Biology subjects

Conway, W.

Publications and source records attributed to Conway, W..

7 recordsLinked to original sources

bioRxiv2026

Eg5 activity and density-driven bundling organize the human metaphase mitotic spindle independently of spindle bipolarity

The mitotic spindle segregates chromosomes through the coordinated actions of microtubules and molecular motors. Classic models propose that microtubules nucleate at spindle poles and grow inward to capture chromosomes; however, recent structural studies show that spindles contain short microtubules that do not span the distance between poles and chromosomes. It is unclear how these short microtubules assemble a bipolar spindle. Using cryo-electron tomography to map microtubule polarity in human metaphase spindles, we find that microtubules form locally antiparallel bundles with consistent 8 nm wall-to-wall spacing. We utilized motor perturbations and centriole depletion, which generated motor-active monopolar spindles, to reveal that the kinesin-5 motor Eg5 organizes local antiparallel overlap independently of spindle bipolarity. We found that bundles are organized by density-driven steric interactions rather than motor-mediated crosslinking. These findings support a self-organized, bottom-up model in which local microtubule-motor interactions within dense bundles generate forces that build the bipolar spindle, challenging pole-centric models. KEY FINDINGSO_LIMicrotubules in metaphase spindles organize in locally antiparallel bundles with consistent 8 nm wall-to-wall spacing. C_LIO_LIThe Eg5 motor generates antiparallel microtubule overlap independently of spindle bipolarity. C_LIO_LIMicrotubule spacing scales with density through steric interactions, not direct motor crosslinking. C_LIO_LIDynein regulates microtubule density to control bundle architecture; balanced Eg5-dynein density regulation maintains spindle bipolarity. C_LI

biophysics↗

bioRxiv2024

Accurate and fast segmentation of filaments and membranes in micrographs and tomograms with TARDIS

Segmentation of macromolecular structures is the primary bottleneck for studying biomolecules and their organization with electron microscopy in 2D/3D - requiring months of manual effort. Transformer-based Rapid Dimensionless Instance Segmentation (TARDIS) is a deep learning framework that automatically and accurately annotates membranes and filaments. Pre-trained TARDIS models can segment electron tomography (ET) reconstructions from both 3D and 2D electron micrographs of cryo and plastic-embedded samples. Furthermore, by implementing a novel geometric transformer architecture, TARDIS is the only method to provide accurate instance segmentations of these structures. Reducing the annotation time for ET data from months to minutes, we demonstrate segmentation of membranes and filaments in over 13,000 tomograms in the CZII Data Portal. TARDIS thus enables quantitative biophysical analysis at scale for the first time. We show this in application to kinetochore-microtubule attachment and viral-membrane interactions. TARDIS can be extended to new biomolecules and applications and open-source at https://github.com/SMLC-NYSBC/TARDIS.

bioinformatics↗

bioRxiv2023

Dual mechanism of kinetochore microtubule detachment

During eukaryotic cell division, microtubules connect to chromosomes by attaching to the kinetochore via the NDC80 complex (NDC80c). Improperly attached kinetochore microtubules (KMTs) are selective detached from the kinetochore. While the proper regulation of KMT detachments is crucial for accurate chromosome segregation, the manner by which KMT detachments are controlled by the biophysical properties of NDC80 and KMTs remains poorly understood. Here, we investigate the mechanism of KMT detachment by combining quantitative measurements of NCD80c-KMT binding and KMT detachments, with FLIM-FRET and photoconversion, and mathematical modeling. We find that individual NDC80c at kinetochores bind noncooperatively to KMTs, with a free energy difference between bound and unbound NDC80c of -1.0 {+/-} 0.1kBT, and we determine that each phosphorylated residue of NDC80c decreases the NDC80c-KMT binding energy by 0.35 {+/-} 0.03kBT. We show that the energetics of NDC80c binding can be related to KMT detachment by a dual detachment mechanism in which KMTs detach from kinetochores when either 1) all NDC80c spontaneously unbind from the KMT or 2) following KMT catastrophe. We find that the affinity of NDC80c for KMTs is reduced at low-tension, non-bioriented kinetochores due to centromere-localized Aurora B phosphorylating the NDC80c, resulting in an elevated detachment rate for the associated KMTs. Taken together, this work leads to a multiscale, bottom-up biophysical model for how the energetics of NDC80c-KMT interactions impact KMT detachments and provides an understanding of the molecular basis of their regulation during mitotic error correction. SIGNIFICANCE STATEMENTChromosome segregation errors can lead to aneuploidy, birth defects and cancer. Accurate cellular division, where one copy of each chromosome is segregated to each daughter cell, depends on regulation of attachments between chromosomes and microtubules in the mitotic spindle. While extensive work has identified proteins involved in suppressing mitotic errors, it is still unclear how incorrect chromosome-microtubule attachments which result in mitotic errors are selectively removed. Here, we employed a combination of quantitative live-cell fluorescence imaging, molecular perturbations, and mathematical modeling to elucidate the biophysical mechanism of how microtubules are selectively detached from misaligned mitotic chromosomes. This work leads to quantitative understanding of how chromosome attachments are regulated and mitotic errors are corrected.

biophysics↗

bioRxiv2021

Three-dimensional structure of the kinetochore-fibers in human mitotic spindles

During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the chromosomes and the rest of the spindle. KMTs in mammalian cells are organized into bundles, so-called kinetochore-fibers (k-fibers), but the ultrastructure of these fibers is currently not well characterized. Here we show by large-scale electron tomography that each k-fiber in HeLa cells in metaphase is composed of approximately nine KMTs, only half of which reach the spindle pole. Our comprehensive reconstructions allowed us to analyze the three-dimensional (3D) morphology of k-fibers and their surrounding MTs in detail. We found that k-fibers exhibit remarkable variation in circumference and KMT density along their length, with the pole-proximal side showing a broadening. Extending our structural analysis then to other MTs in the spindle, we further observed that the association of KMTs with non-KMTs predominantly occurs in the spindle pole regions. Our 3D reconstructions have implications for KMT growth and k-fiber self-organization models as covered in a parallel publication applying complementary live-cell imaging in combination with biophysical modeling (Conway et al., 2022). Finally, we also introduce a new visualization tool allowing an interactive display of our 3D spindle data that will serve as a resource for further structural studies on mitosis in human cells.

cell biology↗

bioRxiv2021

Self-organization of kinetochore-fibers in human mitotic spindles

During eukaryotic cell division, chromosomes are linked to microtubules (MTs) in the spindle by a macromolecular complex called the kinetochore. The bound kinetochore microtubules (KMTs) are crucial to ensuring accurate chromosome segregation. Recent electron tomography reconstructions (Kiewisz et al. 2021) captured the positions and configurations of every MT in human mitotic spindles, revealing that many KMTs in these spindles do not reach the pole. Here, we investigate the processes that give rise to this distribution of KMTs using a combination of analysis of the electron tomography reconstructions, photoconversion experiments, quantitative polarized light microscopy, and biophysical modeling. Our results indicate that in metaphase, KMTs grow away from the kinetochores along well-defined trajectories, continually decreasing in speed as they approach the poles. The locations of KMT minus ends, and the turnover and movements of tubulin in KMTs, are consistent with models in which KMTs predominately nucleate de novo at kinetochores and are inconsistent with substantial numbers of non-KMTs being recruited to the kinetochore in metaphase. Taken together, this work leads to a mathematical model of the self-organization of kinetochore-fibers in human mitotic spindles.

biophysics↗

bioRxiv2021

Metabolic state of human blastocysts measured by fluorescence lifetime imaging microscopy

Mammalian embryos undergo large changes in metabolism over the course of preimplantation development. Embryo metabolism has long been linked to embryo viability, suggesting its potential utility in Assisted Reproductive Technologies (ART) to aid in selecting high quality embryos. However, the metabolism of human embryos remains poorly characterized due to a lack of non-invasive methods to measure their metabolic state. Here, we explore the application of metabolic imaging via fluorescence lifetime imaging microscopy (FLIM) for studying human blastocysts. We use FLIM to measure the autofluorescence of two central coenzymes, NAD(P)H and FAD+, in 215 discarded human blastocysts from 137 patients. We find that FLIM is sensitive enough to detect significant metabolic differences between blastocysts. We show that the metabolic state of human blastocysts changes continually over time, and that variations between blastocyst are partially explained by both the time since fertilization and their developmental stage, but not their morphological grade. We also observe significant metabolic heterogeneity within individual blastocysts, including between the inner cell mass and the trophectoderm, and between the portions of hatching blastocysts within and without the zona pellucida. Taken together, this work reveals novel aspects of the metabolism of human blastocysts and suggests that FLIM is a promising approach to assess embryo viability through non-invasive, quantitative measurements of their metabolism.

cell biology↗

bioRxiv2017

Cell-cycle dependent organization and dynamics of RNA Polymerase I in live human cells

RNA Polymerase I (Pol I) is responsible for over 60% of transcriptional output in human cells, yet basic questions concerning the spatial and temporal organization of the polymerase remain unanswered. Here we investigate how mammalian cells rely on Pol I organization throughout the cell cycle to balance different needs, from complete transcription shut down to massive increase in protein synthesis (and thus ribosomal RNA synthesis) before cell division. In contrast to our previous reports on RNA Polymerase II, Pol I clusters are stable with active transcription, and the presence of transient Pol I clusters correlates with inactive ribosomal transcription. Our results suggest that both stable and transient populations Pol I clusters co-exist in individual living cells, and their relative fraction may directly reflect the global gene expression need of the cell.

biophysics↗