The homotetrameric kinesin-5 KLP61F preferentially crosslinks microtubules into antiparallel orientations

Abstract

The segregation of genetic material during mitosis is coordinated by the mitotic spindle, whose action depends upon the polarity patterns of its microtubules (MTs). Homotetrameric mitotic kinesin-5 motors can crosslink and slide adjacent spindle MTs, but it is unknown whether they or other motors contribute to establishing these MT polarity patterns. Here, we explored whether the Drosophila embryo kinesin-5 KLP61F, which plausibly crosslinks both parallel and antiparallel MTs, displays a preference for parallel or antiparallel MT orientation. In motility assays, KLP61F was observed to crosslink and slide adjacent MTs, as predicted. Remarkably, KLP61F displayed a 3-fold higher preference for crosslinking MTs in the antiparallel orientation. This polarity preference was observed in the presence of ADP or ATP plus AMPPNP, but not AMPPNP alone, which induces instantaneous rigor binding. Also, a purified motorless tetramer containing the C-terminal tail domains displayed an antiparallel orientation preference, confirming that motor activity is not required. The results suggest that, during morphogenesis of the Drosophila embryo mitotic spindle, KLP61F's crosslinking and sliding activities could facilitate the gradual accumulation of KLP61F within antiparallel interpolar MTs at the equator, where the motor could generate force to drive poleward flux and pole-pole separation.

FIGURE 1. Purified KLP61F can crosslink and slide adjacent MTs

(a) Characterization of purified recombinant full length (FL)-KLP61F, headless KLP61F (lacking motor domains), and KLP61F stalk used in these studies. The coomassie-blue-stained SDS-polyacrylamide gel shows the purity of recombinant proteins after gel-filtration (Superose 6 FPLC, GE Pharmacia). Lane 1 shows FL-KLP61F, Lane 2 shows headless KLP61F (stalk + tail), Lane 3 shows KLP61F stalk. (b) Frames from a time-lapse recording showing a relatively short rhodamine-labeled MT sliding sideways (down and left) along a surface-attached Cy5-labeled MT. After 120 s the sliding MT rotates and aligns with the immobilized MT. The two velocities now add, indicating anti-parallel orientation. Scale bar, 1 μm. See also Suppl. Movie. (c) Displacement of the hindmost interaction point of the rhodamine labeled MT along the immobilized MT axis in (b) is plotted versus time. A linear fits reveal two sliding velocities. (d) Histograms of velocities of all measured MTs in aligned and non-aligned configurations shown together with Gaussian fits.

FIGURE 2. KLP61F has a preference for crosslinking MTs into antiparallel orientations

(a) Pure full-length (FL)-KLP61F and motorless KLP61F, but not KLP61F stalk, can crosslink and bundle MTs in 1mM ATP. Fluorescence microscopy shows that headless KLP61F and FL-KLP61F have obvious bundling activity. KLP61F stalk without motor and tail domains, however, did not bundle MTs under the same conditions. Scale bar: 10 μm. (b) Image showing crosslinked pairs of polarity marked MTs. The minus end of the MT is indicated in red, the plus end in green. When two MTs are bundled, the fluorescence intensity doubles. Based on the relative fluorescence intensity and the location of the polarity marks, the orientation of crosslinking can be determined, as indicated. White scalebar: 2 μm (c) Histogram showing the orientation of MT crosslinking by FL-KLP61F in the presence of AMPPNP (n = 124), ATP/AMPPNP (n = 60), and ADP (n = 122), as well as by the motorless KLP61F (n = 49). The errors indicated were calculated from the propagation of the counting errors (square root of the number of counts in each category).