Clustering of centralspindlin is essential for its accumulation to the central spindle and the midbody

Abstract

Cytokinesis in animal cells requires the central spindle and midbody, which contain prominent microtubule bundles. Centralspindlin, a heterotetrameric complex consisting of kinesin-6 and RhoGAP (Rho-family GTPase-activating protein) subunits, is essential for the formation of these structures. Centralspindlin becomes precisely localized to the central spindle, where it promotes the equatorial recruitment of important cytokinetic regulators. These include ECT2, the activator of the small GTPase RhoA, which controls cleavage furrow formation and ingression. Centralspindlin's own RhoGAP domain also contributes to furrow ingression. Finally, centralspindlin facilitates recruitment of the chromosome passenger complex and factors that control abscission. Despite the importance of localized accumulation of centralspindlin, the mechanism by which this motor protein complex suddenly concentrates to the center of interpolar microtubule bundles during anaphase is unclear. Here, we show that centralspindlin travels along central spindle microtubules as higher-order clusters. Clustering of centralspindlin is critical for microtubule bundling and motility along microtubules in vitro and for midbody formation in vivo. These data support a positive feedback loop of centralspindlin clustering and microtubule organization that may underlie its distinctive localization during cytokinesis.

Figure 1

Centralspindlin accumulates to the centre of the central spindle through clustering. (A-E) HeLa cells stably expressing functional HsCYK-4-GFP were observed by live imaging. (A) Schematic of the experimental setup used to observe the movement of HsCYK-4-GFP along the central spindle. (B) Image of a cell in early anaphase. The area in the left panel indicated by the yellow dotted square is shown magnified in the right panel. The plasma membrane is indicated with pink dotted lines. The position of metaphase plate (Figure S1) is indicated by a red dotted line. The white dotted square was used for background correction for fluorescence intensity in D. (C) Kymographs along microtubule tracks indicated in B. (D) Traces of the fluorescent intensity of the particles indicated in C. (E) HsCYK-4-GFP was photobleached in the areas indicated by dotted blue circles in cells in metaphase and anaphase. The ratio of the fluorescence signals in the bleached (yellow square) to the unbleached areas (magenta) was plotted. The time (sec) after photobleaching is shown. (F) Cell lysates were prepared from HeLa cells arrested at prometaphase (0 min) or 90 min after release from nocodazole. MKLP1 in total cell lysates (T) and in supernatants (S) following centrifugation was analysed by Western blotting. (G) Centralspindlin purified from HeLa cells in the presence of 250 mM NaCl was diluted into buffers containing the indicated concentration of salt. Proteins in total inputs (T) and supernatants (S) were detected by Coomassie staining after SDS-PAGE.

Figure 2

In vitro characterization of the clustering of C. elegans centralspindlin. (A-D) Bacterially expressed ZEN-4 proteins were purified in the presence of 250 mM salt and diluted to (A, B) 150 mM or (C, D) 83 mM. Total input (T) and supernatant (S) after centrifugation were analysed by SDS-PAGE. For C, after the first supernatant (S1) was removed, the precipitate (not shown) was resuspended into a high salt buffer and centrifuged to obtain the second supernatant (S2). (E) CYK-4 binding was assayed by reciprocal pull-down from E. coli lysates expressing both ZEN-4 fragments tagged with chitin-binding domain (CBD) and CYK-4 1-120 tagged with glutathione S-transferase (GST). (F) Schematic model of ZEN-4 and deletion constructs used in this study with a summary of their in vitro properties. ZEN-4 is comprised of a motor domain, a neck region containing several helix-breaking proline residues, a 100 amino acid region that forms a parallel coiled coil [20] and a tail domain. A small region (‘clustering element’) is required for clustering. (G) Microtubule-bundling assays with ZEN-4 constructs competent (Z601) or incompetent (Z585) for clustering complexed with CYK-4 1-232 (C232).

Figure 3

Clustering is essential for efficient transport of ZEN-4. (A) Still images from time-lapse observation of the interaction of Z601GFP or Z585GFP at 9 μg/ml with microtubules immobilized on a glass surface, showing plus-end accumulation of Z601GFP. (B) Frames from time-lapse observations showing a particle of Z601GFP (135 ng/ml, green) moving along a microtubule (red). White dot highlights a particle moving in a continuous manner. (C, F, I, L) Kymographs depicting the movement of indicated motor constructs along microtubules. Avidin induces clustering of Z585GFP that has a C-terminal biotin-tag (I). A dimeric Kinesin-1 construct (K432GFP) was also observed for comparison (L-N). The arrowhead in C and I indicates accumulation of particles at the plus-end of microtubule. (D, G, J, M) Traces of photobleaching behaviour of the particles in the corresponding kymographs. (E, H, K, N) Association time plotted against the initial fluorescence intensity of each particle. (O) Summary of association time, velocity and run length. (P) Simulation of distributions of fluorescence intensities of particles containing 2, 4, 8, 12, 16 GFP moieties, assuming 50% of GFPs are fluorescently active.

Figure 4

Clustering of ZEN-4 is essential for cytokinesis. (A) Genetic analysis of the functionality of GFP-tagged wild-type (wt) and self-assembly incompetent (Δ586-603) zen-4 transgenes. The mutant defective in clustering could barely rescue zen-4 null animals to viability. (B) Time-lapse observation of the first cell division of the embryos from zen-4 null hermaphrodite mothers rescued by wt or mutant transgenes. Wild-type ZEN-4 localizes to the central spindle and strongly accumulates to the midbodies (arrows). In contrast, the self-assembly incompetent mutant transiently associates with the central spindle and fails to accumulate to the midbody. The mutant embryos exhibited partial cleavage furrow ingression followed by regression (arrowheads), as seen for depletion of ZEN-4 by RNAi and is a consequence of centralspindlin-independent furrowing. Elapsed time is indicated in min.

Figure 5

(A) Schematic summarizing the effect of clustering of centralspindlin on its motility along microtubules. (B) Positive feedback loop model for the interaction between centralspindlin and microtubules. Red lines and green dots represent microtubules and the centralspindlin heterotetramers, respectively. (C) Model explaining how centralspindlin accumulates and bundles microtubules exclusively in the equatorial region of a normally dividing cell.