Graduate Thesis Or Dissertation

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  • Accurate chromosome segregation and cell cleavage are critical to maintaining genomic integrity. Both events involve the spindle apparatus, but the exact mechanics is as puzzling as the contradicting models proposed in the last two centuries. In this dissertation, current prevailing models of chromosome segregation and cell cleavage are tested using a newly-developed Multimode Microsurgery and Imaging System. The system permits remodeling of the spindle structure in testing the current models and proposing new theories. The mechanics of chromosome segregation is a process coupled to the shortening of kinetochore microtubules (kMTs). Which end shortens and whether the shortening provides poleward forces remain unsolved, since depolymerization may occur at the plus ends by ‘Pac-Man’ activities of a kinetochore and/or the minus ends by Poleward Flux of microtubules (Traction Fibers). Alternatively, the shortening may be secondary to the force-generating Spindle Matrix and/or the non-kMTs. I differentiated these models in grasshopper spermatocytes by revealing dynamics of laser-severed kMTs both in and outside the context of the spindle. I found that the kMTs dynamically maintain their length by poleward flux, polymerizing at the plus ends while depolymerizing at the minus ends without net shortening. Poleward forces are generated when net-shortening of the kMTs occurs at the spindle poles, ‘reeling in’ the attached chromosomes. The mechanics of cleavage furrow induction is a process mediated by spindle microtubules and associated proteins, arguably via Polar Relaxation or Equatorial Stimulation mechanisms. By manipulating distribution of actin filaments in silkworm spermatocytes, I show that ‘relaxation’ can be induced at any region of the cell cortex by any microtubules mechanically brought nearby. The relaxation causes exclusion of cortical actin filaments, which depends on microtubule dynamics but not RhoA activity. ‘Stimulation’ can also be induced at any region of the cell cortex by the plus ends of central spindle microtubules brought nearby. The stimulation occurs as rapid de novo assembly of actin patches at the microtubule overlap and their lateral transport to the cortex, both of which depend on RhoA activity but not microtubule dynamics. I conclude that polar relaxation and equatorial stimulation coexist in cytokinesis, providing cell cleavage with ‘double insurance.’
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