Kinesins in Mitosis and Cytokinesis

1.2.1 Structure of Kinesins

Kinesins have a modular composition and consist of a head domain, also referred to as motor domain, connected to a tail and typically form dimers.^3-4 The head domain is built on a structurally highly conserved β-sheet backbone flanked by α-helices and contains the catalytically active ATPase site as well as the microtubule binding site.

4-5 The connection between head and tail is called neck linker, a short sequence that synchronizes the ATPase cycle of the dimers, and is present in nearly all kinesins. In contrast to the highly conserved head domain, the tail domain differs in length and sequence depending on the kinesin family (Figure 2).

Figure 2: Schematic representation of mitotic kinesins and their structures divided into their families. Adapted by permission from Nature Publishing Group.⁴

The tail is typically build up of α-helical coiled coils to achieve multimerization, interrupted by unstructured, natively unfolded sequences and bears recognition

3 sequences for co-proteins, regulatory kinases, cargo and in some cases another microtubule binding site.^{3-4, 6-7}

1.2.2 Mitosis and Cytokinesis

Mitosis is divided into different distinct phases: Prophase, prometaphase, metaphase, anaphase and telophase.

The main events during prophase are centrosome separation, which is a motor-dependent event, chromosome condensation and nuclear envelope break down (NEBD) (Figure 3, a). Kif11, also known as Eg5, is the only member of the human kinesin-5 family and essential for centrosome separation.^{4, 8-11} It consists of two heterodimers forming a tetramer with two heads at each end functioning as a microtubule cross-linker (Figure 2 and Figure 3, right panel 1).^{8, 12-13} Besides, Eg5 has also microtubule binding sites in the tail (Figure 2) and moves towards the plus end of microtubules, if attached to antiparallel microtubules.^{8, 14-16} Therefore, Eg5 enables the separation of the centrosomes by sliding antiparallel microtubules apart, which originate from different centrosomes (Figure 3, right panel 1).^{8, 14} The Eg5-driven microtubule sliding resists forces that tend to speed up sliding, like pulling forces of dynein on the centrosomal microtubules (Figure 3 right panel 2 and 3).¹⁷

The end of prophase in mammalian cells is reached with NEBD and followed by prometaphase, in which a bipolar mitotic spindle is formed and chromosome congression takes place (Figure 3, b). The mitotic spindle self-organizes through the dynamics of microtubules, which are influenced by motor- and microtubule binding-proteins in order to nucleate, capture, slide and reorient microtubules from both asters.¹⁸ Microtubules originating from opposite poles can either form antiparallel overlaps or capture kinetochores forming parallel bundles known as kinetochore microtubules (K-fibers). The major difference between these kinds of microtubules is their dynamic behavior: non kinetochore microtubules have a half-life of approximately 10 seconds compared to K-fibers with a half-life of several minutes.¹⁹ At the beginning of spindle assembly the activity of Eg5 as well as pulling from cortical myosin and pushing forces from kinetochores are required, and the continued complete separation of the centrosomes take place (Figure 3, right panel 3-5).^20-22 It was found that Kif15 (KLP2) overexpression compensates the loss of Eg5, suggesting

4

that both kinesins are able to slide away overlapping antiparallel microtubules (Figure 3, right panel 6).^23-24 Recent studies revealed that Kif15 preferably associates with parallel microtubules in K-fibers counteracting the forces generated by Eg5.^{23, 25} After assembly of a bipolar spindle its maintenance requires outward sliding forces generated by Eg5 and/or Kif15 and inward forces generated by KifC1, also known as HSET, a member of kinesin-14 family (Figure 3, right panel 4-6).^{24, 26-27} As shown in Figure 2 kinesin-14 family members have their motor domain at the C terminus and are microtubule minus end directed motors (Figure 3, right panel 5). The HSET analogs in Drosophila melanogaster (Ncd) and Schizosaccharomyces pombe (Klp2) are able to stabilize parallel and slide apart antiparallel microtubules, in the opposite direction compared to Eg5.^28-30 These findings highlight the antagonistic activity of Eg5 and HSET during bipolar spindle formation.^{26, 31-33} In parallel to bipolar spindle formation the chromosomes have to be attached to microtubules and aligned in the metaphase plate.³⁴ Multiple kinesins like CENP-E (Kif10), Kif18A, Kif2B and C (MCAK) are necessary for this process (Figure 3, right panel 7 and 8). CENP-E, for example, is a plus end-directed motor, which is able to transport kinetochores, and therefore, the whole chromosome, to the plus ends of microtubules (Figure 3, right panel 7).^35-39 When sister kinetochores are attached to microtubules originating from opposite poles they are able to oscillate between the poles by utilizing microtubule dynamics.

This is achieved by the regulation of microtubules growth and shrinkage and switching between them by kinetochores with the help of kinesins, like Kif18A.

Kif18A, member of the kinesin-8 family, is a plus end-directed motor involved in modulating microtubule plus end dynamics and is localized at kinetochores (Figure 2, Figure 3 right panel 8).^40-49 It is under debate, if Kif18Aposseses a microtubule depolymerization activity. One model suggests that newly arriving Kif18A molecules “bounce” into already plus-tip localized Kif18A ,which than falls off together with one tubulin dimer. The ability to depolymerize microtubules is consistent with the observed higher oscillation amplitude of kinetochores following Kif18A depletion and the resulting severe chromosome congression defect.42, 46, 50-51

The processivity of Kif18A is increased by a C-terminal microtubule binding site, which is also required for the mitotic function of the kinesin.^43-45 Kif2A, Kif2B and MCAK (Kif2C), like Kif18A, are important regulators of kinetochore oscillation

5 (Figure 3, right panel 8). These kinesins are located at both ends of microtubules and act as microtubule depolymerases (Figure 2, Figure 3, right panel 8 and 10).

Figure 3: The first row a-d illustrate the single steps from mitosis and their characteristics are mentioned. The second row 1-12 shows kinesins and dynein and their involvement in spindle assembly and chromosome interactions.

Reprinted by permission of Nature Publishing Group^.4

6

At the minus end of K-fibers, they generate a pulling force on kinetochores whereas at the plus end of microtubules, they seem to be involved in correcting erroneous kinetochore attachments and regulating the speed of kinetochore motility.^{50, 52-55} When the chromosomes are properly aligned metaphase is reached, which is followed by anaphase (Figure 3 c and d). In anaphase, the two sister chromatids are separated and pulled to opposite poles. The motor requirements in human cells for anaphase are still unknown.⁴ In telophase in mammalian cells, the chromosomes decondens and new nuclei are formed.

Afterwards, cytokinesis as the final physical division takes place. A main component of cytokinesis is the contractile ring, consisting of actin and myosin.^56-58 It assembles through actin filament polymerization by GTPase RhoA and myosin motor activity.^59-62 Another key component is the central spindle, which forms during anaphase progression. It consists of bundled antiparallel microtubules and serves as a signaling platform for the positioning of the cleavage furrow.4, 56, 63-64 For the assembly of the central spindle activity of different microtubule-associated proteins (MAPs) like protein regulator of cytokinesis (PRC1), the centralspindlin complex and the chromosomal passenger complex (CPC) are required. Additionally, members of the kinesin-6 family like mitotic kinesin-like protein 2 (Mklp2, Kif20A) and M-phase phosphoprotein 1 (MPP1, Kif20B) contribute to central spindle assembly (Figure 3, right panel 12). PRC1 is localized at the central spindle and contains a conserved central domain, which induces microtubule bundling.^65-66 Further, the N-terminus of PRC1 comprises an oligomerization and a Kif4A binding domain that enhances the localization to the central region.^{65, 67} Despite its interaction with PRC1, Kif4A has a key role in the regulation of microtubule dynamics and therefore controlling the size of the central spindle (Figure 3, right panel 12).^68-69 A dimer of Mklp1 (Kif23) together with a dimer of Cyk4 - a Rho family GTPase-activating protein (GAP) - forms the tetrameric centralspindlin complex and localizes to the center of the central spindle.^70-71 It supports microtubule bundling, RhoA regulation and recruits regulators of abscission.^{60, 72} The third important multi protein complex for central spindle assembly, the CPC, consists of Aurora B, survivin, borealin and inner centromere protein (INCENP) and is not only active at the central spindle during

7 anaphase, but throughout mitosis. Its activity is not only constricted by activation and localization of Aurora B and, related to that phosphorylation of central spindle components, it is also suggested to be involved in microtubule bundling.^73-76 The accumulation of Aurora B and polo-like kinase 1 (PLK1) is achieved by Mklp2.^77-78 MPP1 as well as Mklp2 are essential in the late steps of cytokinesis.^79-80

In summary, kinesins have many talents and functions ranging from their importance for the correct distribution of the genetic material during mitosis to the precise abscission of the daughter cells.