Chromokinesins

During the cell cycle it is critical that the duplicated DNA segregates into two genetically identical daughter cells. This takes place during a short window of time called ‘mitosis’ and is essential for organism survival. An even distribution of the genome during mitosis is mediated by mitotic spindle microtubules, assisted, among others, by motor proteins of the kinesin superfamily. Chromokinesins are members of the kinesin superfamily that harbour a specific DNA-binding domain and were named after the identification and cloning of the first vertebrate Chromokinesin from chicken embryos (hence the gene name “chk”) by Wang and Adler in 1995. In mammals, the best characterized Chromokinesins are Kif4a and Kid, which belong to the kinesin-4 and kinesin-10 family, respectively. More recently, Kif15/Hklp2, a member of the kinesin-12 family, was also found to localize to the chromosome arms during mitosis, but whether it represents a bona fide Chromokinesin remains debatable. Functional analysis of Chromokinesins of the kinesin-4 and kinesin-10 families in several model systems revealed their involvement in chromosome arm orientation and oscillations, consistent with their originally proposed role in the generation of Polar Ejection Forces (PEFs) that assist chromosome congression to the spindle equator. Noteworthy, all Chromokinesins have a chromosome-independent localization on spindle microtubules, and recent works have significantly extended the original portfolio of mitotic processes in which Chromokinesins play additional roles, from error correction and DNA compaction, to the regulation of spindle microtubule dynamics.

The Chromokinesin family and structure

The kinesin superfamily has been implicated in the transport of organelles, proteins and mRNAs to specific cellular destinations, in an ATP and microtubule-dependent manner. Chromokinesins (Figure 1A) are distinct from conventional kinesins due to their ability to associate with chromosomes during mitosis. Their localization on chromatin was originally reported for chk (a kinesin-4 family member) in chicken, and respective orthologues in Xenopus (Xklp1), humans (Kif4a and Kif4b) and C. elegans (Klp19). The putative kinesin-10 ‘No distributive disjunction’ (Nod) in Drosophila female meiosis also associates with DNA, which was subsequently confirmed in other kinesin-10 family members, including Kid in mammals and XKid in Xenopus. More recently, the kinesin-12 Kif15/Hklp2 was also shown to localize to the chromosome arms in human cells, thereby qualifying as a Chromokinesin.

Figure 1. The Chromokinesin Family.

(A) Phylogenetic tree of all Chromokinesins expressed in humans (Hs), mouse (Mm), Chicken (Gg), Xenopus (Xl), D. melanogaster (Dm) and C. elegans (Ce). (B) Schematic representation of the three human Chromokinesins. All Chromokinesins share a conserved N-terminal motor domain (blue), followed by a stalk consisting of coiled-coil regions of different lengths (white). Nuclear localization signals (NLS) in Kif4a and Kid are indicated in dashed lines in the stalk. The chromosome binding domains are represented in green boxes: a zip/basic/leucine zip (ZBZ) domain and a Cystein-Rich (C-R) motif for kinesin-4; an Helix-hairpin-Helix (HhH) motif for kinesin-10 and a region that interacts with the protein Ki67 for kinesin-12. aa, amino acids.

All Chromokinesins share the same structural features of most kinesins: an N-terminal motor domain, an α-helical coiled-coil stalk domain and a C-terminal tail region, which contains a chromatin-interacting domain (Figure 1B). The presence of the motor domain is consistent with the plus-end-directed motility demonstrated for several Chromokinesins, such as Kid, XKid, Kif4a and Xklp1, but they appear to be weakly processive motors under load. Kinesin-4 family members show an extended coiled-coil required for dimerization that prolongs to a leucine zipper motif within the stalk domain, whereas kinesin-10 proteins have only a short coiled-coil, which was thought to compromise dimerization, at least under low concentrations in vitro, but recent evidence from the Nod protein suggest that they might also work as dimers. Kinesin-12 members have an extended coiled-coil region that prolongs into a leucine zipper at the C-terminal, which is essential for dimerization. Specific sequence motifs and protein-protein interactions account for the targeting of the different Chromokinesins to the chromosomes. At the C-terminal, Kinesin-4 family members have a ZBZ (zip/basic/leucine zip) domain and Cysteine-Rich (C-R) motifs critical for chromatin binding. In the case of Kinesin-10 members, the interaction with chromatin is mediated by DNA-binding helix-hairpin-helix (HhH) motifs. Finally, the kinesin-12 Kif15 is a highly processive motor and was shown to target to chromosomes through the interaction with Ki67, a peri-chromosomal layer protein that decorates the chromosome arms during mitosis (Figure 1B).

Spatiotemporal regulation of Chromokinesins

In interphase, kinesin-10 and kinesin-4 are sequestered into the nucleus and the interaction with microtubules is cell-cycle regulated. After nuclear envelope breakdown, phosphorylation by Cyclin B1-Cdk1 on Thr463 close to the second microtubule-binding site on the kinesin-10 stalk domain, reduces Kid’s affinity for microtubules, allowing its localization on chromosomes. After metaphase, degradation of XKid was shown to be necessary for the migration of chromosomes to the poles in Xenopus egg extracts. In somatic cells, while Kid and XKid disappear from the chromosome arms at the metaphase-anaphase transition, a significant pool remains associated with spindle midzone microtubules. At this stage, non-phosphorylated Thr463 allows Kid to bind more strongly to microtubules.

Kinesin-4 re-localization from chromatin to spindle midzone microtubules during anaphase depends on the anti-parallel microtubule-bundling factor PRC1 and the kinesin-6 Mklp2, and is required for central spindle formation. PRC1 activity is largely suppressed until anaphase onset by Cdk1 phosphorylation, which prevents premature central spindle formation. At anaphase onset, PRC1 loses its Cdk1-dependent inhibitory phosphorylation and increases the microtubule bundling capacity, promoting the recruitment of Kif4a that suppresses microtubule dynamic instability. This is restricted to a specific subset of microtubules by an MKlp2-dependent pool of Aurora B at the central spindle, thereby contributing to central spindle size control in anaphase.

Kinesin-12 localizes in the cytoplasm during interphase without interacting with microtubules and it is activated after nuclear envelope breakdown in a TPX2-dependent manner. Human Kif15 assembles into stable tetramers that are highly processive, and can navigate microtubule networks by switching tracks while forming high load-bearing microtubule–microtubule crosslinks when bound to TPX2. The C-terminal domain of TPX2 contributes to the localization of Kif15 to spindle microtubules in cells and suppresses motor walking in vitro.

The roles of Chromokinesins during mitosis

PEFs and chromosome congression

Chromokinesins perform non-redundant functions during mitosis, despite the similarities in protein organization and chromosomal localization (Table 1). During congression to the metaphase plate, chromosomes experience pushing forces along the chromosome arms and away from spindle poles, hence the name ‘PEFs’. In vitro reconstitutions and in vivo measurements have predicted that PEFs range between 0.5–1 pN per microtubule and around 100 pN closer to the centre of the aster where microtubule density is higher. These studies indicated that PEFs could be generated either by individual polymerizing microtubules pushing on chromosome arms or by the action of single kinesin motors on chromosome arms. Chromokinesins have therefore emerged as excellent candidates for powering chromosome motility assisted by PEFs. Kinesin-10 was found to bind more strongly to microtubules, overruling kinesin-4 during cooperative movement associated with chromatin. Functional perturbation of both Chromokinesins in Drosophila and human cells suggested a combined role during chromosome congression, with kinesin-10 providing the major PEF required for arm orientation and oscillation and kinesin-4 mainly regulating microtubule dynamics. Together, these two Chromokinesins are sufficient to promote the random ejection of chromosomes away from the pole. However, their action is not critical for chromosome congression and alternative pathways relying on kinetochore-mediated motility (involving kinesin-7/CENP-E) dominate to guide chromosomes towards the spindle equator. In Xenopus egg extracts, XKid provides the force that pushes chromosome arms towards the spindle equator, playing an important role in the alignment of chromosomes on bipolar spindles assembled in vitro. The Chromokinesin Nod was thought for many years to be an “orphan kinesin” due to its apparent lack of motility. However, very recent studies have now shown that Nod likely works as a dimer with microtubule plus-end-directed motility, consistent with its originally proposed role as PEF generator. In addition, Nod was found to interact with the tip-tracking protein EB1 via a novel type of microtubule tip localization sequence (LxxPTPh motif). Thus, NOD generates PEFs by providing microtubule plus-end directed motility and by associating with EB1 at polymerizing microtubule plus-ends.

Table 1.

Summary of mitotic properties of the three Chromokinesins families. Abbreviations used: Ce, Caenorhabditis elegans; CPC, chromosomal passenger complex; C-R – Cystein-Rich; Dm, Drosophila melanogasterr; Gg, Gallus gallus; HhH – Helix/hairpin/Helix; HMG – High mobility group; Hs, Homo sapiens; Mm, Mus musculus; PRC1 –Protein Required for Cytokinesis 1; Xl, Xenopus laevis; ZBZ - zip/basic/leucine zip; N.A. – not available.

Family	Name	Species	Location	Chromosome targeting	Motility	Oligomeric state	Main mitotic function
Kinesin-4	hKif4a hKif4b	Hs	Xq13.1 5q33.1	ZBZ domains and C-R motifs	N.A.	Dimer	Spindle bipolarity; chromosome condensation and alignment at metaphase; microtubule dynamics.
	Xklp1	Xl	NM_001087550	C2H2 zinc finger domain at C-terminus	Yes	N.A.	Affects spindle microtubules density/polymerization; together with PRC1 controls anaphase midzone MT overlap.
	Kif4a	Mm	X C3; X 43.72 cM	-	Yes	N.A.	Spindle assembly, chromosome alignment and cytokinesis.
	Klp-3B	Dm	3A6-3A6; 1-0.0 cM	-	N.A.	N.A.	Drives spindle pole separation and facilitates chromosome movement.
	Klp-19	Ce	mum-3-unc-49	CPC during meiosis	N.A.	N.A.	Polar ejection forces, important for chromosome segregation.
	Chk	Gg	Chromosome 4	Leucine zipper DNA-binding domain	Yes	N.A.
Kinesin-10	KID/Kif22	Hs	16p11.2	HhH motifs at C-terminal	Yes	Monomer *	Polar ejection force; chromosome arms oscillation and orientation; microtubule bundling.
	Xkid	Xl	NM_203914	HhH motifs at C-terminal	Yes	N.A.	Chromosome alignment.
	Kid	Mm	7 F3; 7 69.29cM	-	Yes	N.A.
	Nod	Dm	10C7-10C8; 1-36 cM	HhH motifs and HMG repeats at C-terminal	Yes	Dimer **	Polar ejection force; stabilization of kinetochore-microtubule attachments.
Kinesin-12	Kif15/Hklp2	Hs	3p21.31	Ki67 interaction	Yes	Tetramer ***	Bipolar spindle assembly/spindle pole separation.

^*at low concentrations in solution;

^**processive only after artificial dimerization; monomer at low concentrations in solution;

^***at physiological ionic strength; dimer at high ionic strength.

The plus-end-directed motor Kif15 can “walk” along the microtubules reaching and binding to the chromosomes. However, interfering with Ki67, the protein that anchors Kif15 to the chromosomes, has no effect in chromosome alignment in HeLa cells and instead generates longer bipolar spindles. This suggests that Kif15, rather than participating in the generation of PEFs on chromosomes, restrains the separation of the spindle poles and regulates bipolar spindle length.

PEFs in kinetochore-microtubule attachments

The attenuation of PEFs after depletion of KLP-19 in nematodes causes kinetochore misorientation, anaphase chromatin bridges and missegregation of holocentric chromosomes. KLP-19-mediated PEFs were proposed to maintain constant tension on pole–kinetochore connections in order to allow proper chromosome orientation, thereby reducing the formation of merotelic attachments (when individual kinetochores erroneously attach to microtubules oriented to opposite spindle poles). Consistent with this hypothesis, depletion of Kif4a has been linked to aneuploidy in mouse cells and functional perturbation of both Kif4a and Kid was implicated in chromosome segregation fidelity in HeLa cells. Depletion of the Drosophila kinesin-4 orthologue Klp3a causes congression defects and lagging anaphase chromosomes. On the other hand, elevated PEFs contribute to the stabilization of erroneous kinetochore-microtubule attachments even in the presence of active Aurora B, and Chromokinesin-mediated PEFs are essential for the stabilization of initial end-on kinetochore-microtubule attachments on mono-oriented chromosomes. More recently, attenuation of PEFs in Indian muntjac gigantic chromosomes by RNAi against Kif4a led to a striking increase in the frequency of lagging chromosomes in anaphase (Figure 2), consistent with a role of PEFs in the modulation of kinetochore-microtubule attachments and chromosome segregation fidelity.

Figure 2. Mitotic roles of kinesin-10 and kinesin-4.

(A) Female Indian muntjac fibroblast depleted of Kid/kinesin-10 by RNAi. Arrows indicate chromosome armorientation problems suggesting a role in PEFs. (B) Female Indian muntjac fibroblast depleted of Kif4a/kinesin-4 by RNAi. Arrow indicate a lagging chromosome during anaphase suggesting a role in error-correction and chromosome segregation fidelity. Time is in h:min. Scale bars = 5 μm.

Chromokinesins on spindle microtubules

Chromokinesins typically localize on chromosome arms, but all the three family members can also associate with microtubules in a chromosome-independent manner. Kinesin-10 and kinesin-12 both co-localize with spindle microtubules and chromosomes until anaphase, whereas kinesin-4 is exclusively on chromosomes until metaphase and relocates to the spindle midzone during anaphase. The mechanism by which they can differently interact with chromatin and microtubules is specific for each Chromokinesin. The affinity of the monomeric motor Kid to microtubules is facilitated by a second ATP-independent microtubule-binding site in the stalk and the interaction with the spindle protein CHICA. Interestingly, even though Kid remains associated with chromosomes in CHICA-depleted cells, chromosomes do not correctly align, suggesting that Kid’s localization at the spindle microtubules is required for chromosome congression or there are other undescribed functions for CHICA. Kid also plays a role in spindle-pole focusing and acts together with NuMA in spindle formation, independently of Kid’s DNA-binding and motor activity. Finally, Kid has microtubule bundling activity, which might assist other motors, such as kinesin-5 or kinesin-12, to separate the spindle poles, while providing robustness to the spindle.

The Kinesin-12 Xklp2 is targeted to microtubule minus ends in Xenopus oocyte spindles assembled in vitro, in a process relying on TPX2. Kif15 silencing weakens spindle bipolarity, sensitizing human cells to kinesin-5 inhibitors, whereas its overexpression is sufficient to restore the assembly of a bipolar spindle in cells treated with kinesin-5 inhibitors. Thus, kinesin-12 proteins play a non-essential role in the establishment and maintenance of spindle bipolarity, in close cooperation with kinesin-5.

Kinesin-4 localizes at the spindle midzone during anaphase where, together with its binding partner PRC1, they play an important role in the organization and control of central spindle length. In vitro reconstitutions in Xenopus egg extracts uncovered a role for PRC1 in bundling antiparallel microtubules and the selective recruitment of Xklp1, which controls the size of overlapping microtubules by lowering tubulin turnover at the growing microtubule plus ends. These observations are in line with loss of function experiments in HeLa cells, where Kif4a was suggested to be involved in the organization of the spindle midzone and midbody, necessary for successful cytokinesis. In agreement, Klp3a in Drosophila, was shown to control mitotic spindle length and to be essential for cytokinesis.

Chromokinesins in DNA condensation and compaction

At the anaphase-telophase transition, neighbouring chromosomes gather and form a compact cluster that is then surrounded by the nuclear envelope to compartmentalize the genomic DNA. In mammalian cells, after anaphase onset, a significant amount of Kid localizes together with microtubules in the interstices between adjacent anaphase chromosomes. Although depletion of Kid causes a short pre-anaphase delay, kinetochore-microtubule attachments are ultimately established and the majority of cells eventually enter anaphase. Loss of function studies in mouse and HeLa cells showed that during anaphase and telophase Kid deficiency leads to an increased rate of lagging chromosomes and the formation of malformed nuclei and multinucleated cells. These observations suggest that Kid helps holding the individual chromosomes together during segregation in order to form a compact chromosome mass at telophase, thus ensuring proper nuclear envelope formation.

Human Kif4a interacts with chromatin associated proteins including condensin I and II complexes and its absence leads to chromosome hypercondensation. This Chromokinesin likely acts as a modulator of chromatin architecture and contributes to higher order organization of metaphase chromosomes. Further studies in DT40 chicken cells, showed that in mitosis, Kif4a, condensin and DNA topoisomerase IIα work together to shape mitotic chromosomes.

Conclusions and Outlook

Although Chromokinesins were originally proposed to be important for the generation of PEFs, these were proved not to be the dominant forces involved in chromosome congression during mitosis in mammals. Instead, Chromokinesins have emerged as important players involved in the stabilization of correct kinetochore-microtubule attachments required for faithful chromosome segregation, proper DNA condensation, chromosome structure and the assembly of a functional bipolar spindle. However, much remains to be uncovered and future work will be necessary to determine the individual roles played by the different Chromokinesins, both at the molecular and physiological levels.