Browsing by Author "Walczak, Claire E."

Now showing 1 - 5 of 5
  • Thumbnail Image
    Item
    MCAK Inhibitors Induce Aneuploidy in Triple-Negative Breast Cancer Models
    (MDPI, 2023-06-23) Smith, John C.; Husted, Stefan; Pilrose, Jay; Ems-McClung, Stephanie C.; Stout, Jane R.; Carpenter, Richard L.; Walczak, Claire E.; Biochemistry and Molecular Biology, School of Medicine
    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. 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 50 k 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.
  • Thumbnail Image
    Item
    Spatial regulation of MCAK promotes cell polarization and focal adhesion turnover to drive robust cell migration
    (American Society for Cell Biology, 2021-04-01) Zong, Hailing; Hazelbaker, Mark; Moe, Christina; Ems-McClung, Stephanie C.; Hu, Ke; Walczak, Claire E.; Biology, School of Science
    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. 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 functions correlate with a spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared with the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream of or in a parallel pathway to MCAK function in migration. Together, our data support a model in which the polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contribute to cell polarity, centrosome positioning, and focal adhesion dynamics, which all help facilitate robust directional migration.
  • Thumbnail Image
    Item
    Structure and dynamics of motor-driven microtubule bundles
    (Royal Society of Chemistry, 2024-07-24) Lemma, Bezia; Lemma, Linnea M.; Ems-McClung, Stephanie C.; Walczak, Claire E.; Dogic, Zvonimir; Needleman, Daniel J.; Medicine, School of Medicine
    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. However, this invariable mesoscopic behavior masks the transition in the microscopic motion of microtubules. Specifically, with increasing attraction, we observe a transition from bi-directional sliding with extension to pure extension with no sliding. Small-angle X-ray scattering shows that the transition in microtubule dynamics is concurrent with a structural rearrangement of microtubules from an open hexagonal to a compressed rectangular lattice. These results demonstrate that bundles of microtubules and molecular motors can display the same mesoscopic extensile behaviors despite having different internal structures and microscopic dynamics. They provide essential information for developing multiscale models of active matter.
  • Thumbnail Image
    Item
    Switch‐1 instability at the active site decouples ATP hydrolysis from force generation in myosin II
    (Wiley, 2021-01) Walker, Benjamin C.; Walczak, Claire E.; Cochran, Jared C.; Medicine, School of Medicine
    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 the ATPase catalytic cycle 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 affects actin binding and productive 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.
  • Thumbnail Image
    Item
    Using FLIM-FRET for Characterizing Spatial Interactions in the Spindle
    (Springer, 2022) Ems-McClung, Stephanie C.; Walczak, Claire E.; Biochemistry and Molecular Biology, School of Medicine
    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. One challenge in elucidating the complexities of spindle assembly is to visualize protein interactions in space and time. The Xenopus egg extract system has been a valuable tool to probe protein function during spindle assembly in vitro. Tagging proteins with fluorescent proteins and utilizing fluorescence-based approaches, such as Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM), have provided visual clues about the mechanics of spindle assembly and its regulators. However, elucidating how spindle assembly factors are spatially regulated is still challenging. Combining the egg extract system and visual FRET approaches provides a powerful tool to probe the processes involved in spindle assembly. Here we describe how a FLIM-FRET biosensor can be used to study protein-protein interactions in spindles assembled in Xenopus egg extracts. This approach should be readily adaptable to a wide variety of proteins to allow for new insights into the regulation of spindle assembly.