Biology subjects

Walczak, C. E.

Publications and source records attributed to Walczak, C. E..

6 recordsLinked to original sources

bioRxiv2025

The Kinesin-14 Tail: Dual microtubule binding domains drive spindle morphogenesis through tight microtubule cross-linking and robust sliding

Proper spindle assembly requires the Kinesin-14 family of motors to organize microtubules (MTs) into the bipolar spindle by cross-linking and sliding anti-parallel and parallel MTs through their motor and tail domains. How they mediate these different activities is unclear. We identified two MT binding domains (MBD1 and MBD2) within the Xenopus Kinesin-14 XCTK2 tail and found that MBD1 MT affinity was weaker than MBD2. Comparable to full-length GFP-XCTK2 wild-type protein (GX-WT), GFP-XCTK2 containing the MBD1 mutations (GX-MBD1mut) stimulated spindle assembly, localized moderately on the spindle, and formed narrow spindles. In contrast, GX-MBD2mut only partially stimulated spindle assembly, localized weakly on the spindle, and formed shorter spindles. Biochemical reconstitution of MT cross-linking and sliding demonstrated that GX-MBD2mut slid anti-parallel MTs faster than GX-WT and GX-MBD1mut. However, GX-WT and GX-MBD1mut statically cross-linked the majority of parallel MTs, whereas GX-MBD2mut equally slid and statically cross-linked parallel MTs without affecting their sliding velocity. These results provide a mechanism by which the two different MT binding domains in the Kinesin-14 tail balance anti-parallel MT sliding velocity (MBD1) and tight parallel MT cross-linking (MBD2), which are important for spindle assembly and localization, and provide a basis for characterizing how molecular motors organize MTs within the spindle. Significance StatementSpindle assembly and organization utilize molecular motors that cross-link and slide anti-parallel and parallel microtubules. How individual motors moderate both active sliding and static cross-linking is not understood. Using biochemical reconstitution, the authors determined that the Kinesin-14 tail contains two independent microtubule binding domains. MBD1 with weaker microtubule binding facilitates faster anti-parallel microtubule sliding, whereas the stronger MBD2 mediates tight parallel microtubule cross-linking, which was important for spindle assembly. These findings provide a mechanism for how Kinesin-14s differentially control microtubule sliding and cross-linking and provide insight into how molecular motors can mediate the dynamic organization of microtubules in the spindle.

cell biology↗

bioRxiv2023

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 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. Identifying agents against targets that limit aneuploidy may be a valuable approach for therapeutic development. One potential target is the microtubule depolymerizing kinesin, MCAK, which limits aneuploidy by regulating microtubule dynamics during mitosis. Using publicly available datasets, we found that MCAK is upregulated in triple negative breast cancer and is associated with poorer prognoses. Knockdown of MCAK in tumor-derived cell lines caused a two- to five-fold reduction in the IC50 for paclitaxel, without affecting normal cells. Using FRET and image-based assays, we screened compounds from the ChemBridge 50k library and discovered three putative MCAK inhibitors. These compounds reproduced the aneuploidy-inducing phenotype of MCAK loss, reduced clonogenic survival of TNBC cells regardless of taxane-resistance, and the most potent of the three, C4, sensitized TNBC cells to paclitaxel. Collectively, our work shows promise that MCAK may serve as both a biomarker of prognosis and as a therapeutic target. Simple SummaryTriple negative breast cancer (TNBC) is the most lethal breast cancer subtype with few treatment options available. Standard of care for TNBC involves the use of taxanes, which are initially effective, but dose limiting toxicities are common, and patients often relapse with resistant tumors. Specific drugs that produce taxane-like effects may be able to improve patient quality of life and prognosis. In this study we identify three novel inhibitors of the Kinesin-13 MCAK. MCAK inhibition induces aneuploidy; similar to cells treated with taxanes. We demonstrate that MCAK is upregulated in TNBC and is associated with poorer prognoses. These MCAK inhibitors reduce the clonogenic survival of TNBC cells, and the most potent of the three inhibitors, C4, sensitizes TNBC cells to taxanes, similar to the effects of MCAK knockdown. This work will expand the field of precision medicine to include aneuploidy-inducing drugs that have the potential to improve patient outcomes.

cancer biology↗

bioRxiv2020

Switch-1 Instability at the Active Site Decouples ATP Hydrolysis from Force Generation in Myosin II

Myosin active site elements (i.e. switch-1) bind both ATP and a divalent metal to coordinate ATP hydrolysis. ATP hydrolysis at the active site is linked via allosteric communication to the actin polymer binding site and lever arm movement, thus coupling the free energy of ATP hydrolysis to force generation. How active site motifs are functionally linked to actin binding and the power stroke is still poorly understood. We hypothesize that destabilizing switch-1 movement at the active site will negatively affect the tight coupling of ATP hydrolysis to force production. Using a metal-switch system, we tested the effect of interfering with switch-1 coordination of the divalent metal cofactor on force generation. We found that while ATPase activity increased, motility was inhibited. Our results demonstrate that a single atom change that affects the switch-1 interaction with the divalent metal directly regulates actin binding and force generation. Even slight modification of the switch-1 divalent metal coordination can decouple ATP hydrolysis from motility. Switch-1 movement is therefore critical for both structural communication with the actin binding site, as well as coupling the energy of ATP hydrolysis to force generation.

biochemistry↗

bioRxiv2020

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, in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These defects are due in part to the loss of the spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared to the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream or in a parallel pathway to MCAK function in migration. Together, our data support a model that places MCAK at a key nexus of a feedback loop, in which polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contributes to cell polarity and directional migration.

cell biology↗

bioRxiv2019

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 /{beta}. Here we examine how the Ran gradient regulates Kinesin-14 function to control spindle organization. We show that Xenopus Kinesin-14, XCTK2, and importin /{beta} form an effector gradient, which is highest at the poles that diminishes toward the chromatin and is inverse of the RanGTP gradient. Importin /{beta} preferentially inhibit XCTK2 anti-parallel microtubule cross-linking and sliding by decreasing the microtubule affinity of the XCTK2 tail domain. This change in microtubule affinity enables RanGTP to target endogenous XCTK2 to the spindle. We propose that these combined actions of the Ran pathway are critical to promote Kinesin-14 parallel microtubule cross-linking at the spindle poles to cluster centrosomes in cancer cells. Furthermore, our work illustrates that RanGTP regulation in the spindle is not simply a switch, but rather generates effector gradients where RanGTP gradually tunes the activities of spindle assembly factors.\n\nSummaryEms-McClung et al. visualize a RanGTP effector gradient of association between XCTK2 and importin /{beta} in the spindle. The importins preferentially inhibit XCTK2-mediate anti-parallel microtubule cross-linking and sliding, which allows XCTK2 to cross-link parallel microtubules and help focus spindle poles.

cell biology↗

bioRxiv2018

A Cyclin A - Myb-MuvB - Aurora B network regulates the choice between mitotic cycles and polyploid endoreplication cycles.

Cells switch to polyploid endoreplication cycles during development, wound healing, and cancer. We used integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between developmental and induced endoreplicating cells (devECs and iECs). We found that while only devECs had a dampened E2F1 transcriptome, repression of a Cyclin A - Myb-MuvB - Aurora B mitotic network promoted endoreplication in both devECs and iECs. Cyclin A associated with and activated Myb-MuvB to induce transcription of mitotic genes, with expression of one, Aurora B, being key for mitotic commitment. Knockdown of Cyclin A, Myb, Aurora B, or downstream cytokinetic proteins induced distinct types of endoreplication, suggesting that repression of different mitotic network steps may explain the known diversity of polyploid cycles. These findings reveal how remodeling of a mitotic network promotes polyploid cycles that contribute to development, wound healing, and cancer.

developmental biology↗