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.

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.

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.

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.

Rat embryonic hippocampus and induced pluripotent stem cell derived cultured neurons recover from laser-induced subaxotomy.

Axonal injury and stress have long been thought to play a pathogenic role in a variety of neurodegenerative diseases. However, a model for studying single-cell axonal injury in mammalian cells and the processes of repair has not been established. The purpose of this study was to examine the response of neuronal growth cones to laser-induced axonal damage in cultures of embryonic rat hippocampal neurons and induced pluripotent stem cell (iPSC) derived human neurons. A 532-nm pulsed [Formula: see text] picosecond laser was focused to a diffraction limited spot at a precise location on an axon using a laser energy/power that did not rupture the cell membrane (subaxotomy). Subsequent time series images were taken to follow axonal recovery and growth cone dynamics. After laser subaxotomy, axons thinned at the damage site and initiated a dynamic cytoskeletal remodeling process to restore axonal thickness. The growth cone was observed to play a role in the repair process in both hippocampal and iPSC-derived neurons. Immunofluorescence staining confirmed structural tubulin damage and revealed initial phases of actin-based cytoskeletal remodeling at the damage site. The results of this study indicate that there is a repeatable and cross-species repair response of axons and growth cones after laser-induced damage.

Laser microbeam targeting of single nerve axons in cell culture.

By focusing a laser with short pulses to a diffraction-limited spot, single nerve axons can be precisely targeted and injured. Subsequent repair can be analyzed using various imaging and biochemical techniques to understand the repair process. In this chapter, we will describe a robotic laser microscope system used to injure nerve axons while simultaneously observing repair using phase and fluorescence microscopy. We provide procedures for controlled laser targeting and an experimental approach for studying axonal repair in embryonic rat hippocampus neurons.

Determination of motility forces on isolated chromosomes with laser tweezers.

Quantitative determination of the motility forces of chromosomes during cell division is fundamental to understanding a process that is universal among eukaryotic organisms. Using an optical tweezers system, isolated mammalian chromosomes were held in a 1064 nm laser trap. The minimum force required to move a single chromosome was determined to be ≈ 0.8-5 pN. The maximum transverse trapping efficiency of the isolated chromosomes was calculated as ≈ 0.01-0.02. These results confirm theoretical force calculations of ≈ 0.1-12 pN to move a chromosome on the mitotic or meiotic spindle. The verification of these results was carried out by calibration of the optical tweezers when trapping microspheres with a diameter of 4.5-15 µm in media with 1-7 cP viscosity. The results of the chromosome and microsphere trapping experiments agree with optical models developed to simulate trapping of cylindrical and spherical specimens.

CtIP maintains stability at common fragile sites and inverted repeats by end resection-independent endonuclease activity.

Chromosomal rearrangements often occur at genomic loci with DNA secondary structures, such as common fragile sites (CFSs) and palindromic repeats. We developed assays in mammalian cells that revealed CFS-derived AT-rich sequences and inverted Alu repeats (Alu-IRs) are mitotic recombination hotspots, requiring the repair functions of carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) and the Mre11/Rad50/Nbs1 complex (MRN). We also identified an endonuclease activity of CtIP that is dispensable for end resection and homologous recombination (HR) at I-SceI-generated "clean" double-strand breaks (DSBs) but is required for repair of DSBs occurring at CFS-derived AT-rich sequences. In addition, CtIP nuclease-defective mutants are impaired in Alu-IRs-induced mitotic recombination. These studies suggest that an end resection-independent CtIP function is important for processing DSB ends with secondary structures to promote HR. Furthermore, our studies uncover an important role of MRN, CtIP, and their associated nuclease activities in protecting CFSs in mammalian cells.

Real-time calcium measurements of live optically trapped microorganisms.

A system has been developed that allows for the real-time measurement of calcium dynamics in swimming sperm. Specifically, the ratiometric dye Indo-I is used as a fluorescent indicator of intracellular calcium dynamics. The dual emissions are collected by a high-sensitivity back-illuminated CCD camera coupled to a Dual-View imaging system. From the CCD, the images are sent to a custom developed algorithm which processes the images and outputs the calcium measurements in real-time. Additionally, sperm velocity and position data are processed and outputted in real-time. The velocity and position data are obtained using a separate coupled red light (>670 nm) phase contrast imaging setup that does not optically interfere with the fluorescent imaging. Using this system the effects of optical trapping on calcium dynamics was determined. Optical trapping of sperm with a decaying focused laser power of 510 mW to 3 mW over 8 seconds causes a statistically insignificant change in calcium dynamics between in-trap and out-of-trap conditions. Progesterone, a calcium activator, was added and sperm were trapped under the 8 second power decay conditions. Progesterone treated sperm has a statistically higher average calcium level than untreated sperm, but shows no statistical difference between progesterone treated in-trap and out-of-trap conditions. Trapping at 16 seconds at 510 mW without decay, which have been shown to decrease sperm motility, shows a statistical difference between baseline pre-trap and in-trap intracellular calcium levels.

An extensive comparison of the effect of anthelmintic classes on diverse nematodes.

Soil-transmitted helminths are parasitic nematodes that inhabit the human intestine. These parasites, which include two hookworm species, Ancylostomaduodenale and Necator americanus, the whipworm Trichuristrichiura, and the large roundworm Ascarislumbricoides, infect upwards of two billion people and are a major cause of disease burden in children and pregnant women. The challenge with treating these diseases is that poverty, safety, and inefficient public health policy have marginalized drug development and distribution to control infection in humans. Anthelmintics (anti-worm drugs) have historically been developed and tested for treatment of non-human parasitic nematodes that infect livestock and companion animals. Here we systematically compare the in vitro efficacy of all major anthelmintic classes currently used in human therapy (benzimidazoles, nicotinic acetylcholine receptor agonists, macrocyclic lactones, nitazoxanide) against species closely related to human parasitic nematodes-Ancylostoma ceylanicum, Trichurismuris, and Ascarissuum--- as well as a rodent parasitic nematode used in veterinary drug discovery, Heligmosomoidesbakeri, and the free-living nematode Caenorhabditis elegans. Extensive in vitro data is complemented with single-dose in vivo data in three rodent models of parasitic diseases. We find that the effects of the drugs in vitro and in vivo can vary greatly among these nematode species, e.g., the efficacy of albendazole is strong on A. ceylanicum but weak on H. bakeri. Nonetheless, certain commonalities of the in vitro effects of the drugs can be seen, e.g., nitazoxanide consistently shows an all-or-nothing response. Our in vitro data suggest that further optimization of the clinical efficacy of some of these anthelmintics could be achieved by altering the treatment routine and/or dosing. Most importantly, our in vitro and in vivo data indicate that the hookworm A. ceylanicum is a particularly sensitive and useful model for anthelmintic studies and should be incorporated early on in drug screens for broad-spectrum human soil-transmitted helminth therapies.

Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells.

Microhomology-mediated end joining (MMEJ) is a major pathway for Ku-independent alternative nonhomologous end joining, which contributes to chromosomal translocations and telomere fusions, but the underlying mechanism of MMEJ in mammalian cells is not well understood. In this study, we demonstrated that, distinct from Ku-dependent classical nonhomologous end joining, MMEJ--even with very limited end resection--requires cyclin-dependent kinase activities and increases significantly when cells enter S phase. We also showed that MMEJ shares the initial end resection step with homologous recombination (HR) by requiring meiotic recombination 11 homolog A (Mre11) nuclease activity, which is needed for subsequent recruitment of Bloom syndrome protein (BLM) and exonuclease 1 (Exo1) to DNA double-strand breaks (DSBs) to promote extended end resection and HR. MMEJ does not require S139-phosphorylated histone H2AX (γ-H2AX), suggesting that initial end resection likely occurs at DSB ends. Using a MMEJ and HR competition repair substrate, we demonstrated that MMEJ with short end resection is used in mammalian cells at the level of 10-20% of HR when both HR and nonhomologous end joining are available. Furthermore, MMEJ is used to repair DSBs generated at collapsed replication forks. These studies suggest that MMEJ not only is a backup repair pathway in mammalian cells, but also has important physiological roles in repairing DSBs to maintain cell viability, especially under genomic stress.

The interaction of CtIP and Nbs1 connects CDK and ATM to regulate HR-mediated double-strand break repair.

CtIP plays an important role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.

Purple sea urchin Strongylocentrotus purpuratus gamete manipulation using optical trapping and microfluidics.

A system has been developed that allows for optical and fluidic manipulation of gametes. The optical manipulation is performed by using a single-point gradient trap with a 40× oil immersion PH3 1.3 NA objective on a Zeiss inverted microscope. The fluidic manipulation is performed by using a custom microfluidic chamber designed to fit into the short working distance between the condenser and objective. The system is validated using purple sea urchin Strongylocentrotus purpuratus gametes and has the potential to be used for mammalian in vitro fertilization and animal husbandry.

Development of a dual joystick-controlled laser trapping and cutting system for optical micromanipulation of chromosomes inside living cells.

A multi-joystick robotic laser microscope system used to control two optical traps (tweezers) and one laser scissors has been developed for subcellular organelle manipulation. The use of joysticks has provided a "user-friendly" method for both trapping and cutting of organelles such as chromosomes in live cells. This innovative design has enabled the clean severing of chromosome arms using the laser scissors as well as the ability to easily hold and pull the severed arm using the laser tweezers.

CtIP is required to initiate replication-dependent interstrand crosslink repair.

DNA interstrand crosslinks (ICLs) are toxic lesions that block the progression of replication and transcription. CtIP is a conserved DNA repair protein that facilitates DNA end resection in the double-strand break (DSB) repair pathway. Here we show that CtIP plays a critical role during initiation of ICL processing in replicating human cells that is distinct from its role in DSB repair. CtIP depletion sensitizes human cells to ICL inducing agents and significantly impairs the accumulation of DNA damage response proteins RPA, ATR, FANCD2, γH2AX, and phosphorylated ATM at sites of laser generated ICLs. In contrast, the appearance of γH2AX and phosphorylated ATM at sites of laser generated double strand breaks (DSBs) is CtIP-independent. We present a model in which CtIP functions early in ICL repair in a BRCA1- and FANCM-dependent manner prior to generation of DSB repair intermediates.

The RING finger protein RNF8 ubiquitinates Nbs1 to promote DNA double-strand break repair by homologous recombination.

Ubiquitination plays an important role in the DNA damage response. We identified a novel interaction of the E3 ubiquitin ligase RNF8 with Nbs1, a key regulator of DNA double-strand break (DSB) repair. We found that Nbs1 is ubiquitinated both before and after DNA damage and is a direct ubiquitination substrate of RNF8. We also identified key residues on Nbs1 that are ubiquitinated by RNF8. By using laser microirradiation and live-cell imaging, we observed that RNF8 and its ubiquitination activity are important for promoting optimal binding of Nbs1 to DSB-containing chromatin. We also demonstrated that RNF8-mediated ubiquitination of Nbs1 contributes to the efficient and stable binding of Nbs1 to DSBs and is important for HR-mediated DSB repair. Taken together, these studies suggest that Nbs1 is one important target of RNF8 to regulate DNA DSB repair.

Organelle size equalization by a constitutive process.

How cells control organelle size is an elusive problem. Two predominant models for size control can be distinguished: (1) induced control, where organelle genesis, maintenance, and disassembly are three separate programs that are activated in response to size change, and (2) constitutive control, where stable size results from the balance between continuous organelle assembly and disassembly. The problem has been studied in Chlamydomonas reinhardtii because the flagella are easy to measure, their size changes only in the length dimension, and the genetics are comparable to yeast. Length dynamics in Chlamydomonas flagella are quite robust: they maintain a length of about 12 µm and recover from amputation in about 90 min with a growth rate that decreases smoothly to zero as the length approaches 12 µm. Despite a wealth of experimental studies, existing data are consistent with both induced and constitutive control models for flagella. Here we developed novel microfluidic trapping and laser microsurgery techniques in Chlamydomonas to distinguish between length control models by measuring the two flagella on a single cell as they equilibrate after amputation of a single flagellum. The results suggest that cells equalize flagellar length by constitutive control.

Rad50 zinc hook is important for the Mre11 complex to bind chromosomal DNA double-stranded breaks and initiate various DNA damage responses.

The Mre11-Rad50-Nbs1 (MRN) complex plays critical roles in checkpoint activation and double-stranded break (DSB) repair. The Rad50 zinc hook domain mediates zinc-dependent intercomplex associations of MRN, which is important for DNA tethering. Studies in yeast suggest that the Rad50 zinc hook domain is essential for MRN functions, but its role in mammalian cells is not clear. We demonstrated that the human Rad50 hook mutants are severely defective in various DNA damage responses including ATM (Ataxia telangiectasia mutated) activation, homologous recombination, sensitivity to IR, and activation of the ATR pathway. By using live cell imaging, we observed that the Rad50 hook mutants fail to be recruited to chromosomal DSBs, suggesting a novel mechanism underlying the severe defects observed for the Rad50 hook mutants. In vitro analysis showed that Zn(2+) promotes wild type but not the hook mutant of MR to bind double-stranded DNA. In vivo, the Rad50 hook mutants are defective in being recruited to chromosomal DSBs in both H2AX-proficient and -deficient cells, suggesting that the Rad50 hook mutants are impaired in direct binding to chromosomal DSB ends. We propose that the Rad50 zinc hook domain is important for the initial binding of MRN to DSBs, leading to ATM activation to phosphorylate H2AX, which recruits more MRN to the DSB-flanking chromosomal regions. Our studies reveal a critical role for the Rad50 zinc hook domain in establishing and maintaining MRN recruitment to chromosomal DSBs and suggest an important mechanism of how the Rad50 zinc hook domain contributes to DNA repair and checkpoint activation.

CtIP protein dimerization is critical for its recruitment to chromosomal DNA double-stranded breaks.

CtIP (CtBP-interacting protein) associates with BRCA1 and the Mre11-Rad50-Nbs1 (MRN) complex and plays an essential role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair. It has been described that CtIP forms dimers in mammalian cells, but the biological significance is not clear. In this study, we identified a conserved motif in the N terminus of CtIP, which is required for dimer formation. We further showed that CtIP mutants impaired in forming dimers are strongly defective in HR, end resection, and activation of the ataxia telangiectasia and Rad3-related pathway, without notable change of CtIP interactions with BRCA1 or Nbs1. In addition to HR, CtIP dimerization is also required for microhomology-mediated end joining. Live cell imaging of enhanced GFP-tagged CtIP demonstrates that the CtIP dimerization mutant fails to be localized to DSBs, whereas placing a heterologous dimerization motif to the dimerization mutant restores CtIP recruitment to DSBs. These studies suggest that CtIP dimer formation is essential for its recruitment to DSBs on chromatin upon DNA damage. Furthermore, DNA damage-induced phosphorylation of CtIP is significantly reduced in the CtIP dimerization mutants. Therefore, in addition to the C-terminal conserved domains critical for CtIP function, the dimerization motif on the N terminus of CtIP is also conserved and essential for its function in DNA damage responses. The severe repair defects of CtIP dimerization mutants are likely due to the failure in localization to chromosomal DSBs upon DNA damage.

Effects of viscosity on sperm motility studied with optical tweezers.

The purpose of this study is to analyze human sperm motility and energetics in media with different viscosities. Multiple experiments were performed to collect motility parameters using customized computer tracking software that measures the curvilinear velocity (VCL) and the minimum laser power (Pesc) necessary to hold an individual sperm in an optical trap. The Pesc was measured by using a 1064 nm Nd:YVO(4) continuous wave laser that optically traps motile sperm at a power of 450 mW in the focused trap spot. The VCL was measured frame by frame before trapping. In order to study sperm energetics under different viscous conditions sperm were labeled with the fluorescent dye DiOC(6)(3) to measure membrane potentials of mitochondria in the sperm midpiece. Fluorescence intensity was measured before and during trapping. The results demonstrate a decrease in VCL but an increase in Pesc with increasing viscosity. Fluorescent intensity is the same regardless of the viscosity level indicating no change in sperm energetics. The results suggest that, under the conditions tested, viscosity physically affects the mechanical properties of sperm motility rather than the chemical pathways associated with energetics.