodal patterns of intracellular calcium and FAK signaling.

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.

Diminished LC3-Associated Phagocytosis by Huntington's Disease Striatal Astrocytes.

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.

Combining quantitative phase microscopy and laser-induced shockwave for the study of cell 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.

Blocking Protein Phosphatase 1 [PP1] Prevents Loss of Tether Elasticity in Anaphase Crane-Fly Spermatocytes.

In normal anaphase cells, telomeres of each separating chromosome pair are connected to each other by tethers. Tethers are elastic at the start of anaphase: arm fragments cut from anaphase chromosomes in early anaphase move across the equator to the oppositely-moving chromosome, telomere moving toward telomere. Tethers become inelastic later in anaphase as the tethers become longer: arm fragments no longer move to their partners. When early anaphase cells are treated with Calyculin A (CalA), an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), at the end of anaphase chromosomes move backward from the poles, with telomeres moving toward partner telomeres. Experiments described herein show that in cells treated with CalA, backwards movements are stopped in a variety of ways, by cutting the tethers of backwards moving chromosomes, by severing arms of backwards moving chromosomes, by severing arms before the chromosomes reach the poles, and by cutting the telomere toward which a chromosome is moving backwards. Measurements of arm-fragment velocities show that CalA prevents tethers from becoming inelastic as they lengthen. Since treatment with CalA causes tethers to remain elastic throughout anaphase and since inhibitors of PP2A do not cause the backwards movements, PP1 activity during anaphase causes the tethers to become inelastic.

Laser-Induced Shockwave (LIS) to Study Neuronal Ca2+ Responses.

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.

Fluid Shear Stress Enhances the Phagocytic Response of Astrocytes.

Astrocytes respond to brain injury at a cellular level by the process of reactive astrogliosis, and are able to adjust their response according to the severity of the insult. Included in the reactive response is the process of phagocytosis, where astrocytes clean up surrounding cellular debris from damaged cells. In this study, we observe the process of phagocytosis by primary cortical astrocytes in the presence of media flow across the apical surface of the cells. Both static and cells under flow conditions respond consistently via phagocytosis of laser-induced cellular debris. We found that astrocytes exposed to shear flow initiate phagocytosis at a consistently faster rate than cells observed under static conditions. Shear forces created by laminar flow were analyzed as well as the flow fields created around astrocyte cells. Results suggest astrocyte phagocytosis is a mechanosensitive response, thus revealing the potential to enhance astrocyte phagocytic cleanup of damaged nervous tissue.

Calcium Dynamics in Astrocytes During Cell Injury.

The changes in intracellular calcium concentration ([Ca2+]) following laser-induced cell injury in nearby cells were studied in primary mouse astrocytes selectively expressing the Ca2+ sensitive GFAP-Cre Salsa6f fluorescent tandem protein, in an Ast1 astrocyte cell line, and in primary mouse astrocytes loaded with Fluo4. Astrocytes in these three systems exhibit distinct changes in [Ca2+] following induced death of nearby cells. Changes in [Ca2+] appear to result from release of Ca2+ from intracellular organelles, as opposed to influx from the external medium. Salsa6f expressing astrocytes displayed dynamic Ca2+ changes throughout the phagocytic response, including lamellae protrusion, cytosolic signaling during vesicle formation, vesicle maturation, and vesicle tract formation. Our results demonstrate local changes in [Ca2+] are involved in the process of phagocytosis in astrocytes responding to cell corpses and/or debris.

Laser Scissors and Tweezers to Study Chromosomes: A Review.

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.

Elastic Tethers Between Separating Anaphase Chromosomes Regulate the Poleward Speeds of the Attached Chromosomes in Crane-Fly Spermatocytes.

Elastic "tethers" connect separating anaphase chromosomes in most (or all) animal cells. We tested whether tethers are involved in coordinating movements of separating anaphase chromosomes in crane-fly spermatocytes. In these cells the coupled movements of separating chromosomes become uncoupled after the tethers are severed by laser microbeam irradiation of the interzone region between the chromosomes (Sheykhani et al., 2017). While this strongly suggests that tethers are involved with coordinating the poleward chromosome movements, the experiments are open to another interpretation: laser irradiations that cut the tethers also might damage something else in the interzone, and those non-tether components might regulate chromosome movements. In the experiments reported herein we distinguish between those two possibilities by disabling the tethers without cutting the interzone. We cut the arms from individual chromosomes, thereby severing the mechanical connection between separating chromosomes, disconnecting them, without damaging components in the interzone. Disabling tethers in this way uncoupled the movements of the separating chromosomes. We thus conclude that tethers are involved in regulating the speeds of separating anaphase chromosomes in crane-fly spermatocytes.

DNA damage induced during mitosis undergoes DNA repair synthesis.

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.

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.

Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair.

TRF2 (TERF2) binds to telomeric repeats and is critical for telomere integrity. Evidence suggests that it also localizes to non-telomeric DNA damage sites. However, this recruitment appears to be precarious and functionally controversial. We find that TRF2 recruitment to damage sites occurs by a two-step mechanism: the initial rapid recruitment (phase I), and stable and prolonged association with damage sites (phase II). Phase I is poly(ADP-ribose) polymerase (PARP)-dependent and requires the N-terminal basic domain. The phase II recruitment requires the C-terminal MYB/SANT domain and the iDDR region in the hinge domain, which is mediated by the MRE11 complex and is stimulated by TERT. PARP-dependent recruitment of intrinsically disordered proteins contributes to transient displacement of TRF2 that separates two phases. TRF2 binds to I-PpoI-induced DNA double-strand break sites, which is enhanced by the presence of complex damage and is dependent on PARP and the MRE11 complex. TRF2 depletion affects non-sister chromatid homologous recombination repair, but not homologous recombination between sister chromatids or non-homologous end-joining pathways. Our results demonstrate a unique recruitment mechanism and function of TRF2 at non-telomeric DNA damage sites.

LSD1 mediated changes in the local redox environment during the DNA damage response.

The redox state of the cell can be affected by many cellular conditions. In this study we show that detectable reactive oxygen species (ROS) are also generated in response to DNA damage by the chromatin remodeling factor and monoamine oxidase LSD1/KDM1A. This raised the possibility that the localized generation of hydrogen peroxide produced by LSD1 may affect the function of proximally located DNA repair proteins. The two major pathways for repair of DNA double-strand breaks (DSBs) are homologous recombination (HR) and non-homologous end joining (NHEJ). Cells were exposed to low levels of ectopic H2O2, DNA breaks generated by laser light, and recruitment kinetics of NHEJ protein Ku80 to DNA damage sites determined. Ku80 recruitment to damage sites was significantly decreased in cells pretreated with H2O2 while HR end binding protein Nbs1 was increased. This suggests that the DNA repair pathway choice has the potential to be modulated by the local redox state. This has implications for chemotherapeutic approaches involving generating DNA damage to target actively dividing cancer cells, which may be more or less effective dependent on the redox state of the targeted cells and the predominant repair pathway required to repair the type of DNA damage generated.

Anaphase Chromosomes in Crane-Fly Spermatocytes Treated With Taxol (Paclitaxel) Accelerate When Their Kinetochore Microtubules Are Cut: Evidence for Spindle Matrix Involvement With Spindle Forces.

Various experiments have indicated that anaphase chromosomes continue to move after their kinetochore microtubules are severed. The chromosomes move poleward at an accelerated rate after the microtubules are cut but they slow down 1-3 min later and move poleward at near the original speed. There are two published interpretations of chromosome movements with severed kinetochore microtubules. One interpretation is that dynein relocates to the severed microtubule ends and propels them poleward by pushing against non-kinetochore microtubules. The other interpretation is that components of a putative "spindle matrix" normally push kinetochore microtubules poleward and continue to do so after the microtubules are severed from the pole. In this study we distinguish between these interpretations by treating cells with taxol. Taxol eliminates microtubule dynamics, alters spindle microtubule arrangements, and inhibits dynein motor activity in vivo. If the dynein interpretation is correct, taxol should interfere with chromosome movements after kinetochore microtubules are severed because it alters the arrangements of spindle microtubules and because it blocks dynein activity. If the "spindle matrix" interpretation is correct, on the other hand, taxol should not interfere with the accelerated movements. Our results support the spindle matrix interpretation: anaphase chromosomes in taxol-treated crane-fly spermatocytes accelerated after their kinetochore microtubules were severed.

A positive-feedback-based mechanism for constriction rate acceleration during cytokinesis in Caenorhabditis elegans.

To ensure timely cytokinesis, the equatorial actomyosin contractile ring constricts at a relatively constant rate despite its progressively decreasing size. Thus, the per-unit-length constriction rate increases as ring perimeter decreases. To understand this acceleration, we monitored cortical surface and ring component dynamics during the first cytokinesis of the Caenorhabditis elegans embryo. We found that, per unit length, the amount of ring components (myosin, anillin) and the constriction rate increase with parallel exponential kinetics. Quantitative analysis of cortical flow indicated that the cortex within the ring is compressed along the axis perpendicular to the ring, and the per-unit-length rate of cortical compression increases during constriction in proportion to ring myosin. We propose that positive feedback between ring myosin and compression-driven flow of cortex into the ring drives an exponential increase in the per-unit-length amount of ring myosin to maintain a high ring constriction rate and support this proposal with an analytical mathematical model.

Phagocytic response of astrocytes to damaged neighboring cells.

This study aims to understand the phagocytic response of astrocytes to the injury of neurons or other astrocytes at the single cell level. Laser nanosurgery was used to damage individual cells in both primary mouse cortical astrocytes and an established astrocyte cell line. In both cases, the release of material/substances from laser-irradiated astrocytes or neurons induced a phagocytic response in near-by astrocytes. Propidium iodide stained DNA originating from irradiated cells was visible in vesicles of neighboring cells, confirming phagocytosis of material from damaged cortical cells. In the presence of an intracellular pH indicator dye, newly formed vesicles correspond to acidic pH fluorescence, thus suggesting lysosome bound degradation of cellular debris. Cells with shared membrane connections prior to laser damage had a significantly higher frequency of induced phagocytosis compared to isolated cells with no shared membrane. The increase in phagocytic response of cells with a shared membrane occurred regardless of the extent of shared membrane (a thin filopodial connection vs. a cell cluster with significant shared membrane). In addition to the presence (or lack) of a membrane connection, variation in phagocytic ability was also observed with differences in injury location within the cell and distance separating isolated astrocytes. These results demonstrate the ability of an astrocyte to respond to the damage of a single cell, be it another astrocyte, or a neuron. This single-cell level of analysis results in a better understanding of the role of astrocytes to maintain homeostasis in the CNS, particularly in the sensing and removal of debris in damaged or pathologic nervous tissue.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Visualizing Spatiotemporal Dynamics of Intercellular Mechanotransmission upon Wounding.

During cell-to-cell communications, the interplay between physical and biochemical cues is essential for informational exchange and functional coordination, especially in multicellular organisms. However, it remains a challenge to visualize intercellular signaling dynamics in single live cells. Here, we report a photonic approach, based on laser microscissors and Förster resonance energy transfer (FRET) microscopy, to study intercellular signaling transmission. First, using our high-throughput screening platform, we developed a highly sensitive FRET-based biosensor (SCAGE) for Src kinase, a key regulator of intercellular interactions and signaling cascades. Notably, SCAGE showed a more than 40-fold sensitivity enhancement than the original biosensor in live mammalian cells. Next, upon local severance of physical intercellular connections by femtosecond laser pulses, SCAGE enabled the visualization of a transient Src activation across neighboring cells. Lastly, we found that this observed transient Src activation following the loss of cell-cell contacts depends on the passive structural support of cytoskeleton but not on the active actomyosin contractility. Hence, by precisely introducing local physical perturbations and directly visualizing spatiotemporal transmission of ensuing signaling events, our integrated approach could be broadly applied to mimic and investigate the wounding process at single-cell resolutions. This integrated approach with highly sensitive FRET-based biosensors provides a unique system to advance our in-depth understanding of molecular mechanisms underlying the physical-biochemical basis of intercellular coupling and wounding processes.