Thursday, March 21, 2013

Measurements of forces produced by the mitotic spindle using optical tweezers

Jessica Ferraro-Gideon, Rozhan Sheykhani, Qingyuan Zhu, Michelle L. Duquette, Michael W. Berns, and Arthur Forer

We used a trapping laser to stop chromosome movements in Mesostoma and crane-fly spermatocytes and to stop inward movements of spindle poles after laser cuts across 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 focussed to a single point using a 63x oil immersion objective. Trap powers of 15 mW to 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 mW to 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. Using appropriate Q’ coefficients, the forces for stopping pole movements were 0.3-2.3 pN, and for stopping chromosome movements in Mesostoma spermatocytes and crane-fly spermatocytes were 2 pN-3pN and 6-10pN, respectively. These forces are close to theoretical calculations of forces causing chromosome movements but 100 times lower than the 700pN measured in grasshopper spermatocytes (Nicklas, 1983).
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