Biology subjects

Draviam, V. M.

Publications and source records attributed to Draviam, V. M..

9 recordsLinked to original sources

bioRxiv2026

Phosphoinositide Variant Fuels 53BP1 Oligomerization and Higher-Order Assembly in the DNA Damage Response

53BP1 nuclear bodies are dynamic structures with properties resembling biomolecular condensates, but the molecular determinants that govern 53BP1 higher-order assembly at DNA double-strand breaks (DSBs) remain to be established. Here, we show that 53BP1 condensation is stimulated by phosphatidylinositol 3-phosphate (PI(3)P) in vitro through a highly specific interaction with its C-terminal tandem BRCT domain (tBRCT). Consequently, mutational inactivation of the 53BP1 tBRCT domain compromised PI(3)P binding and suppressed 53BP1 optodroplet formation in vivo. We further show that rapid 53BP1 clustering following DNA damage precedes its stable assembly on DSB-flanking chromatin, requires its tBRCT, and is suppressed by sequestration of nuclear PI(3)P. Taken together, our findings identify PI(3)P binding as a mechanism that promotes the formation and maturation of 53BP1 condensate-like assemblies on damaged chromatin.

cell biology↗

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↗

bioRxiv2025

Fluorescence recovery in the Super-Resolution regime reveals subcompartments of 53BP1 foci

We combine Lattice Structured Illumination Microscopy (diSIM with [~]60 nm resolution), Lattice Light-sheet microscopy and Fluorescence Recovery After Photobleaching (FRAP) to explore 53BP1 dynamics in Retinal Pigment Epithelial cells. 53BP1 forms liquid condensates during double-strand DNA repair, long-range DNA end-joining and heterochromatin maintenance. Our super-resolution movies reveal differences in 53BP1 foci contour: some foci are compact and stationary while others appear amorphous, dynamically changing shapes. To explore them, we developed FRAP in the Super-Resolution regime (FRAP-SR). 53BP1 foci with an amorphous loose contour display subcompartments that recover 53BP1-eGFP signals rapidly, indicating differential protein mobilities and 53BP1 functions within a single foci. In contrast, 53BP1-eGFP foci with a compact contour recover uniformly as single foci but show higher heterogeneity in 53BP1-eGFP recovery rates compared to foci that recover as multiple subcompartments. In cells released from aphidicolin, amorphous foci show faster 53BP1 recovery compared to compact foci. We discuss the conceptual implications of different 53BP1 mobilities, and how the FRAP-SR method transforms studies of dynamic 60-100 nm structures.

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

Human genetic variations reveal Chromosomal Instability aiding Variants (CIVa) in kinetochore-microtubule associated proteins

The vast majority of Chromosomal Instability (CIN) promoting mutations remain unknown. We assess the prevalence of Chromosomal Instability aiding Variants (CIVa) by collating Loss-of-Function (LoF) variants predicted in 135 chromosome segregation genes from over 150,000 humans, including consanguineous individuals. Surprisingly, we observe heterozygous and homozygous CIVa in Astrin and SKA3 genes that encode evolutionarily conserved microtubule-associated proteins essential for chromosome segregation. By combining high-resolution microscopy and controlled protein expression, we show the naturally occurring Astrin variant, p.Q1012*, as potentially harmful because it fails to localise normally, delays anaphase onset, induces chromosome misalignment and promotes chromosome missegregation. We show that N-terminal frameshift variants in Astrin and SKA3 are likely to generate shorter isoforms that do not compromise chromosome segregation revealing resilient mechanisms to cope with harmful variants. This study provides a framework to predict and stratify naturally occurring CIVa, an important step towards precision medicine for CIN syndromes.

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↗

bioRxiv2020

Cells protect chromosome-microtubule attachments, independent of biorientation, using an Astrin-PP1 and CyclinB-CDK1 feedback loop

Defects in chromosome-microtubule attachment can cause chromosomal instability, associated with infertility and aggressive cancers. Chromosome-microtubule attachment is mediated by a large macromolecular structure, the kinetochore. Kinetochore pairs are bioriented and pulled by microtubules from opposing spindle poles to ensure the equal segregation of chromosomes. Kinetochore-microtubule attachments lacking opposing-pull are detached by Aurora-B/Ipl1; yet, how mono-oriented attachments that are a prerequisite for biorientation, but lacking opposing-pull are spared is unclear. Using an RNAi-mediated screen, we uncover a unique role for the Astrin-SKAP complex in protecting mono-oriented attachments. We provide the first evidence for how a microtubule-end associated protein senses outer-kinetochore changes specific to end-on attachments and assembles into an outer kinetochore crescent to stabilise mature attachments. We find that Astrin-PP1 and Cyclin-B-CDK1 activities counteract each other to preserve mono-oriented attachments. Thus, cells are not only surveying chromosome-microtubule attachment errors, but they are also actively sensing and stabilising mature attachments independent of biorientation.

cell biology↗

bioRxiv2020

Multinucleation Associated DNA Damage causes quiescence despite compromised p53

Nuclear atypia is one of the earliest hallmarks of cancer progression. How distinct forms of nuclear atypia differently impact cell fate is not understood at the molecular level. Here, we perform single-cell tracking studies to determine the immediate and long-term impact of multinucleation or misshapen nuclei and reveal a significant difference between multinucleation and micronucleation, a catastrophic nuclear atypia known to promote genomic rearrangements and tumour heterogeneity. Tracking the fate of newborn cells exhibiting various nuclear atypia shows that multinucleation, unlike other forms of nuclear atypia, blocks proliferation in p53-compromised cells. Because compromised p53 is seen in over 50% of cancers, we explored how multinucleation blocks proliferation and promotes quiescence. Multinucleation increases 53BP1-decorated nuclear bodies (DNA damage repair platforms), along with a heterogeneous reduction in transcription and protein accumulation across the multi-nucleated compartments. Importantly, Multinucleation Associated DNA Damage (MADD) associated 53BP1-bodies remain unresolved for days, despite an intact NHEJ machinery that repairs laser-induced DNA damage within minutes. This persistent MADD signalling blocks the onset of DNA replication and is associated with driving proliferative G1 cells into quiescence, revealing a novel replication stress independent cell cycle arrest caused by mitotic lesions. These findings call for segregating protective and prohibitive nuclear atypia to inform therapeutic approaches aimed at limiting tumour heterogeneity.

cell biology↗

bioRxiv2018

MARK2/Par1b present at retraction fibres corrects spindle off-centering induced by actin disassembly

Tissue maintenance requires adequate cell proliferation and a directed plane of cell division. Retraction fibres can determine the plane of cell division by directing spindle movements; however, retraction fibre components that direct spindle movements remain unclear. We report MARK2/Par1b kinase as a novel component of actin-rich retraction fibres, important for directed spindle movements. A kinase-dead mutant of MARK2 reveals MARK2s ability to monitor actin status. MARK2s localisation at retraction fibres, but not the rest of the cortical membrane or centrosome, is dependent on its kinase activity, highlighting a specialised spatial regulation of MARK2. By subtly perturbing the actin cytoskeleton, we demonstrate MARK2s role in correcting spindle off-centering, induced by lesions in actin assembly. In addition to this mitotic role, we show MARK2s post-mitotic role in ensuring normal G1-S progression and cell proliferation. We propose that MARK2 provides a molecular framework to integrate cortical signals and cytoskeletal changes in both mitosis and interphase.\n\nShort SummaryCoordination of cell proliferation and division is important for tissue maintenance. We report a regulated localisation for MARK2 in mitosis and interphase. We demonstrate its mitotic role in correcting spindle positioning defects and its interphase role in G1-S transition.

cell biology↗