Inhibition of p38-MK2 pathway enhances the efficacy of microtubule inhibitors in breast cancer cells.

Microtubule-targeting agents (MTAs) have been successfully translated from basic research into clinical therapies and have been widely used as first- and second-line chemotherapy drugs for various cancers. However, current MTAs exhibit positive responses only in subsets of patients and are often accompanied by side effects due to their impact on normal cells. This underscores an urgent need to develop novel therapeutic strategies that enhance MTA efficacy while minimizing toxicity to normal tissues.

Confinement in fibrous environments positions and orients mitotic spindles.

Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from neighboring cells or the extracellular matrix (ECM), which can cause rotation of mitotic spindles and, consequently, titling of the metaphase plate (MP). To understand the positioning and orientation of mitotic spindles under confinement by fibers (ECM-confinement), we use flexible ECM-mimicking nanofibers that allow natural rounding of the cell body while confining it to differing levels.

The role of kinetochore dynein in checkpoint silencing is restricted to disassembly of the corona.

During mitosis, kinetochore-microtubule attachments are monitored by a molecular surveillance system known as the spindle assembly checkpoint. The prevailing model posits that dynein evicts checkpoint proteins (e.g.

Mitotic outcomes and errors in fibrous environments.

During mitosis, cells round up and utilize the interphase adhesion sites within the fibrous extracellular matrix (ECM) as guidance cues to orient the mitotic spindles. Here, using suspended ECM-mimicking nanofiber networks, we explore mitotic outcomes and error distribution for various interphase cell shapes. Elongated cells attached to single fibers through two focal adhesion clusters (FACs) at their extremities result in perfect spherical mitotic cell bodies that undergo significant 3-dimensional (3D) displacement while being held by retraction fibers (RFs).

Production and purification of recombinant monoclonal antibodies from human cells based on a primary sequence.

There are challenges to using commercially available antibodies generated in animals, including concerns with reproducibility, high costs, and ethical issues. Here, we present a protocol for generating and purifying recombinant antibodies from human HEK293 suspension culture cells from a primary sequence. We describe the steps to generate antibody heavy and light chain plasmids, followed by transfection of the plasmids into cells and purification of antibodies.

Hyper-active RAS/MAPK introduces cancer-specific mitotic vulnerabilities.

Aneuploidy, the incorrect number of whole chromosomes, is a common feature of tumors that contributes to their initiation and evolution. Preventing aneuploidy requires properly functioning kinetochores, which are large protein complexes assembled on centromeric DNA that link mitotic chromosomes to dynamic spindle microtubules and facilitate chromosome segregation. The kinetochore leverages at least two mechanisms to prevent aneuploidy: error correction and the spindle assembly checkpoint (SAC).

Generation and diversification of recombinant monoclonal antibodies.

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences.

Permitted and restricted steps of human kinetochore assembly in mitotic cell extracts.

Mitotic kinetochores assemble via the hierarchical recruitment of numerous cytosolic components to the centromere region of each chromosome. However, how these orderly and localized interactions are achieved without spurious macromolecular assemblies forming from soluble kinetochore components in the cell cytosol remains poorly understood. We developed assembly assays to monitor the recruitment of green fluorescent protein-tagged recombinant proteins and native proteins from human cell extracts to inner kinetochore components immobilized on microbeads.

BuGZ facilitates loading of spindle assembly checkpoint proteins to kinetochores in early mitosis.

BuGZ is a kinetochore component that binds to and stabilizes Bub3, a key player in mitotic spindle assembly checkpoint signaling. Bub3 is required for kinetochore recruitment of Bub1 and BubR1, two proteins that have essential and distinct roles in the checkpoint. Both Bub1 and BubR1 localize to kinetochores through interactions with Bub3, which are mediated through conserved GLEBS domains in both Bub1 and BubR1.

Lamin A/C deficiency enables increased myosin-II bipolar filament ensembles that promote divergent actomyosin network anomalies through self-organization.

Nuclear envelope proteins influence cell cytoarchitecure by poorly understood mechanisms. Here we show that small interfering RNA-mediated silencing of lamin A/C (LMNA) promotes contrasting stress fiber assembly and disassembly in individual cells and within cell populations. We show that LMNA-deficient cells have elevated myosin-II bipolar filament accumulations, irregular formation of actin comet tails and podosome-like adhesions, increased steady state nuclear localization of the mechanosensitive transcription factors MKL1 and YAP, and induced expression of some MKL1/serum response factor-regulated genes such as that encoding myosin-IIA (MYH9).

The right place at the right time: Aurora B kinase localization to centromeres and kinetochores.

The fidelity of chromosome segregation during mitosis is intimately linked to the function of kinetochores, which are large protein complexes assembled at sites of centromeric heterochromatin on mitotic chromosomes. These key "orchestrators" of mitosis physically connect chromosomes to spindle microtubules and transduce forces through these connections to congress chromosomes and silence the spindle assembly checkpoint. Kinetochore-microtubule attachments are highly regulated to ensure that incorrect attachments are not prematurely stabilized, but instead released and corrected.

The Hec1/Ndc80 tail domain is required for force generation at kinetochores, but is dispensable for kinetochore-microtubule attachment formation and Ska complex recruitment.

The conserved kinetochore-associated NDC80 complex (composed of Hec1/Ndc80, Nuf2, Spc24, and Spc25) has well-documented roles in mitosis including 1) connecting mitotic chromosomes to spindle microtubules to establish force-transducing kinetochore-microtubule attachments and 2) regulating the binding strength between kinetochores and microtubules such that correct attachments are stabilized and erroneous attachments are released. Although the NDC80 complex plays a central role in forming and regulating attachments to microtubules, additional factors support these processes as well, including the spindle and kinetochore-associated (Ska) complex. Multiple lines of evidence suggest that Ska complexes strengthen attachments by increasing the ability of NDC80 complexes to bind microtubules, especially to depolymerizing microtubule plus ends, but how this is accomplished remains unclear.

Hec1/Ndc80 Tail Domain Function at the Kinetochore-Microtubule Interface.

Successful mitotic cell division is critically dependent on the formation of correct attachments between chromosomes and spindle microtubules. Microtubule attachments are mediated by kinetochores, which are large proteinaceous structures assembled on centromeric chromatin of mitotic chromosomes. These attachments must be sufficiently stable to transduce force; however, the strength of these attachments are also tightly regulated to ensure timely, error-free progression through mitosis.

Aurora B kinase is recruited to multiple discrete kinetochore and centromere regions in human cells.

Aurora B kinase has a critical role in regulating attachments between kinetochores and spindle microtubules during mitosis. Early in mitosis, kinase activity at kinetochores is high to promote attachment turnover, and in later mitosis, activity decreases to ensure attachment stabilization. Aurora B localizes prominently to inner centromeres, and a population of the kinase is also detected at kinetochores.

Effectors of the spindle assembly checkpoint are confined within the nucleus of Saccharomyces cerevisiae.

The spindle assembly checkpoint (SAC) prevents erroneous chromosome segregation by delaying mitotic progression when chromosomes are incorrectly attached to the mitotic spindle. This delay is mediated by mitotic checkpoint complexes (MCCs), which assemble at unattached kinetochores and repress the activity of the anaphase promoting complex/cyclosome (APC/C). The cellular localizations of MCCs are likely critical for proper SAC function, yet remain poorly defined.

Aurora A Kinase Function at Kinetochores.

One of the most important regulatory aspects of chromosome segregation is the ability of kinetochores to precisely control their attachment strength to spindle microtubules. Central to this regulation is Aurora B, a mitotic kinase that phosphorylates kinetochore substrates to promote microtubule turnover. A critical target of Aurora B is the kinetochore protein Ndc80/Hec1, which is a component of the NDC80 complex, the primary force-transducing link between kinetochores and microtubules.

Use of Confocal Microscopy to Evaluate Equine Zygote Development After Sperm Injection of Oocytes Matured In Vivo or In Vitro.

Confocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were matured in vivo or in vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated with α/β tubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging.

Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore-microtubule dynamics.

Precise regulation of kinetochore-microtubule attachments is essential for successful chromosome segregation. Central to this regulation is Aurora B kinase, which phosphorylates kinetochore substrates to promote microtubule turnover. A critical target of Aurora B is the N-terminal "tail" domain of Hec1, which is a component of the NDC80 complex, a force-transducing link between kinetochores and microtubules.

Sensitivity to Inhibition Defines an Alternative Classification of Glioblastoma.

Glioblastoma multiforme (GBM) remains a mainly incurable disease in desperate need of more effective treatments. In this study, we develop evidence that the mitotic spindle checkpoint molecule may offer a predictive marker for aggressiveness and effective drug response. A subset of GBM tumor isolates requires to suppress lethal kinetochore-microtubule attachment defects.

Spindle assembly checkpoint signaling and sister chromatid cohesion are disrupted by HPV E6-mediated transformation.

Aneuploidy, a condition that results from unequal partitioning of chromosomes during mitosis, is a hallmark of many cancers, including those caused by human papillomaviruses (HPVs). E6 and E7 are the primary transforming proteins in HPV that drive tumor progression. In this study, we stably expressed E6 and E7 in noncancerous RPE1 cells and analyzed the specific mitotic defects that contribute to aneuploidy in each cell line.

"Wait anaphase" signals are not confined to the mitotic spindle.

The spindle assembly checkpoint ensures the faithful inheritance of chromosomes by arresting mitotic progression in the presence of kinetochores that are not attached to spindle microtubules. This is achieved through inhibition of the anaphase-promoting complex/cyclosome by a kinetochore-derived "wait anaphase" signal known as the mitotic checkpoint complex. It remains unclear whether the localization and activity of these inhibitory complexes are restricted to the mitotic spindle compartment or are diffusible throughout the cytoplasm.

Cofilin Regulates Nuclear Architecture through a Myosin-II Dependent Mechanotransduction Module.

Structural features of the nucleus including shape, size and deformability impact its function affecting normal cellular processes such as cell differentiation and pathological conditions such as tumor cell migration. Despite the fact that abnormal nuclear morphology has long been a defining characteristic for diseases such as cancer relatively little is known about the mechanisms that control normal nuclear architecture. Mounting evidence suggests close coupling between F-actin cytoskeletal organization and nuclear morphology however, mechanisms regulating this coupling are lacking.

Real-time quantification of single RNA translation dynamics in living cells.

Although messenger RNA (mRNA) translation is a fundamental biological process, it has never been imaged in real time in vivo with single-molecule precision. To achieve this, we developed nascent chain tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify protein synthesis dynamics at the single-mRNA level. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 seconds.

Superresolved multiphoton microscopy with spatial frequency-modulated imaging.

Superresolved far-field microscopy has emerged as a powerful tool for investigating the structure of objects with resolution well below the diffraction limit of light. Nearly all superresolution imaging techniques reported to date rely on real energy states of fluorescent molecules to circumvent the diffraction limit, preventing superresolved imaging with contrast mechanisms that occur via virtual energy states, including harmonic generation (HG). We report a superresolution technique based on spatial frequency-modulated imaging (SPIFI) that permits superresolved nonlinear microscopy with any contrast mechanism and with single-pixel detection.

Measuring Kinetochore-Microtubule Attachment Stability in Cultured Cells.

Duplicated sister chromatids connect to the mitotic spindle through kinetochores, large proteinaceous structures built at sites of centromeric heterochromatin. Kinetochores are responsible for harnessing the forces generated by microtubule polymerization and depolymerization to power chromosome movements. The fidelity of chromosome segregation relies on proper kinetochore function, as precise regulation of the attachment between kinetochores and microtubules is essential to prevent mitotic errors, which are linked to the initiation and progression of cancer and the formation of birth defects (Godek et al.