UC San Diego

Mitosis is the process by which cells, after having duplicated their DNA content, segregate chromosomes equally into two identical daughter cells. Mitosis is a very short part of a normal cell cycle (usually 24+hours) and ranges from 30 minutes to an hour depending on cell type and environmental conditions. During this incredibly short amount of time, the cell undergoes several complex re-arrangements, biomechanically and biochemically. Microtubules, 20 nm width dimer polymers, play an essential role as the building blocks that provides the cytoskeleton and mitotic spindle for the cell, provide the force that segregates chromosomes (anaphase), to satisfaction of tension and attachment based checkpoints (metaphase-anaphase transition). To elucidate the key role microtubules have in mitosis, drugs such as taxol and nocodazole have been used to impart catastrophic global damage to the mitotic spindle and study the effects on cellular division. However, catastrophic global damage can not answer specific questions regarding highly spatially localized damage and temporally transient damage. In elucidating the role of microtubules, chromosomes and other key biological structures, there is the need for a transient perturbation on the mitotic process. To study the effects of transient perturbation on mitosis, a Laser microscope system (Robolase) was developed to deliver spatially localized (0̃.4 um) and temporally-specific disruption inside living cells (nanosurgery). Specifically, the affect of ablating chromosome tips, mitotic spindles, and chromatid are examined, and the relationship between damaged sites and pathways controlling the progression of the cell cycle and DNA damage pathways are examined. In conclusion, an optically based method for studying mitosis with transient perturbation has been developed and used to determine that chromosome tip disruption affects cytokinetic progression, prolonged disruption of mitotic spindle reveals force sensing in the metaphase spindle, and double-strand breaks of DNA recruit CENP-A in addition to known DNA damage proteins