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.

CtIP links DNA double-strand break sensing to resection.

In response to DNA double-strand breaks (DSBs), cells sense the DNA lesions and then activate the protein kinase ATM. Subsequent DSB resection produces RPA-coated ssDNA that is essential for activation of the DNA damage checkpoint and DNA repair by homologous recombination (HR). However, the biochemical mechanism underlying the transition from DSB sensing to resection remains unclear. Using Xenopus egg extracts and human cells, we show that the tumor suppressor protein CtIP plays a critical role in this transition. We find that CtIP translocates to DSBs, a process dependent on the DSB sensor complex Mre11-Rad50-NBS1, the kinase activity of ATM, and a direct DNA-binding motif in CtIP, and then promotes DSB resection. Thus, CtIP facilitates the transition from DSB sensing to processing: it does so by binding to the DNA at DSBs after DSB sensing and ATM activation and then promoting DNA resection, leading to checkpoint activation and HR.

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.

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.

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.

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.

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.

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.

Comparison of glycolysis and oxidative phosphorylation as energy sources for mammalian sperm motility, using the combination of fluorescence imaging, laser tweezers, and real-time automated tracking and trapping.

The combination of laser tweezers, fluorescent imaging, and real-time automated tracking and trapping (RATTS) can measure sperm swimming speed and swimming force simultaneously with mitochondrial membrane potential (MMP). This approach is used to study the roles of two sources of ATP in sperm motility: oxidative phosphorylation, which occurs in the mitochondria located in the sperm midpiece and glycolysis, which occurs along the length of the sperm tail (flagellum). The relationships between (a) swimming speed and MMP and (b) swimming force and MMP are studied in dog and human sperm. The effects of glucose, oxidative phosphorylation inhibitors and glycolytic inhibitors on human sperm motility are examined. The results indicate that oxidative phosphorylation does contribute some ATP for human sperm motility, but not enough to sustain high motility. The glycolytic pathway is shown to be a primary source of energy for human sperm motility.

Use of laser tweezers to analyze sperm motility and mitochondrial membrane potential.

We combine laser tweezers with custom computer tracking software and robotics to analyze the motility [swimming speed, VCL (curvilinear velocity), and swimming force in terms of escape laser power (Pesc)] and energetics [mitochondrial membrane potential (MP)] of individual sperm. Domestic dog sperm are labeled with a cationic fluorescent probe, DiOC2(3), that reports the MP across the inner membrane of the mitochondria located in the sperm's midpiece. Individual sperm are tracked to calculate VCL. Pesc is measured by reducing the laser power after the sperm is trapped using laser tweezers until the sperm is capable of escaping the trap. The MP is measured every second over a 5-s interval during the tracking phase (sperm is swimming freely) and continuously during the trapping phase. The effect of the fluorescent probe on sperm motility is addressed. The sensitivity of the probe is measured by assessing the effects of a mitochondrial uncoupling agent (CCCP) on MP of free swimming sperm. The effects of prolonged exposed to the laser tweezers on VCL and MP are analyzed. The system's capabilities are demonstrated by measuring VCL, Pesc, and MP simultaneously for individual sperm. This combination of imaging tools is useful to quantitatively assess sperm quality and viability.

The use of optical tweezers to study sperm competition and motility in primates.

Optical trapping is a non-invasive biophysical tool which has been widely applied to study physiological and biomechanical properties of cells. Using laser 'tweezers' in combination with custom-designed computer tracking algorithms, the swimming speeds and the relative swimming forces of individual sperm can be measured in real time. This combination of physical and engineering tools has been used to examine the evolutionary effect of sperm competition in primates. The results demonstrate a correlation between mating type and sperm motility: sperm from polygamous (multi-partner) primate species swim faster and with greater force than sperm from polygynous (single partner) primate species. In addition, sperm swimming force linearly increases with swimming speed for each species, yet the regression relating the two parameters is species specific. These results demonstrate the feasibility of using these tools to study rapidly moving (microm s(-1)) biological cells.

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.

Laser nanosurgery of single microtubules reveals location-dependent depolymerization rates.

In this study, 532-nm picosecond and 800-nm femtosecond lasers are used in combination with fluorescently labeled tubulin to further elucidate microtubule depolymerization and the effect lasers may have on the resulting depolymerization. Depolymerization rates of targeted single microtubules are dependent on location with respect to the nucleus. Microtubules located near the nucleus exhibit a significantly faster depolymerization rate when compared to microtubule depolymerization rates near the periphery of the cell. Microtubules cut with the femtosecond laser depolymerize at a slower rate than unirradiated controls (p=0.002), whereas those cut with the picosecond laser depolymerize at the same rate as unirradiated controls (p=0.704). Our results demonstrate the ability of both the picosecond and femtosecond lasers to cut individual microtubules. The differences between the two ablation results are discussed.

Real-time automated tracking and trapping system for sperm.

We have developed a microscope system for real-time single sperm tracking with an automated laser tweezers escape power assay. Phase contrast images of swimming sperm are digitized to the computer at video rate. The custom algorithm creates a region of interest centered about a sperm in response to a mouse click and performs all subsequent tasks autonomously. Microscope stage movement responds to feedback from video analysis of swimming sperm to center the sperm with respect to the field of view. For escape power assays, sperm are automatically relocated to the laser trap focus where they are held for a user-defined duration at fixed power, or held as laser power is gradually reduced. The sperm's position is automatically monitored to measure the laser power at which the sperm escapes the trap. Sperm are tracked for extended durations before and after laser trap experiments. Motility measurements including the curvilinear velocity and the absolute position of the sperm relative to the cell chamber are calculated and written to the hard drive at video rate. Experimental throughput is increased over 30 times compared to off-line data analysis. The efficacy of the "track and trap" algorithm is validated through examples and comparisons with the manually collected data.

Computer-based tracking of single sperm.

This paper describes a robust single sperm tracking algorithm (SSTA) that can be used in laser optical trapping and sperm motility studies. The algorithm creates a region of interest (ROI) centered about a sperm selected by the user. SSTA contrast enhances the ROI image and implements a modified four-class thresholding method to extract the tracked sperm as it transitions in and out of focus. The nearest neighbor method is complemented with a speed-check feature to aid tracking in the presence of additional sperm or other particles. SSTA has a collision-detection feature for real or perceived collision or near-miss cases between two sperm. Subsequent postcollision analysis employs three criteria to distinguish the tracked sperm in the image. The efficacy of SSTA is validated through examples and comparisons to commercially available computer-aided sperm tracking systems.

Analysis of sperm motility using optical tweezers.

This study examines the use of optical trapping as a quantitative measure of sperm motility. The effects of laser trap duration and laser trapping power on sperm motility are described between sperm swimming force, swimmimg speed, and speed of progression (SOP) score. Sperm (SOP scores of 2-4) were trapped by a continuous-wave 1064 nm single-point gradient laser trap. Trap duration effects were quantified for 15, 10, and 5 seconds at 420 mW laser power. Laser power effects were quantified at powers of 420 mW, 350 mW, 300 mW, and 250 mW for five seconds. Swimming force, swimming speed, and SOP score relationships were examined at a trap duration and trapping power shown to minimally affect sperm motility. Swimming forces were measured by trapping sperm and subsequently decreasing laser power until the sperm escaped the trap. Swimming trajectories were calculated by custom-built software, and SOP scores were assigned by three qualified sperm scoring experts. A ubiquitous class of sperm were identified that swim with relatively high forces that are uncorrelated to swimming speed. It is concluded that sperm swimming forces measured by optical trapping provide new and valuable quantitative information to assess sperm motility.