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.

Laser microsurgery directed between sister telomeres reduced strain in anaphase PtK2 chromosomes. Optical trapping of severed chromosome ends suggests this strain arises from a physical element which links sister chromatids during early anaphase.

Originally described in crane-fly spermatocytes, tethers physically link and transmit force between the ends of separating chromosomes. Optical tweezers and laser scissors were used to sever the tether between chromosomes, create chromosome fragments attached to the tether which move toward the opposite pole, and to trap the tethered fragments. Laser microsurgery in the intracellular space between separating telomeres reduced chromosome strain in half of tested chromosome pairs. When the telomere-containing region was severed from the rest of the chromosome body, the resultant fragment either traveled towards the proper pole (poleward), towards the sister pole (cross-polar), or movement ceased. Fragment travel towards the sister pole varied in distance and always ceased following a cut between telomeres, indicating the tether is responsible for transferring a cross-polar force to the fragment. Optical trapping of cross-polar traveling fragments places an upper boundary on the tethering force of ~1.5 pN.

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.

We used a trapping laser to stop chromosome movements in Mesostoma and crane-fly spermatocytes and inward movements of spindle poles after laser cuts across Potorous tridactylus (rat kangaroo) kidney (PtK2) cell half-spindles. Mesostoma spermatocyte kinetochores execute oscillatory movements to and away from the spindle pole for 1-2 h, so we could trap kinetochores multiple times in the same spermatocyte. The trap was focused to a single point using a 63× oil immersion objective. Trap powers of 15-23 mW caused kinetochore oscillations to stop or decrease. Kinetochore oscillations resumed when the trap was released. In crane-fly spermatocytes trap powers of 56-85 mW stopped or slowed poleward chromosome movement. In PtK2 cells 8-mW trap power stopped the spindle pole from moving toward the equator. Forces in the traps were calculated using the equation F = Q'P/c, where P is the laser power and c is the speed of light. Use of appropriate Q' coefficients gave the forces for stopping pole movements as 0.3-2.3 pN and for stopping chromosome movements in Mesostoma spermatocytes and crane-fly spermatocytes as 2-3 and 6-10 pN, respectively. These forces are close to theoretical calculations of forces causing chromosome movements but 100 times lower than the 700 pN measured previously in grasshopper spermatocytes.

We describe the general occurrence in animal cells of elastic components ("tethers") that connect individual chromosomes moving to opposite poles during anaphase. Tethers, originally described in crane-fly spermatocytes, exert force on chromosome arms opposite to the direction the anaphase chromosomes move. We show that they exist in a broad range of animal cells. Thus tethers are previously unrecognised components of general mitotic mechanisms that exert force on chromosomes and they need to be accounted for in general models of mitosis in terms of forces on chromosomes and in terms of what their roles might be.