Biology subjects

Efstathiou, C.

Publications and source records attributed to Efstathiou, C..

4 recordsLinked to original sources

bioRxiv2025

Dynein synergises with EB1 to facilitate cortex-microtubule encounter and proper spindle positioning in metaphase

To rotate the bulky mitotic spindle, astral microtubules are pulled by cortex-bound dynein. We investigate whether dynein regulates astral microtubules, independent of its classical microtubule-pulling role by combining super-resolution live-cell microscopy with optogenetic tools to disrupt and measure astral microtubule distributions and spindle movements. Localised photoinactivation of EB1 reveals that microtubule ends reaching distinct cortical regions differently influence spindle movements. Using a dynein inhibitor gradient, we separate dyneins role in spindle positioning (maintaining pole-to-cortex distance) from its role in directing spindle movements. EB1 photoinactivation of whole spindles uncovers a previously unrecognised synergistic role for dynein and EB1 in maintaining astral microtubules reaching the cortex. We demonstrate the EB1-dependent role for dynein in maintaining astral microtubule growth is independent of its classical cortical roles by analysing dynamic changes in spindle position, displacement rates and cortical dynein localisation and by depleting dyneins cortical platform, LGN. Our finding that Dynein synergises with EB1 to regulate astral microtubule, independent of cortical pulling, has implications for how spindle positioning is correctly established in cells. HIGHLIGHTSO_LISystematic quantification of spindle movements and microtubule lengths reveals dyneins dual role in microtubule growth and pulling. C_LIO_LIMicrotubules reaching distinct cortex regions differently regulate spindle movements. C_LIO_LIEB1 photoinactivation shows dynein promotes EB1-independent astral microtubule growth. C_LIO_LIReduced dynein causes shorter asters and asymmetric spindle positioning, without reducing spindle movements. C_LIO_LINoncortical dynein regulates cortex-microtubule encounter; cortical dynein controls spindle movement. C_LI

cell biology↗

bioRxiv2024

Multi-SpinX: An Advanced Framework for Automated Tracking of Mitotic Spindles and Kinetochores in Multicellular Environments

SpinX, an AI-guided spindle tracking software, allows the 3-dimensional (3D) tracking of metaphase spindle movements in mammalian cells. Using over 900 images of dividing cells, we create the Multi-SpinX framework to significantly expand SpinXs applications: a) to track spindles and cell cortex in multicellular environments, b) to combine two object tracking (spindle with kinetochores marked by centromeric probes) and c) to extend spindle tracking beyond metaphase to prometaphase and anaphase stages where spindle morphology is different. We have used a human-in-the-loop approach to assess our optimisation steps, to manually identify challenges and to build a robust computational pipeline for segmenting kinetochore pairs and spindles. Spindles of both H1299 and RPE1 cells have been assessed and validated for use through Multi-SpinX, and we expect the tool to be versatile in enabling quantitative studies of mitotic subcellular dynamics.

cell biology↗

bioRxiv2022

Synchronisation of apical constriction and cell cycle progression is a conserved behaviour of pseudostratified neuroepithelia informed by their tissue geometry

Neuroepithelial cells balance tissue growth requirement with the morphogenetic imperative of closing the neural tube. They apically constrict to generate mechanical forces which elevate the neural folds, but are thought to apically dilate during mitosis. However, we previously reported that mitotic neuroepithelial cells in the mouse posterior neuropore have smaller apical surfaces than non-mitotic cells. Here, we document progressive apical enrichment of non-muscle myosin-II in mitotic, but not non-mitotic, neuroepithelial cells with smaller apical areas. Live-imaging of the chick posterior neuropore confirms apical constriction synchronised with mitosis, reaching maximal constriction by anaphase, before division and re-dilation. Mitotic apical constriction amplitude is significantly greater than interphase constrictions. To investigate conservation in humans, we characterised early stages of iPSC differentiation through dual SMAD-inhibition to robustly produce pseudostratified neuroepithelia with apically enriched actomyosin. These cultured neuroepithelial cells achieve an equivalent apical area to those in mouse embryos. iPSC-derived neuroepithelial cells have large apical areas in G2 which constrict in M phase and retain this constriction in G1/S. Given that this differentiation method produces anterior neural identities, we studied the anterior neuroepithelium of the elevating mouse mid-brain neural tube. Instead of constricting, mid-brain mitotic neuroepithelial cells have larger apical areas than interphase cells. Tissue geometry differs between the apically convex early midbrain and flat posterior neuropore. Culturing human neuroepithelia on equivalently convex surfaces prevents mitotic apical constriction. Thus, neuroepithelial cells undergo high-amplitude apical constriction synchronised with cell cycle progression but the timing of their constriction if influenced by tissue geometry.

cell biology↗

bioRxiv2021

SpinX: Time-resolved 3D Analysis of Mitotic Spindle Dynamics using Deep Learning Techniques and Mathematical Modelling

Time-lapse microscopy movies have transformed the study of subcellular dynamics. However, manual analysis of movies can introduce bias and variability, obscuring important insights. While automation can overcome such limitations, spatial and temporal discontinuities in time-lapse movies render methods such as object segmentation and tracking difficult. Here we present SpinX, a framework for reconstructing gaps between successive frames by combining Deep Learning and mathematical object modelling. By incorporating expert feedback through selective annotations, SpinX identifies subcellular structures, despite confounding neighbour-cell information, non-uniform illumination and variable marker intensities. The automation and continuity introduced allows precise 3-Dimensional tracking and analysis of spindle movements with respect to the cell cortex for the first time. We demonstrate the utility of SpinX using distinct spindle markers and drug treatments. In summary, SpinX provides an exciting opportunity to study spindle dynamics in a sophisticated way, creating a framework for step changes in studies using time-lapse microscopy.

cell biology↗