Measurement of the Force that Centers the Mitotic Spindle in the Early C. elegans Embryo using Magnetic Tweezers

Abstract

Because the plane of cell division bisects the mitotic spindle, the positioning of the spindle plays a key role in specifying the size of the daughter cells and in segregating cytoplasmic constituents. Little is known, however, about the mechanical processes underlying spindle positioning. To study this mechanism, we built an apparatus whereby calibrated magnetic forces are applied to the spindle via super-paramagnetic beads inserted into the cytoplasm of one- and two-cell C. elegans embryos.At metaphase in the one-cell embryo, a 20 pN force displaced the mitotic spindle pole of one-cell embryos in metaphase approximately 1 μm away from the anterior-posterior axis over 10-20 seconds. By tracking the bead displacement, we found that the spindle behaved roughly as a damped spring with a spring constant of 22 ± 13 pN/μm and a drag coefficient of 138 ± 71 pN·s/μm (mean ± SD, 26 traces from 19 cells). The stiffness was five-fold higher during anaphase, indicating that the centering forces increase during the cell cycle. The stiffness was two-fold higher in the two-cell embryo, consistent with a centering mechanism that scales inversely with cell size. Finally, gpr-1/2 RNAi knockdown had no significant effect on the viscoelastic properties of either metaphase or anaphase spindles.Taken together, our results constrain molecular models of centering. The gpr-1/2 results rule out a role for cortical forces pulling on the spindle via astral microtubules. On the other hand, the results are consistent with centering being mediated by astral microtubules either pushing against the cortex or being pulled by cytoplasmic factors.