Structure and dynamics of motor-driven microtubule bundles.

Connecting the large-scale emergent behaviors of active cytoskeletal materials to the microscopic properties of their constituents is a challenge due to a lack of data on the multiscale dynamics and structure of such systems. We approach this problem by studying the impact of depletion attraction on bundles of microtubules and kinesin-14 molecular motors. For all depletant concentrations, kinesin-14 bundles generate comparable extensile dynamics.

Correction: Smith et al. MCAK Inhibitors Induce Aneuploidy in Triple-Negative Breast Cancer Models. 2023, , 3309.

The authors alerted the Editorial Office of the mistake on 5 August 2023 and the final documents were sent for evaluation on 12 December 2023 [...

MCAK Inhibitors Induce Aneuploidy in Triple-Negative Breast Cancer Models.

Standard of care for triple-negative breast cancer (TNBC) involves the use of microtubule poisons such as paclitaxel, which are proposed to work by inducing lethal levels of aneuploidy in tumor cells. While these drugs are initially effective in treating cancer, dose-limiting peripheral neuropathies are common. Unfortunately, patients often relapse with drug-resistant tumors.

MCAK Inhibitors Induce Aneuploidy in Triple Negative Breast Cancer Models.

Unlabelled: Standard of care for triple negative breast cancer (TNBC) involves the use of microtubule poisons like paclitaxel, which are proposed to work by inducing lethal levels of aneuploidy in tumor cells. While these drugs are initially effective in treating cancer, dose-limiting peripheral neuropathies are common. Unfortunately, patients often relapse with drug resistant tumors.

Importin α/β promote Kif18B microtubule association and enhance microtubule destabilization activity.

Tight regulation of microtubule (MT) dynamics is necessary for proper spindle assembly and chromosome segregation. The MT destabilizing Kinesin-8, Kif18B, controls astral MT dynamics and spindle positioning. Kif18B interacts with importin α/β as well as with the plus-tip tracking protein EB1, but how these associations modulate Kif18B is not known.

Using FLIM-FRET for Characterizing Spatial Interactions in the Spindle.

Proper spindle assembly and the attachment of chromosomes to the spindle are key for the accurate segregation of chromosomes to daughter cells. Errors in these processes can lead to aneuploidy, which is a hallmark of cancer. Understanding the mechanisms that drive spindle assembly will provide fundamental insights into how accurate chromosome segregation is achieved.

Spatial regulation of MCAK promotes cell polarization and focal adhesion turnover to drive robust cell migration.

The asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK (mitotic centromere-associated kinesin), in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement.

Self-straining of actively crosslinked microtubule networks.

Cytoskeletal networks are foundational examples of active matter and central to self-organized structures in the cell. In vivo, these networks are active and densely crosslinked. Relating their large-scale dynamics to the properties of their constituents remains an unsolved problem.

In Vitro FRET- and Fluorescence-Based Assays to Study Protein Conformation and Protein-Protein Interactions in Mitosis.

Proper cell division and the equal segregation of genetic material are essential for life. Cell division is mediated by the mitotic spindle, which is composed of microtubules (MTs) and MT-associated proteins that help align and segregate the chromosomes. The localization and characterization of many spindle proteins have been greatly aided by using GFP-tagged proteins in vivo, but these tools typically do not allow for understanding how their activity is regulated biochemically.

RanGTP induces an effector gradient of XCTK2 and importin α/β for spindle microtubule cross-linking.

High RanGTP around chromatin is important for governing spindle assembly during meiosis and mitosis by releasing the inhibitory effects of importin α/β. Here we examine how the Ran gradient regulates Kinesin-14 function to control spindle organization. We show that Xenopus Kinesin-14, XCTK2, and importin α/β form an effector gradient that is highest at the poles and diminishes toward the chromatin, which is opposite the RanGTP gradient.

Aurora A activation in mitosis promoted by BuGZ.

Protein phase separation or coacervation has emerged as a potential mechanism to regulate biological functions. We have shown that coacervation of a mostly unstructured protein, BuGZ, promotes assembly of spindle and its matrix. BuGZ in the spindle matrix binds and concentrates tubulin to promote microtubule (MT) assembly.

The far C-terminus of MCAK regulates its conformation and spindle pole focusing.

To ensure proper spindle assembly, microtubule (MT) dynamics needs to be spatially regulated within the cell. The kinesin-13 MCAK is a potent MT depolymerase with a complex subcellular localization, yet how MCAK spatial regulation contributes to spindle assembly is not understood. Here we show that the far C-terminus of MCAK plays a critical role in regulating MCAK conformation, subspindle localization, and spindle assembly in Xenopus egg extracts.

The Ran-GTP gradient spatially regulates XCTK2 in the spindle.

Ran is a small GTP binding protein that was originally identified as a regulator of nucleocytoplasmic transport [1] and subsequently found to be important for spindle formation [2-5]. In mitosis, a gradient of Ran-GTP emanates from chromatin and diminishes toward spindle poles [6]. Ran-GTP promotes spindle self-organization through the release of importin-bound spindle assembly factors (SAFs), which stimulate microtubule (MT) nucleation and organization and regulate MT dynamics [7-9].

Aurora B inhibits MCAK activity through a phosphoconformational switch that reduces microtubule association.

Background: Proper spindle assembly and chromosome segregation rely on precise microtubule dynamics, which are governed in part by the kinesin-13 MCAK. MCAK microtubule depolymerization activity is inhibited by Aurora B-dependent phosphorylation, but the mechanism of this inhibition is not understood.

Results: Here, we develop the first Förster resonance energy transfer (FRET)-based biosensor for MCAK and show that MCAK in solution exists in a closed conformation mediated by an interaction between the C-terminal domain (CT) and the neck.

Kif18A uses a microtubule binding site in the tail for plus-end localization and spindle length regulation.

The mitotic spindle is a macromolecular structure utilized to properly align and segregate sister chromatids to two daughter cells. During mitosis, the spindle maintains a constant length, even though the spindle microtubules (MTs) are constantly undergoing polymerization and depolymerization [1]. Members of the kinesin-8 family are important for the regulation of spindle length and for chromosome positioning [2-9].

Kif18B interacts with EB1 and controls astral microtubule length during mitosis.

Regulation of microtubule (MT) dynamics is essential for proper spindle assembly and organization. Kinesin-8 family members are plus-end-directed motors that modulate plus-end MT dynamics by acting as MT depolymerases or as MT plus-end capping proteins. In this paper, we show that the human kinesin-8 Kif18B functions during mitosis to control astral MT organization.

Proper organization of microtubule minus ends is needed for midzone stability and cytokinesis.

Successful cytokinesis is critical for maintaining genome stability and requires the assembly of a robust central spindle to specify the cleavage furrow position, to prevent separated chromosomes from coming back together, and to contribute to midbody abscission. A proper central spindle is assembled and maintained by a number of microtubule-associated and molecular motor proteins that sort microtubules into bundles with their plus ends overlapping at the center. The mechanisms by which different factors organize the central spindle microtubules remain unclear.

Kinesin-13s in mitosis: Key players in the spatial and temporal organization of spindle microtubules.

Dynamic microtubules are essential for the process of mitosis. Thus, elucidating when, where, and how microtubule dynamics are regulated is key to understanding this process. One important class of proteins that directly regulates microtubule dynamics is the Kinesin-13 family.

TgICMAP1 is a novel microtubule binding protein in Toxoplasma gondii.

The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules.

Kinesin-14 family proteins HSET/XCTK2 control spindle length by cross-linking and sliding microtubules.

Kinesin-14 family proteins are minus-end directed motors that cross-link microtubules and play key roles during spindle assembly. We showed previously that the Xenopus Kinesin-14 XCTK2 is regulated by Ran via the association of a bipartite NLS in the tail of XCTK2 with importin alpha/beta, which regulates its ability to cross-link microtubules during spindle formation. Here we show that mutation of the nuclear localization signal (NLS) of human Kinesin-14 HSET caused an accumulation of HSET in the cytoplasm, which resulted in strong microtubule bundling.

Aurora A phosphorylates MCAK to control ran-dependent spindle bipolarity.

During mitosis, mitotic centromere-associated kinesin (MCAK) localizes to chromatin/kinetochores, a cytoplasmic pool, and spindle poles. Its localization and activity in the chromatin region are regulated by Aurora B kinase; however, how the cytoplasmic- and pole-localized MCAK are regulated is currently not clear. In this study, we used Xenopus egg extracts to form spindles in the absence of chromatin and centrosomes and found that MCAK localization and activity are tightly regulated by Aurora A.

Aurora B phosphorylates multiple sites on mitotic centromere-associated kinesin to spatially and temporally regulate its function.

Chromosome congression and segregation require the proper attachment of microtubules to the two sister kinetochores. Disruption of either Aurora B kinase or the Kinesin-13 mitotic centromere-associated kinesin (MCAK) increases chromosome misalignment and missegregation due to improper kinetochore-microtubule attachments. MCAK localization and activity are regulated by Aurora B, but how Aurora B phosphorylation of MCAK affects spindle assembly is unclear.

The interplay of the N- and C-terminal domains of MCAK control microtubule depolymerization activity and spindle assembly.

Spindle assembly and accurate chromosome segregation require the proper regulation of microtubule dynamics. MCAK, a Kinesin-13, catalytically depolymerizes microtubules, regulates physiological microtubule dynamics, and is the major catastrophe factor in egg extracts. Purified GFP-tagged MCAK domain mutants were assayed to address how the different MCAK domains contribute to in vitro microtubule depolymerization activity and physiological spindle assembly activity in egg extracts.

Full-length dimeric MCAK is a more efficient microtubule depolymerase than minimal domain monomeric MCAK.

MCAK belongs to the Kinesin-13 family, whose members depolymerize microtubules rather than translocate along them. We defined the minimal functional unit of MCAK as the catalytic domain plus the class specific neck (MD-MCAK), which is consistent with previous reports. We used steady-state ATPase kinetics, microtubule depolymerization assays, and microtubule.

Catastrophic kinesins: piecing together their mechanism by 3D reconstruction.

Kin Is, kinesins with an internal catalytic domain, de-polymerize microtubules from both ends, and the KIF2C crystal structure presented by ([this issue of Cell]) provides provocative evidence to support the theory that the highly conserved sequences are critical structural elements in these catastrophic kinesins.