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Open Access Publications from the University of California

Scholarly Works (212 results)

  • Article
  • Peer Reviewed
UC Irvine Previously Published Works (2020)
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.
    Cover page: Laser Scissors and Tweezers to Study Chromosomes: A Review
    • Thesis
    • Peer Reviewed
    UC San Diego Electronic Theses and Dissertations (2017)

    Reproductive success is dependent on the ability of spermatozoa to reach and fertilize the oocyte. Although there are numerous assisted reproductive technologies that were developed for human use, not all are as effective in animals due to large variations in reproductive biology. The use of drugs and excessive handling on nondomestic animals may induce additional stress. Thus, there is a need for novel noninvasive, drug-free fertility treatments that are applicable across a range of species. Sperm swimming speed and swimming force are two measures of sperm quality; improvement of these qualities may increase the chances of successful reproduction. It is believed that red light irradiation stimulates cytochrome c oxidase in the mitochondria to increase ATP production. However, stimulation of the electron transport chain has been shown to increase reactive oxygen species, which may have negative effects on sperm DNA. In this thesis, the efficacy of red light irradiation for improving sperm swimming speed and swimming force and its effect on DNA are assessed. Human spermatozoa were irradiated with a monochromatic, coherent 633 nm laser for 30 minutes. Swimming speed was measured using a wavelet-based tracking algorithm implemented in LabVIEW. Swimming force was measured using optical trapping and position tracking. Oxidative DNA damage was assessed using single-cell imaging following immunofluorescent staining for double-strand breaks and an enzyme-linked immunosorbent assay for oxidized guanine. Both sperm swimming speed and swimming force increased after irradiation without significant DNA damage.

      Cover page: Analysis of Spermatozoa Motility and DNA Integrity Following Irradiation with 633 nm Light
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (1976)
      Behavior of the centriolar duplexes during early mitosis was investigated. In general, both duplexes are capable of migrating about the cell as a unit with little change in their center to center spacing prior to separation to form the spindle poles. This duplex separation may occur at any point within the mid-prophase-prometaphase period. If it is delayed to prometaphase, transitory monopolar spindles were observed.
        Cover page: Centriole behavior in early mitosis of rat kangaroo cells (PTK2)Creative Commons 'BY' version 4.0 license
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2020)
        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.
          Cover page: Elastic Tethers Between Separating Anaphase Chromosomes Regulate the Poleward Speeds of the Attached Chromosomes in Crane-Fly Spermatocytes
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (1983)
          One of the two nucleoli of tetraploid PtK2 WA cells in early prophase was irradiated with an ultraviolet (UV) laser microbeam. The daughter cells that maintained the nucleolar deficiency were isolated and cloned. Five nucleolar deficient sublines of PtK2 WA were established, thus providing an experimental system to study the ribosomal gene-nucleolar organizer complex.
            Cover page: Establishment of nucleolar deficient sublines of PtK2 (Potorous tridactylis) by ultraviolet laser microirradiationCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (1973)
            Mitotic chromosomes of rat kangaroo cells were irradiated with a green argon laser microbeam without prior dye sensitization. Deoxyribonucleic acid-negative chromosome paling was observed. The irradiated cells were isolated and cloned into viable populations.
              Cover page: Cloning of Rat Kangaroo (PTK2) Cells Following Laser Microirradiation of Selected Mitotic ChromosomesCreative Commons 'BY' version 4.0 license
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2014)
              Low-level light therapy has been shown to improve in vitro wound healing. However, well-defined parameters of different light sources for this therapy are lacking. The goal of this study was (1) to determine if the wavelengths tested are effective for in vitro wound healing and (2) to compare a laser and a light-emitting diode (LED) source at similar wavelengths. We show four wavelengths, delivered by either a laser or LED array, improved in vitro wound healing in A549, U2OS, and PtK2 cells. Improved wound healing occurred through increased cell migration demonstrated through scratch wound and transwell assays. Cell proliferation was tested by the (3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay and was found generally not to be involved in the wound healing process. The laser and LED sources were found to be comparable when equal doses of light were applied. The biological response measured was similar in most cases. We conclude that the laser at 652 (5.57  mW/cm2, 10.02  J/cm2) and 806 nm (1.30  mW/cm2, 2.334  J/cm2) (full bandwidth 5 nm), and LED at 637 (5.57  mW/cm2, 10.02  J/cm2) and 901 nm (1.30  mW/cm2, 2.334  J/cm2) (full bandwidth 17 and 69 nm respectively) induce comparable levels of cell migration and wound closure.
                Cover page: Comparison of laser and diode sources for acceleration of in vitro wound healing by low-level light therapyCreative Commons 'BY' version 4.0 license
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2021)
                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.
                  Cover page: Blocking Protein Phosphatase 1 [PP1] Prevents Loss of Tether Elasticity in Anaphase Crane-Fly Spermatocytes
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2005)
                  We have engineered a robotic laser ablation and tweezers microscope that can be operated via the internet using most internet accessible devices, including laptops, desktop computers, and personal data assistants (PDAs). The system affords individual investigators the ability to conduct micromanipulation experiments (cell surgery or trapping) from remote locations (i.e., between the US and Australia). This system greatly expands the availability of complex and expensive research technologies via investigator-networking over the internet. It serves as a model for other "internet-friendly" technologies leading to large scale networking and data-sharing between investigators, groups, and institutions on a global scale. The system offers three unique features: (1) the freedom to operate the system from any internet-capable computer, (2) the ability to image, ablate, and/or trap cells and their organelles by "remote-control," and (3) the security and convenience of controlling the system in the laboratory on the user's own personal computer and not on the host machine. Four "proof of principle" experiments were conducted: (1) precise control of microscope movement and live cell visualization, (2) subcellular microsurgery on the microtubule organizing center of live cells viewed under phase contrast and fluorescence microscopy, (3) precise targeting of multiple sites within single red blood cells, and (4) optical trapping of 10 microm diameter polystyrene microspheres.
                    Cover page: Internet‐based robotic laser scissors and tweezers microscopyCreative Commons 'BY' version 4.0 license
                    • Article
                    • Peer Reviewed
                    UC Irvine Previously Published Works (1978)
                    Potorous tridactylis (PTK2) cells growing in culture were treated with psoralen derivatives and dividing cells were located by phase-contrast microscopy. Psoralens, light-sensitive DNA-photoadducting drugs, were reacted with mitotic chromosomes through exposure to 365-nm light from an argon laser microbeam system. It was found that following mitosis and photoreaction, cells without nuclear envelopes were produced when psoralen-treated cells received 60 light pulses over their entire chromosome complement. These 'non-nuclear membrane' cells were found to incorporate [3H]uridine and, to a lesser extent, [3H]thymidine by autoradiography. Reduction of the light exposure by half (30 near-u.v. pulses) over the entire chromosome complement in the presence of psoralen also produced non-nuclear-membrane cells as seen by light microscopy. Further examination of these cells (30 light pulses) by single-cell electron microscopy revealed that unlike the high light exposure (60 near-u.v. pulses), the low light dosage resulted in cells with membrane patches associated with their chromatin. Since neither actinomycin D nor cycloheximide impeded nuclear envelope reformation, the psoralen-DNA reaction is concluded to produce non-nuclear-membrane cells by a mechanism other than transcription or translation inhibition. The association of Golgi with areas of nuclear membrane patches gives indirect evidence of a possible Golgi contribution to the reformation of the nuclear envelope after mitosis. It is concluded that DNA plays a role in envelope reformation.
                      Cover page: Chromatin influence on the function and formation of the nuclear envelope shown by laser-induced psoralen photoreactionCreative Commons 'BY' version 4.0 license
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