Article KIF4 Regulates Midzone Length during Cytokinesis (2011)

Graduate Program in Biological and Biomedical SciencesHarvard Medical School, Boston, MA 02115, USASummaryBackground: Midzones, also called central spindles, are anarray of antiparallel microtubules that form during cytokinesisbetween the separated chromosomes. Midzones can beconsidered to be platforms that recruit speciﬁc proteins andorchestrate cytokinetic events, such as sister nuclei beingkept apart, furrow ingression, and abscission. Despite thisimportant role, many aspects of midzone biology remain un-known, including the dynamic organization of midzone micro-tubules. Investigating midzone microtubule dynamics hasbeen difﬁcult in part because their plus ends are interdigitatedandburiedinadensematrix,makingthemdifﬁculttoobserve.Result: We employed monopolar cytokinesis to reveal thatmidzone plus ends appear to be nondynamic. We identiﬁedthe chromokinesin KIF4 as a negative regulator of midzoneplus-end dynamics whose activity controls midzone lengthbutnotstability.KIF4isrequiredtoterminatemidzoneelonga-tion in late anaphase. In the absence of KIF4, midzones elon-gate abnormally, and their overlap regions are unfocused.Electron-dense material and midbodies are both absent fromthe elongated midzones, and actin ﬁlaments from the furrowcortex are not disassembled after ingression.Conclusion: KIF4-mediated midzone length regulationappears to occur by terminating midzone elongation ata speciﬁc time during cytokinesis, making midzones andmitotic spindles differ in their dynamics and length-regulatingmechanisms.IntroductionMidzones, also called central spindles, are a set of microtu-bules speciﬁcally formed between segregated chromosomesaftercytokinesisonset[1].Servingasaplatformtoorchestratecrucial cytokinetic events, such as keeping segregated sisterchromatids away from the equator by appropriate distanceandsignalingthecortextocorrectlypositionacleavagefurrowon the division plane, midzones have thus drawn signiﬁcantinterest for their regulations to those cytokinetic events[2, 3]. However, the dynamic behavior of midzone microtu-bules per se and how their length is regulated are poorlyunderstood. Midzones are antiparallel arrays with plus endsoriented toward the center [4], like the metaphase spindlesthey follow during cell division. It has thus been natural toassumethatthesetwoarespatiallyorganizedandlengthregu-lated according to similar principles [5–8]. In this study, weinvestigated midzone dynamics and length regulation andfound that they are in fact very different from those of mitoticspindles.In normal midzones, the overlapping, antiparallel plus endsare buried in a dense matrix that excludes antibodies againsttubulin and stains darkly by thin section electron microcopy(EM) [9, 10], making it difﬁcult to observe their dynamics. Mo-nopolar cytokinesis, in contrast, is a good complementarysystem for studying midzone microtubule plus ends, becauseplus ends of monopolar midzones are exposed in the cyto-plasm [11]. Monopolar midzones resemble normal bipolarmidzones in protein composition, competency to initiatefurrows, and presence of electron-dense material coatingmicrotubules near plus ends [10]. HeLa cells are arrested inmonopolar mitosis with the Kinesin-5 inhibitor S-trityl-L-cysteine (STLC) [12] and are forced into cytokinesis with theCdk1 inhibitor RO-3306 [13] in a synchronized fashion. Afteran early Aurora B-dependent polarization step that breakssymmetry, midzone microtubules assemble and polarizetoward a region of the plasma membrane that recruits furrowproteins. Midzone plus ends ﬁrst terminate in or near thecortex, but as the cortex recruits furrow proteins, changesshape, and protrudes, the plus ends of midzone bundles areleft isolated in the cytoplasm several microns from the cortex(Figure 1)[11]. In this study, we used monopolar cytokinesisas a tool to identify proteins that are involved in midzonedynamics and length regulations, and we then returned tonormal bipolar cytokinesis, probing their functions in normalcell division.ResultsKIF4 Negatively Regulates Microtubule Plus-EndDynamics in Monopolar CytokinesisWe overcame the obstacle of observing midzone plus-enddynamics by employing monopolar cytokinesis, in which theplus ends of monopolar midzones are exposed in the cyto-plasm [11]. To identify proteins that regulate monopolarmidzone length, we depleted a list of cytokinetic proteins thatare either midzone associated or cytokinesis regulators (Fig-ure1B).DepletionofPRC1,MKLP1,RacGAP1,orEct2blockedthe early polarization step; neither midzones nor furrowsformed. Depletion of other proteins, including CLASPs, whichregulatemidzonedynamicsinyeast[14],hadnoobviouseffect.Uniquely, depletion of the chromokinesin KIF4 allowed polari-zation and midzone assembly, but midzones no longer termi-nated coherently in the cytoplasm. As shown in thetime-lapsemovies of monopolar HeLa cells expressing GFP-b-tubulin(Figure 1C; see also Movie S1 and Movie S2 available online)and GFP-EB3, a marker for growing microtubule plus ends(Figure S1, Movie S3, and Movie S4), midzones in control cellsterminated in cytoplasm with plus ends that appeared to benondynamic. However, KIF4-depleted midzone plus endscontinued to grow and entered the protruding furrow region.A furrow could still be assembled, but it was typically lessrobust. These data conﬁrm that PRC1, MKLP1, RacGAP1,and Ect2 are essential for midzone assembly and the earlypolarization step. KIF4, in contrast, is required later, to inhibit