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Cho, N. H.

Publications and source records attributed to Cho, N. H..

14 recordsLinked to original sources

Sound-Driven Motion of Human Cochlear Structures Depends on Organ of Corti Integrity

Optical coherence tomography (OCT) was used to image and measure the passive sound-driven displacements of cochlear structures in situ through the intact round window membrane of fresh human cadaveric specimens at 12 to 30 hours postmortem. We find that the anatomical integrity of the organ of Corti (OoC) affects cochlear structural motion. OoC with high integrity (erect with supporting cells) have small osseous spiral lamina (OSL) motion. Conversely, OoC with low integrity (flattened without supporting cells) have large OSL motion. For the ratio of motion between the basilar membrane (BM) and OSL (BM/OSL), OoC with high integrity has large BM/OSL motion while a cochlear partition without OoC has small BM/OSL motion. Furthermore, the high-integrity OoC has reticular lamina surface motion that is lower than the BM, whereas low-integrity OoC has similar reticular lamina and BM motion. This relationship between OoC anatomical integrity and cochlear structural motion will have consequential effects on sound transduction.

neuroscience↗

Midzone bundles of the mammalian anaphase spindle are mechanically coupled both locally and globally

Robust chromosome segregation requires the anaphase spindle to both preserve and remodel its structure under force. How it does so remains unclear as probing mechanics during anaphases short lifetime is challenging. Here, we use microneedles to pull on mammalian anaphase midzone bundles and ask how they respond to and transmit force across space and time. We find that midzone bundles locally transmit force to each other in the spindles short axis over multiple timescales. Along the spindles long axis, midzone bundles globally transmit force: rather than bundles sliding apart or detaching under force, the spindle shortens. This reveals strong anchorage and resistance to outward sliding, and that spindle elongation requires all midzone bundles to elongate. This global force transmission is stronger for short-lived forces and is strictly PRC1-dependent, indicating limited mechanistic redundancy. In sum, the anaphase spindle acts as a single mechanical unit over short timescales to resist and transmit force, while remodeling over long timescales to segregate chromosomes. SummaryUsing microneedle manipulation, Mullin-Bernstein et al. show that microtubule bundles of the mammalian anaphase spindle are mechanically coupled, transmitting force both laterally and between spindle poles. These strong connections may help ensure coordinated and error-free chromosome segregation.

cell biology↗

Anatomical integrity of the human cochlea estimated with optical coherence tomography for future clinical application

The human cochlea is encased within the otic capsule, the densest bone in the body, posing significant challenges for anatomical imaging of cochlear structures. Because of difficult access and fragility of cochlear structures, our understanding of intracochlear anatomy has historically relied on postmortem histology. We thus have a limited understanding of human cochlear anatomy in its native, unfixed state. Clinical diagnostics for hearing loss, such as audiometry and otoacoustic emissions, offer functional assessments but fail to elucidate the often diverse underlying structural pathologies with any degree of precision. To address the critical need for assessing the human cochlear anatomy and associated pathologies without the risk of traumatizing cochlear structures, we imaged fresh cochleae in situ soon after death through the intact round window membrane with Optical Coherence Tomography (OCT) without inserting instruments inside or opening the cochlea. Micron-resolution OCT cross-sectional images of the human intracochlear structures were acquired and compared with corresponding histology systematically to aid in the identification of fine structural features and possible pathologies. With OCT imaging, we observed varied anatomy of the organ of Corti, and developed a cochlear "integrity" rating system to differentiate healthy appearing cochleae from various pathological states. These results demonstrate the capability of OCT to non-traumatically visualize cochlear integrity, highlighting its potential as a diagnostic tool. This work shows promise in translating the ability to determine the likelihood of existing or lack of hair cells and supporting cells in live patients, which would enable appropriate targeted treatments.

physiology↗

Mechanical force locally damages, remodels and stabilizes the lattice of spindle microtubules

To segregate chromosomes at cell division, the spindle must maintain its structure under force. How it does so remains poorly understood. To address this question, we use microneedle manipulation to apply local force to spindle microtubule bundles, kinetochore-fibers (k-fibers), inside mammalian cells. We show that local load directly fractures k-fibers, and that newly created plus-ends often have arrested dynamics, resisting depolymerization. Force alone, without fracture, is sufficient for spindle microtubule stabilization, as revealed by laser ablating k-fibers under local needle force. Doublecortin, which binds a compacted microtubule lattice, is lost around the force application site, suggesting local force-induced structural remodeling. In turn, EB1, which recognizes GTP-tubulin, is locally enriched at stabilization sites, both before and after force-induced fracture. Together, our findings support a model where force-induced damage leads to local spindle microtubule lattice remodeling and stabilization, which we propose reinforces the spindle where it experiences critical loads.

cell biology↗

NuMA mechanically reinforces the spindle independently of its partner dynein

Both motor and non-motor proteins organize microtubules to build the spindle and maintain it against opposing forces. NuMA, a long microtubule binding protein, is essential to spindle structure and function. NuMA recruits the motor dynein to spindle microtubule minus-ends to actively cluster them, but whether NuMA performs other spindle roles remains unknown. Here, we show that NuMA acts independently of dynein to passively reinforce the mammalian spindle. NuMA that cannot bind dynein is sufficient to protect spindle poles against fracture under external force. In contrast, NuMA with a shorter coiled-coil or disrupted self-interactions cannot protect spindle poles, and NuMA turnover differences cannot explain mechanical differences. In vitro, NuMAs C-terminus self-interacts and bundles microtubules without dynein, dependent on residues essential to pole protection in vivo. Together, this suggests that NuMA reinforces spindle poles by crosslinking microtubules, using its long coiled-coiled and self-interactions to reach multiple, far-reaching pole microtubules. We propose that NuMA acts as a mechanical "multitasker" targeting contractile motor activity and separately crosslinking microtubules, both functions synergizing to drive spindle mechanical robustness.

cell biology↗

Structural and functional insights into activation and regulation of the dynein-dynactin-NuMA complex

During cell division, NuMA orchestrates the focusing of microtubule minus-ends in spindle poles and cortical force generation on astral microtubules by interacting with dynein motors, microtubules, and other cellular factors. Here, we used in vitro reconstitution, cryo-electron microscopy, and live cell imaging to understand the mechanism and regulation of NuMA. We determined the structure of the processive dynein/dynactin/NuMA complex (DDN) and showed that the NuMA N-terminus drives dynein motility in vitro and facilitates dynein-mediated transport in live cells. The C-terminus of NuMA directly binds to and suppresses the dynamics of the microtubule minus-end. Full-length NuMA is autoinhibited for its interactions with dynein and microtubules, but mitotically phosphorylated NuMA activates dynein in vitro and interphase cells. Together with dynein, activated full-length NuMA focuses microtubule minus-ends into aster-like structures. These results provide critical insights into the activation of NuMA and dynein for their mitotic functions.

biophysics↗

Torques within and outside the human spindle balance twist at anaphase

At each cell division, nanometer-scale motors and microtubules give rise to the micron-scale spindle. Many mitotic motors step helically around microtubules in vitro, and most are predicted to twist the spindle in a left-handed direction. However, the human spindle exhibits only slight global twist, raising the question of how these molecular torques are balanced. Here, using lattice light sheet microscopy, we find that anaphase spindles in the epithelial cell line MCF10A have a high baseline twist, and we identify factors that both increase and decrease this twist. The midzone motors KIF4A and MKLP1 are redundantly required for left-handed twist at anaphase, and we show that KIF4A generates left-handed torque in vitro. The actin cytoskeleton also contributes to left-handed twist, but dynein and its cortical recruitment factor LGN counteract it. Together, our work demonstrates that force generators regulate twist in opposite directions from both within and outside the spindle, preventing strong spindle twist during chromosome segregation.

cell biology↗

Kinetochore-fiber lengths are maintained locally but coordinated globally by poles in the mammalian spindle

At each cell division, nanometer-scale components self-organize to build a micron-scale spindle. In mammalian spindles, microtubule bundles called kinetochore-fibers attach to chromosomes and focus into spindle poles. Despite evidence suggesting that poles can set spindle length, their role remains poorly understood. In fact, many species do not have spindle poles. Here, we probe the poles contribution to mammalian spindle length, dynamics, and function by inhibiting dynein to generate spindles whose kinetochore-fibers do not focus into poles, yet maintain a metaphase steady-state length. We find that unfocused kinetochore-fibers have a mean length indistinguishable from control, but a broader length distribution, and reduced length coordination between sisters and neighbors. Further, we show that unfocused kinetochore-fibers, like control, can grow back to their steady-state length if acutely shortened by drug treatment or laser ablation: they recover their length by tuning their end dynamics, albeit slower due to their reduced baseline dynamics. Thus, kinetochore-fiber dynamics are regulated by their length, not just pole-focusing forces. Finally, we show that spindles with unfocused kinetochore-fibers can segregate chromosomes but fail to correctly do so. We propose that mammalian spindle length emerges locally from individual k-fibers while spindle poles globally coordinate k-fibers across space and time.

cell biology↗

Motion of the Cochlear Reticular Lamina Varies Radially Across Outer-Hair-Cell Rows

The basilar membrane (BM) is connected to the reticular lamina (RL) through three rows of Y-shaped structures consisting of an outer hair cell (OHC) and a Deiters cell (DC) with a phalangeal process (PhP) that forms part of the RL mosaic surface. Morphological differences in the anatomy of the Y-shaped structures across the three OHC rows suggest differences in motion across the rows. Here we report OoC transverse motions measured across several radial locations for the gerbil basal region corresponding to ~45 kHz. Cross-sectional imaging and vibrometry measurements were made using a high-resolution (2.23 um axially in water) spectral-domain optical-coherence-tomography (SD-OCT) system. The stimuli were pure tones (2-63 kHz) at ear-canal sound pressure levels (SPLs) of 30-95 dB SPL in anesthetized gerbils (N=9) with healthy cochleae. We report displacements at the RL regions of OHC rows 1-3 (RL1-3), at the OHC-DC junctions of OHC rows 1-3 (OHC-DC-junction1-3), and at the arcuate zone, arcuate-pectinate junction, and pectinate zone of the BM (BMAZ, BMAPJ, and BMPZ, respectively). The in vivo BM displacements showed classic compressive nonlinearity and traveling-wave delays. The RL gain was similar to the BM gain at low frequencies (<20 kHz), but increased with frequency. Near the best frequency (BF), the RL gain was greater than the high-level BM gain by 40 {+/-}5 dB (mean{+/-}std), and had greater compressive nonlinearity. RL motion varied radially, and the RL3 gain was significantly greater than that of RL1 by 10 {+/-}1 dB (p<0.001). In contrast, the OHC-DC-junction gain varied little radially across OHCs. At low frequencies the OHC-DC-junction gain was constant across SPLs, and 14 {+/-} 3 dB greater than the BM gain. As the frequency increased, the OHC-DC-junction gain decreased to a level similar to the BM gain at BF. The RL2, 3 phase was advanced by 0.25-0.375 cycles relative to the BM phase at low frequencies, but the RL2, 3 phase lead decreased as the frequency increased, became similar to the BM phase at BF, and lagged behind the BM phase by 0.25-0.5 cycles above BF. The OHC-DC-junction phases were mostly similar to the BM phase at low frequencies, but became delayed relative to the BM as the frequency increased, typically by 0.25-0.5 cycles near BF and by up to 1 cycle above BF. Our results show the most detailed picture of motion around the three OHC rows yet published, indicating that RL motion varied radially. Surprisingly, there was little motion difference across the three OHC rows in the OHC-DC-junction region, indicating that the tops of the DCs move in unison. Our data show a rich array of OoC amplitude and phase variations that are not explained by current theories.

physiology↗

Watching Death in the Gerbil Cochlea Using Optical Coherence Tomography (OCT)

Because it is difficult to directly observe the morphology of the living cochlea, our ability to infer the mechanical functioning of the living ear has been limited. Nearly all of our knowledge about cochlear morphology comes from postmortem tissue that was fixed and processed using procedures that possibly distort the structures and fluid spaces of the organ of Corti. In this study, optical coherence tomography was employed to obtain in vivo and postmortem micron-scale volumetric images of the high-frequency hook region of the gerbil cochlea through the round-window membrane. The anatomical structures and fluid spaces of the organ of Corti were segmented and quantified in vivo and over a 90-minute postmortem period. The results show that some aspects of the organ of Corti are significantly altered over the course of death, such as the volumes of the fluid spaces, whereas the dimensions of other features change very little. We postulate that the fluid space of the outer tunnel and its surrounding tectal cells form a resonant structure that can affect the motion of the reticular lamina and thereby have a profound effect on outer-hair-cell transduction and thus cochlear amplification. In addition, the in vivo fluid pressure of the inner spiral sulcus is postulated to effectively inflate the connected sub-tectorial gap between the tectorial membrane and the reticular lamina. This gap height decreases after death, which is hypothesized to reduce and disrupt hair-cell transduction

neuroscience↗

OpenCell: proteome-scale endogenous tagging enables the cartography of human cellular organization

Elucidating the wiring diagram of the human cell is a central goal of the post-genomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery, and uncovers that RNA-binding proteins form a specific sub-group defined by unique interaction and localization properties. Furthermore, we discover that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions. Paired with a fully interactive website opencell.czbiohub.org, we provide a resource for the quantitative cartography of human cellular organization.

cell biology↗

Opposing motors provide mechanical and functional robustness in the human spindle

At each cell division, the spindle self-organizes from microtubules and motors. How the spindles diverse motors, often acting redundantly or in opposition, collectively give rise to its emergent architecture, mechanics, and function is unknown. In human spindles, the motors dynein and Eg5 generate contractile and extensile stress, respectively. Inhibiting dynein or its targeting factor NuMA leads to unfocused, turbulent spindles and inhibiting Eg5 leads to monopoles, yet bipolar spindles form when both are inhibited together. What, then, are the roles of these opposing motors? Here we generate NuMA/dynein- and Eg5-doubly inhibited spindles that not only attain a typical metaphase shape and size, but also undergo anaphase. However, these spindles have reduced microtubule dynamics and are mechanically fragile, fracturing under force. Further, they exhibit lagging chromosomes and dramatic left-handed twist at anaphase. Thus, while these opposing motor activities are not required for the spindles shape, they are essential to its mechanical and functional robustness. Together, this work suggests a design principle whereby opposing active stresses provide robustness to force-generating cellular structures.

cell biology↗

Prostate Epithelial Genes Define Docetaxel-Sensitive Prostate Cancer Molecular Subtype

BACKGROUND & OBJECTIVESAnalysis of the transcriptomic landscape of prostate adenocarcinoma shows multidimensional gene expression variability. Understanding cancer transcriptome complexity can provide biological insight and therapeutic guidance. To avoid potential confounding factors, such as stromal contamination and stress-related material degradation, we utilized a set of genes expressed by prostate epithelial cells from single-cell transcriptome data of the human prostate gland. MATERIALS & METHODSAnalyzing publicly available bulk and single-cell RNA sequencing data, we defined 1,629 genes expressed by prostate epithelial cells. Consensus clustering and CIBERSORT deconvolution were used for class discovery and proportion estimate analysis. The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) dataset served as a training set. The resulting clusters were analyzed in association with clinical, pathologic, and genomic characteristics and impact on survival. RESULTSTCGA-PRAD tumors were separated into four subtypes: A (30.0%), B (26.0%), C (14.7%), D (4.2%), and mixed (25.0%). Subtype A was characterized by low frequency of ETS-family fusions and high expression of KLK3, which encodes prostate-specific antigen (PSA). Subtype B showed the highest expression of ACP3, encoding PAP (prostatic acid phosphatase). Subtypes C and D were commonly associated with advanced T/N stages, high Gleason grades, and p53 or PIK3CA mutations. In silico drug-sensitivity screening suggested that subtype B is likely sensitive to docetaxel and paclitaxel. Serum PSA/PAP ratio was predictive of a radiographic response to docetaxel in metastatic castration-resistant prostate cancer patients. CONCLUSIONWe propose four prostate adenocarcinoma subtypes with distinct transcriptomic, genomic, and pathologic characteristics. PSA/PAP ratio in advanced cancer may aid in determining which patients would benefit from maximized androgen receptor inhibition or early use of antimicrotubule agents. Molecular subtypes and biomarkers must be validated in a prospective cohort study.

cancer biology↗

Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments

CRISPR/Cas technologies have transformed our ability to add functionality to the genome by knock-in of payload via homology-directed repair (HDR). However, a systematic and quantitative profiling of the knock-in integration landscape is still lacking. Here, we present a framework based on long-read sequencing and an integrated computational pipeline (knock-knock) to analyze knock-in repair outcomes across a wide range of experimental parameters. Our data uncover complex repair profiles, with perfect HDR often accounting for a minority of payload integration events, and reveal markedly distinct mis-integration patterns between cell-types or forms of HDR templates used. Our analysis demonstrates that the two sides of a given double-strand break can be repaired by separate pathways and identifies a major role for sequence micro-homology in driving donor mis-integration. Altogether, our comprehensive framework paves the way for investigating repair mechanisms, monitoring accuracy, and optimizing the precision of genome engineering.

genomics↗