Benjamin H. Kwok

Microtubule cross-linking triggers the directional motility of kinesin-5

Although assembly of the mitotic spindle is known to be a precisely controlled process, regulation of the key motor proteins involved remains poorly understood. In eukaryotes, homotetrameric kinesin-5 motors are required for bipolar spindle formation. Eg5, the vertebrate kinesin-5, has two modes of motion: an adenosine triphosphate (ATP)–dependent directional mode and a diffusive mode that does not require ATP hydrolysis. We use single-molecule experiments to examine how the switching between these modes is controlled. We find that Eg5 diffuses along individual microtubules without detectable directional bias at close to physiological ionic strength. Eg5s motility becomes directional when bound between two microtubules. Such activation through binding cargo, which, for Eg5, is a second microtubule, is analogous to known mechanisms for other kinesins. In the spindle, this might allow Eg5 to diffuse on single microtubules without hydrolyzing ATP until the motor is activated by binding to another microtubule. This mechanism would increase energy and filament cross-linking efficiency.

The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex

The direct interaction between the ERM protein Moesin and microtubules is required for spindle organization in metaphase and cell shape transformation after anaphase onset.

Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis

Aurora B phosphorylation of the Polo kinase activation loop disrupts its binding to Map205 and central spindle microtubules, allowing it to be recruited to the site of cytokinesis.

Adaptive Resistance to an Inhibitor of Chromosomal Instability in Human Cancer Cells

Karyotype diversity is a hallmark of solid tumors that contributes to intratumor heterogeneity. This diversity is generated by persistent chromosome mis-segregation associated with chromosomal instability (CIN). CIN correlates with tumor relapse and is thought to promote drug resistance by creating a vast genomic landscape through which karyotypically unique clones survive lethal drug selection. We explore this proposition using a small molecule (UMK57) that suppresses chromosome mis-segregation in CIN cancer cells by potentiating the activity of the kinesin-13 protein MCAK. Sublethal doses of UMK57 destabilize kinetochore-microtubule (k-MT) attachments during mitosis to increase chromosome segregation fidelity. Surprisingly, chromosome mis-segregation rebounds in UMK57-treated cancer cells within a few days. This rapid relapse is driven by alterations in the Aurora B signaling pathway that hyper-stabilize k-MT attachments and is reversible following UMK57 removal. Thus, cancer cells display adaptive resistance to therapies targeting CIN through rapid and reversible changes to mitotic signaling networks.

Structural maintenance of chromosome (SMC) proteins link microtubule stability to genome integrity.

Background: SMC proteins organize chromosome architecture, whereas microtubules form the structural framework for chromosome movement in mitosis. Results: SMC proteins bind to and stabilize microtubules to promote proper mitotic spindle formation. Conclusion: SMC proteins can provide a functional link between microtubules and DNA to ensure faithful chromosome segregation. Significance: SMC-microtubule interactions are essential to establish a robust system for maintaining genome integrity. Structural maintenance of chromosome (SMC) proteins are key organizers of chromosome architecture and are essential for genome integrity. They act by binding to chromatin and connecting distinct parts of chromosomes together. Interestingly, their potential role in providing connections between chromatin and the mitotic spindle has not been explored. Here, we show that yeast SMC proteins bind directly to microtubules and can provide a functional link between microtubules and DNA. We mapped the microtubule-binding region of Smc5 and generated a mutant with impaired microtubule binding activity. This mutant is viable in yeast but exhibited a cold-specific conditional lethality associated with mitotic arrest, aberrant spindle structures, and chromosome segregation defects. In an in vitro reconstitution assay, this Smc5 mutant also showed a compromised ability to protect microtubules from cold-induced depolymerization. Collectively, these findings demonstrate that SMC proteins can bind to and stabilize microtubules and that SMC-microtubule interactions are essential to establish a robust system to maintain genome integrity.

DHTP is an allosteric inhibitor of the kinesin-13 family of microtubule depolymerases.

The kinesin‐13 family of microtubule depolymerases is a major regulator of microtubule dynamics. RNA interference‐induced knockdown studies have highlighted their importance in many cell division processes including spindle assembly and chromosome segregation. Since microtubule turnovers and most mitotic events are relatively rapid (in minutes or seconds), developing tools that offer faster control over protein functions is therefore essential to more effectively interrogate kinesin‐13 activities in living cells. Here, we report the identification and characterization of a selective allosteric kinesin‐13 inhibitor, DHTP. Using high resolution microscopy, we show that DHTP is cell permeable and can modulate microtubule dynamics in cells.

High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non-cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy.

Ternary complex of Kif2A-bound tandem tubulin heterodimers represents a kinesin-13-mediated microtubule depolymerization reaction intermediate

Kinesin-13 proteins are major microtubule (MT) regulatory factors that catalyze removal of tubulin subunits from MT ends. The class-specific “neck” and loop 2 regions of these motors are required for MT depolymerization, but their contributing roles are still unresolved because their interactions with MT ends have not been observed directly. Here we report the crystal structure of a catalytically active kinesin-13 monomer (Kif2A) in complex with two bent αβ-tubulin heterodimers in a head-to-tail array, providing a view of these interactions. The neck of Kif2A binds to one tubulin dimer and the motor core to the other, guiding insertion of the KVD motif of loop 2 in between them. AMPPNP-bound Kif2A can form stable complexes with tubulin in solution and trigger MT depolymerization. We also demonstrate the importance of the neck in modulating ATP turnover and catalytic depolymerization of MTs. These results provide mechanistic insights into the catalytic cycles of kinesin-13.The kinesin-13 family of microtubule (MT) depolymerases are major regulators of MT dynamics. Here the authors provide insights into the MT depolymerization mechanism by solving the crystal structure of a kinesin-13 monomer (Kif2A) in complex with two bent αβ-tubulin heterodimers.

Multimodal and Polymorphic Interactions between Anillin and Actin: Their Implications for Cytokinesis

Cytokinesis of animal cells requires the assembly of a contractile ring, which promotes daughter cell splitting. Anillin is a conserved scaffold protein involved in organizing the structural components of the contractile ring including filamentous actin (F-actin), myosin, and septins and in forming the subsequent midbody ring. Like other metazoan homologs, Drosophila anillin contains a conserved domain that can bind and bundle F-actin, but the importance and molecular details of its interaction with F-actin remain unclear. Here, we show that in a depletion-and-rescue assay in Drosophila S2 cells, anillin lacking the entire actin-binding domain (ActBD) exhibits defective cortical localization during mitosis and a greatly diminished ability to support cytokinesis. Using in vitro binding assays and electron microscopy on recombinant fragments, we determine that the anillin ActBD harbors three distinct actin-binding sites (ABS 1-3). We show that each ABS binds to a distinct place on F-actin. Importantly, ABS1 and ABS3 partially overlap on the surface of actin and, therefore, interact with F-actin in a mutually exclusive fashion. Although ABS2 and ABS3 are sufficient for bundling, ABS1 contributes to the overall F-actin bundling activity of anillin and enables anillin to switch between two actin-bundling morphologies and promote the formation of three-dimensional F-actin bundles. Finally, we show that in live S2 cells, ABS2 and ABS3 are each required and together sufficient for the robust cortical localization of the ActBD during cytokinesis. Collectively, our structural, biochemical, and cell biological data suggest that multiple anillin-actin interaction modes promote the faithful progression of cytokinesis.