Dynamic partitioning of mitotic kinesin-5 cross-linkers between microtubule-bound and freely diffusing states

Abstract

The dynamic behavior of homotetrameric kinesin-5 during mitosis is poorly understood. Kinesin-5 may function only by binding, cross-linking, and sliding adjacent spindle microtubules (MTs), or, alternatively, it may bind to a stable "spindle matrix" to generate mitotic movements. We created transgenic Drosophila melanogaster expressing fluorescent kinesin-5, KLP61F-GFP, in a klp61f mutant background, where it rescues mitosis and viability. KLP61F-GFP localizes to interpolar MT bundles, half spindles, and asters, and is enriched around spindle poles. In fluorescence recovery after photobleaching experiments, KLP61F-GFP displays dynamic mobility similar to tubulin, which is inconsistent with a substantial static pool of kinesin-5. The data conform to a reaction-diffusion model in which most KLP61F is bound to spindle MTs, with the remainder diffusing freely. KLP61F appears to transiently bind MTs, moving short distances along them before detaching. Thus, kinesin-5 motors can function by cross-linking and sliding adjacent spindle MTs without the need for a static spindle matrix.

Figure 1.

Localization and function of KLP61F-GFP in D. melanogaster embryo mitosis. (A) Micrographs from a time-lapse video of a representative spindle showing KLP61F-GFP (left), rhodamine-tubulin (center), and double-label fluorescence (right) at various stages of mitosis. The plots (far right) are line scans extending pole to pole along an ipMT (10 pixels wide; ∼0.129 μm/pixel) for KLP61F (green) and tubulin (red). The y axis shows normalized fluorescence intensity. Bar, 5 μm. (B) Spindle pole dynamics in wild-type embryos, GFP-KLP61F rescued mutant embryos, and anti-KLP61F microinjected wild-type embryos showing how bipolar spindles collapse into monoasters after the loss of KLP61F function. Pole–pole separation dynamics are very similar in wild-type and rescued mutant embryos.

Figure 2.

Kymography and FSM of KLP61F-GFP dynamics. (A) Kymographs of tubulin and KLP61F distribution from mid-metaphase (0 s) until telophase. In anaphase B, KLP61F moves toward the spindle equator before tubulin. Kymographs show the mean total intensity per pixel along the pole (P)–pole axis. Red and dark blue indicate the highest and lowest intensity, respectively. (B) Plot of normalized integrated fluorescence intensity of KLP61F and tubulin within a 2-μm-wide region along the pole–pole axis of the same spindle near the pole and equator during the preanaphase B–to–anaphase B transition (0 s = late metaphase). Arrows in A and B indicate anaphase B onset, i.e., when chromatids have separated and the spindle elongates at a characteristic linear rate (from 13 to 17 μm). (C, left) A frame from an FSM video showing KLP61F-GFP speckles on spindles. Kymographs from same video (center) display many speckles as “dots” rather than lines, which indicates transient binding of KLP61F to spindle MTs. (right) Tracings (overlaid on kymograph) showing a subset of speckles that appear as lines due to KLP61F motility (see text).

Figure 3.

FRAP analysis of KLP61F-GFP in D. melanogaster embryo spindles. KLP61F turns over rapidly. Plots show corrected (see Materials and methods) normalized fluorescence recovery of a 2-μm-wide region at the equator and pole during preanaphase B and anaphase B. (insets) Representative plots for individual experiments. Each curve was fitted with a single exponential (black line). Error bars represent standard deviation (see Table I).

Figure 4.

Localization of wild-type and bimC-box mutant KLP61F. Distribution of KLP61FT933A-GFP and KLP61FT933D-GFP point mutants compared with wild-type KLP61F-GFP during metaphase (A) and anaphase B (B) in embryos injected with rhodamine-tubulin to mark MTs. The mutants barely associate with spindle MTs. (C) Localization of wild-type KLP61F-GFP to spindles is MT dependent. (top) Prometaphase KLP61F-GFP embryos containing rhodamine tubulin were imaged, transferred to a microinjection microscope, and injected with 25 mg/ml colchicine. (bottom) 3D projections 3 min later, after transfer back to the spinning disc microscope, show that spindle MTs were depolymerized, which results in a complete loss of KLP61F-GFP. Bars, 5 μm.