K-fiber bundles in the mitotic spindle are mechanically reinforced by Kif15

Abstract

<jats:title>Abstract</jats:title><jats:p>The mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes into two eventual daughter nuclei. In mammalian cells, microtubule bundles called kinetochore-fibers (k-fibers) anchor chromosomes within the spindle. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here, we investigate the physical and molecular basis of k-fiber bundle cohesion. We sever k-fibers using laser ablation, thereby detaching them from poles and testing the contribution of pole-localized force generation to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often, but not always, splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced in response to ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the putative k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15 microtubule binding reduces k-fiber mechanical integrity. In contrast, inhibition of its motor activity but not its microtubule binding does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule crosslinker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors.</jats:p>

Force by minus-end motors Dhc1 and Klp2 collapses the S. pombe spindle after laser ablation

“…We performed live-cell spinning disk confocal imaging with laser ablation at room temperature of 22 C as previously described (46)(47)(48)(49). Briefly, we performed GFP imaging on a Nikon Ti-E stand on an Andor Dragonfly spinning disk confocal fluorescence microscope; spinning disk dichroic Chroma ZT405/488/561/640rpc; 488-nm (50 mW) diode laser (75 ms-120 ms exposures) with Borealis attachment (Andor); emission filter Chroma ET525/50m; and an Andor iXon3 camera.…”

Automated tracking of S. pombe spindle elongation dynamics

“…We performed live‐cell imaging of S. pombe expressing GFP‐Atb2p as previously described 42 . Briefly, we imaged on a Nikon Ti‐E stand on an Andor Dragonfly spinning disk confocal fluorescence microscope, equipped with spinning disk dichroic Chroma ZT405/488/561/640 rpc; 488 nm (50 mW) diode laser with Borealis attachment (Andor) and emission filter Chroma ET525/50 m. The system is equipped with an Andor Zyla CMOS camera and an Andor iXon EMCCD camera; we used both cameras without binning.…”

“…We performed live-cell imaging of S. pombe expressing GFP-Atb2p as previously described. 42 We collected z-stacks containing eight planes each 1 m apart at intervals of 15 or 30 s (depending on the particular acquisition). We used Andor Fusion software to control the data acquisition.…”

Automated tracking ofS. pombespindle elongation dynamics

“…Live cell imaging. Live-cell imaging of S. pombe expressing GFP-Atb2p was performed as described (42) on a Nikon Ti-E stand on an Andor Dragonfly spinning disk confocal fluorescence microscope; spinning disk dichroic Chroma ZT405/488/561/640rpc; 488 nm (50 mW) diode laser with Borealis attachment (Andor); emission filter Chroma ET525/50m; and an Andor Zyla camera. Example images shown in the text were collected with a 60x 1.49 TIRF Nikon objective.…”