Papers by Stephanie Ems-Mcclung
arXiv (Cornell University), Dec 3, 2018
Cancers, Jun 23, 2023
Triple-negative breast cancer (TNBC) is the most lethal breast cancer subtype with few treatment ... more Triple-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.
Journal of clinical and translational science, Apr 1, 2023
T cell responses. Our work shows that STING activation, which primarily targets innate immunity m... more T cell responses. Our work shows that STING activation, which primarily targets innate immunity myeloid cells 'upstream' of T cells in the antitumor immunity cycle, can cure ICB-refractory GBM tumors in an adaptive immunity-dependent manner.
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Aug 27, 2010
Aurora-B is a component of the Chromosomal Passenger Complex (CPC) required for correct spindle-k... more Aurora-B is a component of the Chromosomal Passenger Complex (CPC) required for correct spindle-kinetochore attachments during chromosome segregation and for cytokinesis. The chromatin factors that recruit the CPC to centromeres are unknown, however. Here we show that phosphorylation of Histone-H3 Thr-3 (H3T3ph) by Haspin is necessary for CPC accumulation at centromeres, and that the CPC subunit Survivin binds directly to H3T3ph. A non-binding Survivin-D70A/D71A mutant does not support centromeric CPC concentration and both Haspin depletion and Survivin-D70A/D71A mutation diminish centromere localization of MCAK and mitotic checkpoint signaling in taxol. Survivin-D70A/D71A mutation and microinjection of H3T3ph-specific antibody both compromise centromeric Aurora-B functions but do not prevent cytokinesis. Therefore, H3T3ph generated by Haspin positions the CPC at centromeres to regulate selected targets of Aurora-B during mitosis. The CPC (containing Aurora-B, INCENP, Survivin and Borealin) is found on chromosome arms in prophase, concentrates at inner centromeres during prometaphase, and transfers to the central spindle at anaphase (1). Aurora-B phosphorylates several substrates at these locations, including Histone-H3 Ser-10 (H3S10ph) on chromosome arms, Mitotic Centromere-Associated Kinesin (MCAK) at inner centromeres, Centromere Protein-A Ser-7 (CENP-AS7ph) at outer centromeres and the KNL1/Mis12 complex/Ndc80 network at kinetochores (1-6). Current models suggest that centromeric Aurora-B responds to lack of tension across sister kinetochores that are incorrectly attached to the spindle. Bipolar kinetochore attachment forces may pull kinetochore substrates away from inner centromeric Aurora-B, leading to substrate dephosphorylation, selective stabilization of correct microtubule attachments, and eventually to satisfaction of the spindle checkpoint (Fig. S1) (7). Despite its central importance, it is not understood how Aurora-B accumulates at centromeres (8-10). Immunofluorescence microscopy of mitotic cells shows that H3T3ph and Aurora-B localize similarly at inner centromeres (Fig. 1A, Fig. S2). We therefore tested whether Haspin, which is responsible for generating H3T3ph in mitosis (11,12), is required for CPC localization. Upon Haspin RNAi in HeLa cells, a marked reduction in Aurora-B at centromeres (>5-fold)
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Aug 31, 2011
When not transporting cargo, kinesin-1 is autoinhibited by binding of a tail region to the motor ... more When not transporting cargo, kinesin-1 is autoinhibited by binding of a tail region to the motor domains, but the mechanism of inhibition is unclear. We report the crystal structure of a motor domain dimer in complex with its tail domain at 2.2 Å and compare it with a structure of the motor domain alone at 2.7 Å. These structures indicate that neither an induced conformational change nor steric blocking is the cause of inhibition. Instead, the tail cross-links the motor domains at a second position, in addition to the coiled-coil. This 'double lockdown', by crosslinking at two positions, prevents the movement of the motor domains that is needed to undock the neck linker and release ADP. This autoinhibition mechanism could extend to some other kinesins.
Molecular Biology of the Cell, Apr 1, 2021
The asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell... more 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.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
arXiv (Cornell University), Sep 14, 2022
Faculty Opinions recommendation of GTPgammaS microtubules mimic the growing microtubule end structure recognized by end-binding proteins (EBs)
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, May 16, 2011
A Hierarchy of Instabilities in an Active Material
Bulletin of the American Physical Society, Mar 9, 2018
Bulletin of the American Physical Society, Mar 5, 2019
A network of mutually propelled rods: theory and experiment
Bulletin of the American Physical Society, Nov 18, 2018
bioRxiv (Cold Spring Harbor Laboratory), Mar 30, 2022
Tight regulation of microtubule dynamics and microtubule organization is necessary for proper spi... more Tight regulation of microtubule dynamics and microtubule organization is necessary for proper spindle assembly and chromosome segregation. The microtubule destabilizing Kinesin-8, Kif18B, controls astral microtubule dynamics and spindle positioning. Kif18B interacts with importin a/b as well as with the plus-tip tracking protein EB1, but how these associations modulate Kif18B activity or function is not known. We mapped the key binding sites on Kif18B, made residue-specific mutations, and assessed their impact on Kif18B function. Blocking EB1 interaction disrupted Kif18B MT plus-end accumulation and inhibited its ability to control astral microtubule length in cells. Blocking importin a/b interaction reduced Kif18B plus-end accumulation on astral MTs but did not inhibit its ability to control astral MT length. In vitro, importin a/b increased Kif18B binding to the microtubule lattice, which enhanced Kif18B accumulation at microtubule plus ends, and stimulated microtubule destabilization, suggesting that the importins spatially regulate Kif18B. In contrast, EB1 promoted microtubule destabilization without increasing lattice binding in vitro, which suggests that EB1 and importin a/b have distinct roles in the regulation of astral microtubule dynamics. We propose that Ran-regulation is important not only to control motor function near chromatin but also provides a spatial control mechanism to modulate microtubule binding of NLS-containing spindle assembly factors. .
Standard of care for triple negative breast cancer (TNBC) involves the use of microtubule poisons... more 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 IC50for paclitaxel, without affecting normal cells. Using FRET and image-based assays, we screened compounds from the ChemBridge 5...
Using FLIM-FRET for Characterizing Spatial Interactions in the Spindle
Methods in Molecular Biology, 2022
Proper spindle assembly and the attachment of chromosomes to the spindle are key for the accurate... more 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.
gondiiCortical Microtubules of Toxoplasma Components of a Protein Complex Coating Novel Thioredoxin-Like Proteins Are
Molecular Biology of the Cell, Apr 1, 2023
Tight regulation of microtubule (MT) dynamics is necessary for proper spindle assembly and chromo... more 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. We mapped the key binding sites on Kif18B, made residue-specific mutations, and assessed their impact on Kif18B function. Blocking EB1 interaction disrupted Kif18B MT plus-end accumulation and inhibited its ability to control MT length on monopolar spindles in cells. Blocking importin α/β interaction disrupted Kif18B localization without affecting aster size. In vitro, importin α/β increased Kif18B MT association by increasing the on-rate and decreasing the off-rate from MTs, which stimulated MT destabilization. In contrast, EB1 promoted MT destabilization without increasing lattice binding in vitro, which suggests that EB1 and importin α/β have distinct roles in the regulation of Kif18B-mediated MT destabilization. We propose that importin α/β spatially modulate Kif18B association with MTs to facilitate its MT destabilization activity. Our results suggest that Ran regulation is important not only to control molecular motor function near chromatin but also to provide a spatial control mechanism to modulate MT binding of nuclear localization signal–containing spindle assembly factors.
Proper cell division and the equal segregation of genetic material are essential for life. Cell d... more 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. With the recent explosion of the pallet of GFP-derived fluorescent proteins, fluorescence-based biosensors are becoming useful tools for the quantitative analysis of protein activity and protein-protein interactions. Here, we describe solution-based Förster resonance energy transfer (FRET) and fluorescence assays that can be used to quantify protein-protein interactions and to characterize protein conformations of MT-associated proteins involved in mitosis.
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... more 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. Toget...
Faculty Opinions recommendation of Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature
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Papers by Stephanie Ems-Mcclung