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.

Impacts of H2O2, SARM1 inhibition, and high NAm concentrations on Huntington's disease laser-induced degeneration.

Axonal degeneration is a key component of neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease, and amyotrophic lateral sclerosis. Nicotinamide, an NAD+ precursor, has long since been implicated in axonal protection and reduction of degeneration. However, studies on nicotinamide (NAm) supplementation in humans indicate that NAm has no protective effect. Sterile alpha and toll/interleukin receptor motif-containing protein 1 (SARM1) regulates several cell responses to axonal damage and has been implicated in promoting neuronal degeneration. SARM1 inhibition seems to result in protection from neuronal degeneration while hydrogen peroxide has been implicated in oxidative stress and axonal degeneration. The effects of laser-induced axonal damage in wild-type and HD dorsal root ganglion cells treated with NAm, hydrogen peroxide (H2O2), and SARM1 inhibitor DSRM-3716 were investigated and the cell body width, axon width, axonal strength, and axon shrinkage post laser-induced injury were measured.

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.

Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation.

In this study the femtosecond near-IR and nanosecond green lasers are used to induce alterations in mitotic chromosomes. The subsequent double-strand break responses are studied. We show that both lasers are capable of creating comparable chromosomal alterations and that a phase paling observed within 1-2 s of laser exposure is associated with an alteration of chromatin as confirmed by serial section electron microscopy, DAPI, γH2AX and phospho-H3 staining. Additionally, the accumulation of dark material observed using phase contrast light microscopy (indicative of a change in refractive index of the chromatin) ∼ 34 s post-laser exposure corresponds spatially to the accumulation of Nbs1, Ku and ubiquitin. This study demonstrates that chromosomes selectively altered in mitosis initiate the DNA damage response within 30 s and that the accumulation of proteins are visually represented by phase-dark material at the irradiation site, allowing us to determine the fate of the damage as cells enter G1. These results occur with two widely different laser systems, making this approach to study DNA damage responses in the mitotic phase generally available to many different labs. Additionally, we present a summary of most of the published laser studies on chromosomes in order to provide a general guide of the lasers and operating parameters used by other laboratories.

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.

Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells.

Proper recognition and repair of DNA damage is critical for the cell to protect its genomic integrity. Laser microirradiation ranging in wavelength from ultraviolet A (UVA) to near-infrared (NIR) can be used to induce damage in a defined region in the cell nucleus, representing an innovative technology to effectively analyze the in vivo DNA double-strand break (DSB) damage recognition process in mammalian cells. However, the damage-inducing characteristics of the different laser systems have not been fully investigated. Here we compare the nanosecond nitrogen 337 nm UVA laser with and without bromodeoxyuridine (BrdU), the nanosecond and picosecond 532 nm green second-harmonic Nd:YAG, and the femtosecond NIR 800 nm Ti:sapphire laser with regard to the type(s) of damage and corresponding cellular responses. Crosslinking damage (without significant nucleotide excision repair factor recruitment) and single-strand breaks (with corresponding repair factor recruitment) were common among all three wavelengths. Interestingly, UVA without BrdU uniquely produced base damage and aberrant DSB responses. Furthermore, the total energy required for the threshold H2AX phosphorylation induction was found to vary between the individual laser systems. The results indicate the involvement of different damage mechanisms dictated by wavelength and pulse duration. The advantages and disadvantages of each system are discussed.

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.

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.

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.

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.

Recruitment of DNA damage recognition and repair pathway proteins following near-IR femtosecond laser irradiation of cells.

An 800-nm 200-fs laser is used to produce DNA damage in rat kangaroo (PtK1) and human cystic fibrosis pancreatic adenoma carcinoma (CFPAC-1) cells. Immunofluorescence staining for DNA repair factors in irradiated cells displays localization of gammaH2AX, Nbs1, and Rad50 to the site of irradiation 3 to 30 min following laser exposure. It is concluded that the 200-fs near-infrared laser is an excellent source for the production and study of spatially defined regions of DNA damage.

Red light improves spermatozoa motility and does not induce oxidative DNA damage.

The ability to successfully fertilize ova relies upon the swimming ability of spermatozoa. Both in humans and in animals, sperm motility has been used as a metric for the viability of semen samples. Recently, several studies have examined the efficacy of low dosage red light exposure for cellular repair and increasing sperm motility. Of prime importance to the practical application of this technique is the absence of DNA damage caused by radiation exposure. In this study, we examine the effect of 633 nm coherent, red laser light on sperm motility using a novel wavelet-based algorithm that allows for direct measurement of curvilinear velocity under red light illumination. This new algorithm gives results comparable to the standard computer-assisted sperm analysis (CASA) system. We then assess the safety of red light treatment of sperm by analyzing, (1) the levels of double-strand breaks in the DNA, and (2) oxidative damage in the sperm DNA. The results demonstrate that for the parameters used there are insignificant differences in oxidative DNA damage as a result of irradiation.

Laser-induced shockwave paired with FRET: a method to study cell signaling.

Cells within the body are subject to various forces; however, the details concerning the way in which cells respond to mechanical stimuli are not well understood. We demonstrate that laser-induced shockwaves (LIS) combined with biosensors based on fluorescence resonance energy transfer (FRET) is a promising new approach to study biological processes in single live cells. As "proof-of-concept," using a FRET biosensor, we show that in response to LIS, cells release intracellular calcium. With the parameters used, cells retain their morphology and remain viable. LIS combined with FRET permits observation of the cells immediate response to a sudden shear force.

The role of actin and myosin in PtK2 spindle length changes induced by laser microbeam irradiations across the spindle.

This study investigates spindle biomechanical properties to better understand how spindles function. In this report, laser microbeam cutting across mitotic spindles resulted in movement of spindle poles toward the spindle equator. The pole on the cut side moved first, the other pole moved later, resulting in a shorter but symmetric spindle. Intervening spindle microtubules bent and buckled during the equatorial movement of the poles. Because of this and because there were no detectable microtubules within the ablation zone, other cytoskeletal elements would seem to be involved in the equatorial movement of the poles. One possibility is actin and myosin since pharmacological poisoning of the actin-myosin system altered the equatorial movements of both irradiated and unirradiated poles. Immunofluorescence microscopy confirmed that actin, myosin and monophosphorylated myosin are associated with spindle fibers and showed that some actin and monophosphorylated myosin remained in the irradiated regions. Overall, our experiments suggest that actin, myosin and microtubules interact to control spindle length. We suggest that actin and myosin, possibly in conjunction with the spindle matrix, cause the irradiated pole to move toward the equator and that cross-talk between the two half spindles causes the unirradiated pole to move toward the equator until a balanced length is obtained.

Neuronal growth cones respond to laser-induced axonal damage.

Although it is well known that damage to neurons results in release of substances that inhibit axonal growth, release of chemical signals from damaged axons that attract axon growth cones has not been observed. In this study, a 532 nm 12 ns laser was focused to a diffraction-limited spot to produce site-specific damage to single goldfish axons in vitro. The axons underwent a localized decrease in thickness ('thinning') within seconds. Analysis by fluorescence and transmission electron microscopy indicated that there was no gross rupture of the cell membrane. Mitochondrial transport along the axonal cytoskeleton immediately stopped at the damage site, but recovered over several minutes. Within seconds of damage nearby growth cones extended filopodia towards the injury and were often observed to contact the damaged site. Turning of the growth cone towards the injured axon also was observed. Repair of the laser-induced damage was evidenced by recovery of the axon thickness as well as restoration of mitochondrial movement. We describe a new process of growth cone response to damaged axons. This has been possible through the interface of optics (laser subcellular surgery), fluorescence and electron microscopy, and a goldfish retinal ganglion cell culture model.