Swap and stop – kinetochores play error correction with microtubules

Correct chromosome segregation in mitosis relies on chromosome biorientation, in which sister kinetochores attach to microtubules from opposite spindle poles prior to segregation. To establish biorientation, aberrant kinetochore–microtubule interactions must be resolved through the error correction process. During error correction, kinetochore–microtubule interactions are exchanged (swapped) if aberrant, but the exchange must stop when biorientation is established. In this article, we discuss recent findings in budding yeast, which have revealed fundamental molecular mechanisms promoting this “swap and stop” process for error correction. Where relevant, we also compare the findings in budding yeast with mechanisms in higher eukaryotes. Evidence suggests that Aurora B kinase differentially regulates kinetochore attachments to the microtubule end and its lateral side and switches relative strength of the two kinetochore–microtubule attachment modes, which drives the exchange of kinetochore–microtubule interactions to resolve aberrant interactions. However, Aurora B kinase, recruited to centromeres and inner kinetochores, cannot reach its targets at kinetochore–microtubule interface when tension causes kinetochore stretching, which stops the kinetochore–microtubule exchange once biorientation is established

Microtubules originate asymmetrically at the somatic golgi and are guided via Kinesin2 to maintain polarity within neurons

Neurons contain polarised microtubule arrays essential for neuronal function. How microtubule nucleation and polarity are regulated within neurons remains unclear. We show that γ-tubulin localises asymmetrically to the somatic Golgi within Drosophila neurons. Microtubules originate from the Golgi with an initial growth preference towards the axon. Their growing plus ends also turn towards and into the axon, adding to the plus-end-out microtubule pool. Any plus ends that reach a dendrite, however, do not readily enter, maintaining minus-end-out polarity. Both turning towards the axon and exclusion from dendrites depend on Kinesin-2, a plus-end-associated motor that guides growing plus ends along adjacent microtubules. We propose that Kinesin-2 engages with a polarised microtubule network within the soma to guide growing microtubules towards the axon; while at dendrite entry sites engagement with microtubules of opposite polarity generates a backward stalling force that prevents entry into dendrites and thus maintains minus-end-out polarity within proximal dendrites

Mechanical and geometrical constraints control kinesin-based microtubule guidance

Proper organization of microtubule networks depends on microtubule-associated proteins and motors that use different spatial cues to guide microtubule growth [1–3]. For example, it has been proposed that the uniform minus-end-out microtubule organization in dendrites of Drosophila neurons is maintained by steering of polymerizing microtubules along the stable ones by kinesin-2 motors bound to growing microtubule plus ends [4]. To explore the mechanics of kinesin-guided microtubule growth, we reconstituted this process in vitro. In the presence of microtubule plus-end tracking EB proteins, a constitutively active kinesin linked to the EB-interacting motif SxIP effectively guided polymerizing microtubules along other microtubules both in cells and in vitro. Experiments combined with modeling revealed that at angles larger than 90, guidance efficiency is determined by the force needed for microtubule bending. At angles smaller than 90, guidance requires microtubule growth, and guidance efficiency depends on the ability of kinesins to maintain contact between the two microtubules despite the geometrical constraints imposed by microtubule length and growth rate. Our findings provide a conceptual framework for understanding microtubule guidance during the generation of different types of microtubule arrays

KAP, the accessory subunit of kinesin-2, binds the predicted coiled-coil stalk of the motor subunits

Kinesin-2 is an anterograde motor involved in intraflagellar transport and certain other intracellular transport processes. It consists of two different motor subunits and an accessory protein KAP (kinesin accessory protein). The motor subunits were shown to bind each other through the coiled-coil stalk domains, while KAP was proposed to bind the tail domains of the motor subunits. Although several genetic studies established that KAP plays an important role in kinesin-2 functions, its exact role remains unclear. Here, we report the results of a systematic analysis of the KAP binding sites by using recombinant Drosophila kinesin-2 subunits as well as the endogenous proteins. These show that at least one of the coiled-coil stalks is sufficient to bind the N-terminal region of DmKAP. The soluble complex involving the recombinant kinesin-2 fragments is reconstituted in vitro at high salt concentrations, suggesting that the interaction is primarily nonionic. Furthermore, independent distant homology modeling indicated that DmKAP may bind along the coiled-coil stalks through a combination of predominantly hydrophobic interactions and hydrogen bonds. These observations led us to propose that KAP would stabilize the motor subunit heterodimer and help assemble a greater kinesin-2 complex in vivo

End-binding proteins sensitize microtubules to the action of microtubule-targeting agents

Microtubule-targeting agents (MTAs) are widely used for treatment of cancer and other diseases, and a detailed understanding of the mechanism of their action is important for the development of improved microtubule-directed therapies. Although there is a large body of data on the interactions of different MTAs with purified tubulin and microtubules, much less is known about how the effects of MTAs are modulated by microtubule-associated proteins. Among the regulatory factors with a potential to have a strong impact on MTA activity are the microtubule plus end-tracking proteins, which control multiple aspects of microtubule dynamic instability. Here, we reconstituted microtubule dynamics in vitro to investigate the influence of end-binding proteins (EBs), the core components of the microtubule plus end-tracking protein machinery, on the effects that MTAs exert onmicrotubule plus-end growth. We found that EBs promote microtubule catastrophe induction in the presence of all MTAs tested. Analysis of microtubule growth times supported the view that catastrophes are microtubule age dependent. This analysis indicated that MTAs affect microtubule aging in multiple ways: destabilizing MTAs, such as colchicine and vinblastine, accelerate aging in an EB-dependent manner, whereas stabilizing MTAs, such as paclitaxel and peloruside A, induce not only catastrophes but also rescues and can reverse the aging process

Biochemical and Molecular Dynamic Simulation Analysis of a Weak Coiled Coil Association between Kinesin-II Stalks

<div><h3>Definition</h3><p>Kinesin-2 refers to the family of motor proteins represented by conserved, heterotrimeric kinesin-II and homodimeric Osm3/Kif17 class of motors.</p> <h3>Background</h3><p>Kinesin-II, a microtubule-based anterograde motor, is composed of three different conserved subunits, named KLP64D, KLP68D and DmKAP in <em>Drosophila</em>. Although previous reports indicated that coiled coil interaction between the middle segments of two dissimilar motor subunits established the heterodimer, the molecular basis of the association is still unknown.</p> <h3>Methodology/Principal Findings</h3><p>Here, we present a detailed heterodimeric association model of the KLP64D/68D stalk supported by extensive experimental analysis and molecular dynamic simulations. We find that KLP64D stalk is unstable, but forms a weak coiled coil heteroduplex with the KLP68D stalk when coexpressed in bacteria. Local instabilities, relative affinities between the C-terminal stalk segments, and dynamic long-range interactions along the stalks specify the heterodimerization. Thermal unfolding studies and independent simulations further suggest that interactions between the C-terminal stalk fragments are comparatively stable, whereas the N-terminal stalk reversibly unfolds at ambient temperature.</p> <h3>Conclusions/Significance</h3><p>Results obtained in this study suggest that coiled coil interaction between the C-terminal stalks of kinesin-II motor subunits is held together through a few hydrophobic and charged interactions. The N-terminal stalk segments are flexible and could uncoil reversibly during a motor walk. This supports the requirement for a flexible coiled coil association between the motor subunits, and its role in motor function needs to be elucidated.</p> </div

KLP64D and KLP68D stalks heterodimerize through coiled-coil interaction.

<p>A) Expression and affinity purification of the kinesin-II stalk fragments. <i>i)</i> Coomasssie stained gel shows affinity purified products of the individual and combinatorial expression. Arrowheads indicate the KLP64DS bands and the arrow indicates the His-KLP68DS. <i>ii)</i> mAb-KLP64D staining of the western blot of a gel similar to the one presented in (<i>i</i>) show that recombinant His-KLP64DS (lane 3) migrates in two distinct forms in SDS-PAGE. <i>iii)</i> Anti-KLP68D staining of a similar blot revealed the corresponding KLP68DS band (arrows). B) Gel filtration analysis of the affinity purified His-KLP68D/64D-S heterodimer using HiLoad 16/60 Superdex™ 75 pg column: <i>i</i>) fraction-wise absorption profiles of the affinity purified protein (black line), the pooled P2 (red), and the P3 (green) fractions. Coomassie staining of the SDS-PAGE gels of representative fractions from the P1, P2 and P3 sets. Labeled dashed lines mark the position of respective bands on a coomassie stained gels. <i>ii</i>) Gel filtration analysis of His-KLP64D/68D-S indicates that the heterodimer elutes in the P2 range. C) Mean residue molar ellipticity ([Θ]) values of the purified His-KLP64D/68D-S (black curve) and His-KLP68D/64D-S (red curve) heterodimers measured in the far-UV wavelengths by using a circular dichroism (CD) spectrometer at 5°C (278K). D) Plot of [Θ]₂₂₂ values of His-KLP64D/68D-S and H-KLP68D/64D-S at different temperatures. E) Stability curves of His-KLP64D/68D-S (black squares) and His-KLP68D/64D-S (red squares), each point in the stability curve represents the free energy of unfolding (ΔG) at corresponding temperature. The solid line represents the non-linear curve fits to Gibbs-Helmholtz equation (Eq. 6).</p