Length control of the metaphase spindle.
BACKGROUND
The pole-to-pole distance of the metaphase spindle is reasonably constant in a given cell type; in the case of vertebrate female oocytes, this steady-state length can be maintained for substantial lengths of time, during which time microtubules remain highly dynamic. Although a number of molecular perturbations have been shown to influence spindle length, a global understanding of the factors that determine metaphase spindle length has not been achieved.


RESULTS
Using the Drosophila S2 cell line, we depleted or overexpressed proteins that either generate sliding forces between spindle microtubules (Kinesin-5, Kinesin-14, dynein), promote microtubule polymerization (EB1, Mast/Orbit [CLASP], Minispindles [Dis1/XMAP215/TOG]) or depolymerization (Kinesin-8, Kinesin-13), or mediate sister-chromatid cohesion (Rad21) in order to explore how these forces influence spindle length. Using high-throughput automated microscopy and semiautomated image analyses of >4000 spindles, we found a reduction in spindle size after RNAi of microtubule-polymerizing factors or overexpression of Kinesin-8, whereas longer spindles resulted from the knockdown of Rad21, Kinesin-8, or Kinesin-13. In contrast, and differing from previous reports, bipolar spindle length is relatively insensitive to increases in motor-generated sliding forces. However, an ultrasensitive monopolar-to-bipolar transition in spindle architecture was observed at a critical concentration of the Kinesin-5 sliding motor. These observations could be explained by a quantitative model that proposes a coupling between microtubule depolymerization rates and microtubule sliding forces.


CONCLUSIONS
By integrating extensive RNAi with high-throughput image-processing methodology and mathematical modeling, we reach to a conclusion that metaphase spindle length is sensitive to alterations in microtubule dynamics and sister-chromatid cohesion, but robust against alterations of microtubule sliding force.
Goshima
Gohta
G
Physiology Course 2004, Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA.
Wollman
Roy
R
Stuurman
Nico
N
Scholey
Jonathan M
JM
Vale
Ronald D
RD
D000818:Animals; D002460:Cell Line; D002842:Chromatids / Q000378:metabolism; D004330:Drosophila; D005260:Female; D007091:Image Processing, Computer-Assisted; D008677:Metaphase / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008954:Models, Biological; D020409:Molecular Motor Proteins / Q000378:metabolism; D009865:Oocytes / Q000166:cytology*; D034622:RNA Interference; D008941:Spindle Apparatus / Q000502:physiology*
D008869:Microtubule-Associated Proteins; D020409:Molecular Motor Proteins