Biology subjects

Lakadamyali, M.

Publications and source records attributed to Lakadamyali, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Reprogramming Dedifferentiation Regulatory Networks Preserves Human Chondrocyte Phenotypes

Chondrocyte-based cartilage repair strategies such as autologous chondrocyte implantation (ACI) require extensive in vitro expansion to obtain clinically relevant cell numbers. However, this expansion step progressively drives chondrocyte dedifferentiation, reducing matrix-forming capacity and contributing to variable repair outcomes. To better understand this process, we used single-nucleus multiome profiling (snRNA-Seq + snATAC-Seq) to define the transcriptional and chromatin accessibility programs underlying human chondrocyte dedifferentiation during expansion. Multiome integration across passages revealed a continuous dedifferentiation trajectory accompanied by coordinated remodeling of gene expression and chromatin accessibility, identifying chromatin destabilization as an early regulatory event during phenotype loss. Guided by these regulatory signatures, we screened available small-molecule inhibitors targeting candidate pathways and found that Fludarabine most consistently preserved chondrocyte identity during early expansion. Fludarabine was associated with suppression of STAT1-related programs and early stabilization of the chromatin landscape prior to broader transcriptional recovery. Functionally, treated cells demonstrated enhanced matrix-forming capacity in chondrogenic pellet culture and significantly increased nascent protein synthesis in 3D hydrogel culture, with biosynthetic output approaching unexpanded controls by day 21. Together, these findings identify chromatin stability as a key regulatory determinant of expansion-associated chondrocyte dedifferentiation and establish a pharmacologic strategy to preserve chondrocyte functional potency during cell manufacturing for cartilage repair.

bioengineering↗

bioRxiv2026

Expansion Microscopy reveals changes in cellular and extracellular structures between healthy and perturbed tendons

Tendons transmit mechanical forces between muscles and bones through their highly aligned, collagen-rich extracellular matrix. When damaged, resident cells help restore this matrix. However, in tendinopathies, this repair response fails, leading to loss of proper tendon function. How altered mechanical states reshape tendon cell and matrix architecture remains poorly understood because existing methods do not readily capture tendon structure across relevant length scales. Here, we combine Fluorescent Labeling of Abundant Reactive Entities (FLARE) with Expansion Microscopy (ExM) to visualize cellular and extracellular structures in native tendon tissue. FLARE-ExM resolves the dense fibrillar matrix across multiple tendon types, including elastic fibers and glycan-rich cellular protrusions. In a tendon resection model, acute loss-of-tension was associated with increased fibril width and expansion of carbohydrate- and protein-rich regions. In ruptured human Achilles tendon, FLARE-ExM revealed extracellular disorganization and disrupted cellular architecture. These results establish FLARE-ExM as a useful approach for studying how mechanical perturbation remodels tendon architecture across physiological and disease contexts.

physiology↗

bioRxiv2026

Active transport of tRNAs facilitates distributed protein synthesis

Large, polarized cells such as cardiomyocytes, skeletal myofibers, and neurons rely on localized protein synthesis to sustain size, remodel and adapt to stress. The subcellular architecture of these cells is also inherently unfavorable for long-range, diffusion-based transport, which may promote their heavy reliance on active transport mechanisms for the localization of RNA and proteins. Transfer RNAs (tRNAs) function as essential regulators of protein synthesis by linking transcription and translation. Since their discovery in the 1950s, tRNA subcellular distribution has been assumed to occur through passive diffusion. Here, we report that there are pools of tRNAs that depend on the microtubule network for distribution in cardiomyocytes, skeletal myofibers and neurons. Employing dual-color live and fixed-cell super-resolution imaging, we demonstrate that active transport of tRNAs involves hitchhiking on the exterior of endolysosomal vesicles (ELV). We establish that leucyl-tRNA synthetase (LeuRS), the tRNA-binding protein that charges leucine to its cognate tRNA and interacts with Rag GTP on the surface of ELVs, is essential for tRNA transport. Disruption of LeuRS-ELV interactions is sufficient to impair long-range, microtubule-dependent tRNA transport, without affecting mRNA or rRNA transport. We also show that preventing tRNA transport is sufficient to impair translation at sites distal from the nucleus as well as globally impair protein synthesis, ultimately reducing cell size. These findings redefine tRNAs as actively trafficked cargo and establish their directed transport as a fundamental layer of translation regulation required for myocyte homeostasis. Highlights1. Pools of tRNAs require an intact microtubule network for distribution in large, complex cells. 2. tRNAs undergo active transport by hitchhiking on endolysosomal vesicles trafficked by microtubule motors. 3. The tRNA synthetase LeuRS functions as a tRNA-specific adaptor linking tRNAs to endolysosomal vesicles, and disrupting this interaction selectively impairs tRNA, but not mRNA or rRNA transport. 4. Loss of tRNA transport reduces distal translation, global protein synthesis, and cell size.

cell biology↗

bioRxiv2025

Sparse delivery of Synaptobrevin-2 to neurons using extracellular vesicles

Synaptobrevin-2 (Syb2) is an essential SNARE protein for neurotransmitter release and communication in the nervous system. We previously showed that Syb2 is also exchanged among neurons via extracellular vesicles (EVs). Host neurons can rapidly incorporate exogenous Syb2 into their synaptic vesicle cycle to support neurotransmitter release, however the endocytic mechanism of Syb2-containing EVs is unknown. Here, we use a fusion of Syb2 with the pH-sensitive GFP (Syb2-pHluorin) to track the incorporation of Syb2-containing EVs into neurons and the trafficking of exogenous Syb2 to synaptic vesicles at synapses. We determined that Syb2-containing EVs are endocytosed via Clathrin- and Dynamin-independent pathways. Moreover, Syb2-containing EVs are directly uptaken by axons and are rapidly incorporated into functional synaptic vesicles. These Syb2-pHluorin positive synaptic vesicles are endocytosed with either ultrafast (<1s) or fast ([~]1-3s) kinetics during synaptic transmission, suggesting limited diffusion and high fidelity in the fast retrieval of synaptic vesicle molecules immediately after fusion. This work introduces a novel application of EVs as vehicles to deliver fluorescent molecules in a neuron-specific, targeted manner to investigate protein transport mechanisms without overexpression artifacts. Our findings expand our understanding of the mechanisms EVs use to enter neurons.

cell biology↗

bioRxiv2025

SWING domains prime chromatin for nuclear body mediated gene regulation

Nuclear speckles have long been recognized as RNA-rich nuclear bodies, yet their role in genome organization and gene regulation remains incompletely understood. Using a rapid dTAG-mediated degradation system to simultaneously deplete SON and SRRM2--the core structural components of nuclear speckles--we identify a novel class of genomic regions, which we term SWING regions. Upon speckle disruption, SWING regions relocate to the nuclear periphery and acquire repressive histone marks such as H3K9me3, accompanied by gene downregulation, particularly of genes involved in developmental pathways. Consistent with this, depletion of Lamin A reduces lamina association of SWING regions and enhances their association with nuclear speckles, supporting a bidirectional balance between these two nuclear compartments. Notably, human mutations in SON and SRRM2 are associated with neurodevelopmental disorders characterized by intellectual disability and global developmental delay. Patient-derived cells bearing such mutations similarly exhibit SWING-region relocalization and gene repression, underlining a role for speckles in developmental gene regulation. We also report that drug-induced speckle rejuvenation partially rescues aberrant SWING-region localization to the nuclear lamina in patient-derived cells and iPSCs with acute depletion of SON. These findings identify SWING regions as an intermediate chromatin state positioned between nuclear speckles and the lamina, maintained by opposing functions from both structures. Our work reveals a mechanism underlying the contribution of nuclear bodies to 3D genome organization, highlights the importance of nuclear speckles and SWING regions in developmental regulation, and provides a potential therapeutic intervention in speckle dysfunction. Impact StatementIdentification of nuclear speckles as determinants of specific 3D genome organization. Demonstration of functional interactions between opposing nuclear structures (speckle versus lamina) through SWING regions. Establishment of developmental and human disease relevance of speckle-mediated genome organization. Providing potential avenues for therapeutic intervention in speckleopathies.

genetics↗

bioRxiv2025

Determination of α-Synuclein Protein Interactions by μMap Photo-proximity Labeling

Fibrillar aggregates of the natively disordered protein -synuclein (S) are hallmarks of Parkinsons disease and related neurodegenerative disorders termed synucleinopathies. Here, we used micromap ({micro}Map) photo-proximity labeling to determine the interactomes of S monomers and fibrils in mouse brain lysate to better understand both the loss of healthy function and gain of toxic function aspects of synucleinopathies. Several S variants were synthesized and characterized, showing that the small size (1 kDa) of the Ir catalyst attached through a Cys-maleimide linkage makes it minimally-perturbing to S, with a narrow labeling radius that allows one to identify interactome differences between different regions of S. Monomer and fibril interactomes were compared to each other and to previous proximity labeling data sets for validation and several examples of further investigations are demonstrated, including Western blotting, super-resolution microscopy, and {micro}Map in primary neurons.

biochemistry↗

bioRxiv2025

O-SNAP: A comprehensive pipeline for spatial profiling of chromatin architecture

We present O-SNAP (Objective Single-Molecule Nuclear Architecture Profiler), a comprehensive pipeline for the automated extraction, comparison, and classification of nuclear features from single-molecule localization microscopy (SMLM) data. O-SNAP quantifies 144 interpretable, biologically grounded spatial features describing chromatin organization or histone mark distributions at nanoscale resolution. The pipeline includes modules for pairwise comparison of features using volcano plots, feature set enrichment analysis, robust feature selection and classification of cell states, and pseudotime trajectory inference. We validate O-SNAP across diverse biological contexts, including fibroblast-to-stem cell reprogramming, tendon disease, histone variant sensitivity to oxidative stress, and chondrocyte de-differentiation, demonstrating its ability to detect subtle changes in nanoscale chromatin organization across diverse biological transitions.

biophysics↗

bioRxiv2024

Chromatin Compaction Follows a Power Law Scaling with Cell Size from Interphase Through Mitosis

Coordination of mitotic chromosome compaction with cell size is crucial for proper genome segregation during mitosis. During development, DNA content remains constant but cell size evolves, necessitating a mechanism that scales chromosome compaction with cell size. In this study, we examined chromatin compaction in the developing Drosophila nervous system by analyzing the large neuronal stem cells and their smaller progeny, the ganglion mother cells. Using super-resolution 3D Stochastic Optical Reconstruction Microscopy and quantitative time-lapse fluorescence microscopy, we observed that nanoscale chromatin density during interphase scales with nuclear volume according to a power law. This scaling relationship is disrupted by inhibiting histone deacetylase activity, indicating that molecular cues rather than mechanical constraints primarily regulate chromatin compaction. Notably, this power law dependency is maintained into mitosis but the scaling exponent decreases, suggesting phase separation of chromatin events during mitotic compaction. We propose that the scaling of mitotic chromosome size relative to cell size depends on the power law behaviour of interphase chromatin volume, and that scaling of mitotic chromatin compaction is an emergent property of linear polymers undergoing phase separation with their solvent. Statement of SignificanceUnderstanding how chromatin compaction changes with cell size is essential for understanding the mechanisms ensuring accurate genome segregation during cell division. In this study, we combine Voronoi tessellation analysis of Stochastic Optical Reconstruction Microscopy and image processing methods based on fluorescence intensity spectrum analysis of live confocal images to measure chromatin volume in interphase and mitosis in cells of different sizes. This work reveals that chromatin density in Drosophila neuronal cells follows a power law relationship with nuclear volume from interphase through mitosis, suggesting a fundamental principle underlying chromosome scaling across different cell sizes. We show that this scaling is regulated by molecular cues, such as histone deacetylase activity, and is conserved through a potential phase separation during mitosis, providing new insights into the biophysical processes that govern chromatin organization. This work broadens our understanding of chromosome biology and will have implications for understanding size-dependent chromatin dynamics in other organisms.

cell biology↗

bioRxiv2024

Polymer Model Integrates Super-Resolution Imaging and Epigenomic Sequencing to Elucidate the Role of Epigenetic Reactions in Shaping 4D Chromatin Organization

Chromatin, with its complex spatial and temporal organization, plays a crucial role in regulating gene expression. Recent advancements in super-resolution microscopy have revealed that nanoscale domains of heterochromatin (repressed segments) embedded within a euchromatin (active segments) background are fundamental units of 3D chromatin organization. In tissue-resident cells, the size of these heterochromatin domains varies with the microenvironment, particularly its stiffness, and chromatin organization is also influenced by pharmacological and epigenetic drugs. However, the mechanisms governing heterochromatin domain size under various conditions and their impact on gene expression remain unclear. To address this knowledge gap, we have developed a dynamic, next-generation sequencing informed chromatin copolymer model. Our model simulates the spatiotemporal evolution of chromatin, driven by passive diffusion and active epigenetic reactions, which interconvert euchromatin and heterochromatin. By integrating chromatin-chromatin interaction energetics and diffusion-reaction dynamics, we predict the formation of nanoscale heterochromatin-rich domains and establish a scaling relationship between their size and the modulation of epigenetic reaction rates. Additionally, our model predicts that epigenetic and chromatin compaction changes in response to changes in global reaction rates occur predominantly at domain boundaries. We validated these predictions via Hi-C contact map analysis and super-resolution imaging of hyperacetylated melanoma cells. Subsequent RNA-seq analysis suggested a pivotal role of these epigenetic shifts in influencing the metastatic potential of these cells. We further validated our mesoscale findings against chromatin rearrangement in hMSCs, which exhibit sensitivity of epigenetic reaction rates to changes in microenvironmental stiffness. Finally, we evaluated the effects of cycling of epigenetic reaction rates in silico, mimicking the cellular transition to different extracellular conditions, and back again. This finding reveals a cell-type invariant mechanism driven by domain boundaries, whereby chromatin organization guides epigenetic memory formation. Our findings show that chromatin reorganization in response to changes in epigenetic reaction rates resulting from alterations in the microenvironment, drug exposure and disease progression impacts both immediate cellular responses and long-term epigenetic memory.

biophysics↗

bioRxiv2024

A retrograde transit filter mediated by optineurin controls mitostasis in distal axons

The mechanisms of mitochondrial homeostasis in neurons-- mitostasis--are enigmatic and disease-prone, given a neurons large synaptic pool of mitochondria. To better understand mitostasis in mature mouse motor axons, we used ex vivo optical pulse-chase measurements of mitochondrial volume flux and identified a reiterative degradation system near presynaptic terminals and distal paranodes, which accounts for 75% of mitochondrial turnover in synapses. This distal filter system captures dysfunctional mitochondria from the retrograde stream and redirects them for lysosomal degradation. It depends on optineurin, a motor neuron disease-related mitophagy adaptor, but not on PINK1 and parkin, implicating a non-canonical mitophagy pathway. In presymptomatic motor neuron disease models, where retrograde transport is disrupted, the fraction of removed mitochondria is increased. Thus, we identify a new mitostasis system in distal axons with a cascade of checkpoints for local mitophagy, which normally maintains mitochondrial mass balance but is disrupted early in degenerative axonopathies.

cell biology↗

bioRxiv2024

Revealing the Biophysics of Lamina-Associated Domain Formation by Integrating Theoretical Modeling and High-Resolution Imaging

The interactions between chromatin and the nuclear lamina orchestrate cell type-specific gene activity by forming lamina-associated domains (LADs) which preserve cellular characteristics through gene repression. However, unlike the interactions between chromatin segments, the strength of chromatin-lamina interactions and their dependence on cellular environment are not well understood. Here, we develop a theory to predict the size and shape of peripheral heterochromatin domains by considering the energetics of chromatin-chromatin interactions, the affinity between chromatin and the nuclear lamina and the kinetics of methylation and acetylation9in human mesenchymal stem cells (hMSCs). Through the analysis of super-resolution images of peripheral heterochromatin domains using this theoretical framework, we determine the nuclear lamina-wide distribution of chromatin-lamina affinities. We find that the extracted affinity is highly spatially heterogeneous and shows a bimodal distribution, indicating regions along the lamina with strong chromatin binding and those exhibiting vanishing chromatin affinity interspersed with some regions exhibiting a relatively diminished chromatin interactions, in line with the presence of structures such as nuclear pores. Exploring the role of environmental cues on peripheral chromatin, we find that LAD thickness increases when hMSCs are cultured on a softer substrate, in correlation with contractility-dependent translocation of histone deacetylase 3 (HDAC3) from the cytosol to the nucleus. In soft microenvironments, chromatin becomes sequestered at the nuclear lamina, likely due to the interactions of HDAC3 with the chromatin anchoring protein LAP2{beta} ,increasing chromatin-lamina affinity, as well as elevated levels of the intranuclear histone methylation. Our findings are further corroborated by pharmacological interventions that inhibit contractility, as well as by manipulating methylation levels using epigenetic drugs. Notably, in the context of tendinosis, a chronic condition characterized by collagen degeneration, we observed a similar increase in the thickness of peripheral chromatin akin to that of cells cultured on soft substrates consistent with theoretical predictions. Our findings underscore the pivotal role of the microenvironment in shaping genome organization and highlight its relevance in pathological conditions.

biophysics↗

bioRxiv2024

Super-Resolution Imaging Uncovers Nanoscale Tau Aggregate Hyperphosphorylation Patterns in Human Alzheimers Disease Brain Tissue

Tau aggregation plays a critical role in Alzheimers Disease (AD), where tau neurofibrillary tangles (NFTs) are a key pathological hallmark. While much attention has been given to NFTs, emerging evidence underscores nano-sized pre-NFT tau aggregates as potentially toxic entities in AD. By leveraging DNA-PAINT super-resolution microscopy, we visualized and quantified nanoscale tau aggregates (nano-aggregates) in human postmortem brain tissues from intermediate and advanced AD, and Primary Age-Related Tauopathy (PART). Nano-aggregates were predominant across cases, with AD exhibiting a higher burden compared to PART. Hyperphosphorylated tau residues (p-T231, p-T181, and p-S202/T205) were present within nano-aggregates across all AD Braak stages and PART. Moreover, nano-aggregates displayed morphological differences between PART and AD, and exhibited distinct hyperphosphorylation patterns in advanced AD. These findings suggest that changes in nano-aggregate morphology and hyperphosphorylation patterns may exacerbate tau aggregation and AD progression. The ability to detect and profile nanoscale tau aggregates in human brain tissue opens new avenues for studying the molecular underpinnings of tauopathies.

biophysics↗

bioRxiv2024

Multiplexed DNA-PAINT Imaging of the Heterogeneity of Late Endosome/Lysosome Protein Composition

Late endosomes/lysosomes (LELs) are crucial for numerous physiological processes and their dysfunction is linked to many diseases. Proteomic analyses have identified hundreds of LEL proteins, however, whether these proteins are uniformly present on each LEL, or if there are cell-type dependent LEL sub-populations with unique protein compositions is unclear. We employed a quantitative, multiplexed DNA-PAINT super-resolution approach to examine the distribution of six key LEL proteins (LAMP1, LAMP2, CD63, TMEM192, NPC1 and LAMTOR4) on individual LELs. While LAMP1 and LAMP2 were abundant across LELs, marking a common population, most analyzed proteins were associated with specific LEL subpopulations. Our multiplexed imaging approach identified up to eight different LEL subpopulations based on their unique membrane protein composition. Additionally, our analysis of the spatial relationships between these subpopulations and mitochondria revealed a cell-type specific tendency for NPC1-positive LELs to be closely positioned to mitochondria. Our approach will be broadly applicable to determining organelle heterogeneity with single organelle resolution in many biological contexts. SummaryThis study develops a multiplexed and quantitative DNA-PAINT super-resolution imaging pipeline to investigate the distribution of late endosomal/lysosomal (LEL) proteins across individual LELs, revealing cell-type specific LEL sub-populations with unique protein compositions, offering insights into organelle heterogeneity at single-organelle resolution.

cell biology↗

bioRxiv2024

Piggybacking functionalized DNA nanostructures into live cell nuclei

DNA origami (DO) are promising tools for in vitro or in vivo applications including drug delivery; biosensing, detecting biomolecules; and probing chromatin sub-structures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing visualizing and controlling important biological processes in live cells. Here we present an approach to deliver DO strucures into live cell nuclei. We show that labelled DOs do not undergo detectable structural degradation in cell culture media or human cell extracts for 24 hr. To deliver DO platforms into the nuclei of human U2OS cells, we conjugated 30 nm long DO nanorods with an antibody raised against the largest subunit of RNA Polymerase II (Pol II), a key enzyme involved in gene transcription. We find that DOs remain structurally intact in cells for 24hr, including within the nucleus. Using fluorescence microscopy we demonstrate that the electroporated anti-Pol II antibody conjugated DOs are efficiently piggybacked into nuclei and exihibit sub-diffusive motion inside the nucleus. Our results reveal that functionalizing DOs with an antibody raised against a nuclear factor is a highly effective method for the delivery of nanodevices into live cell nuclei.

bioengineering↗

bioRxiv2023

ECLiPSE: A Versatile Classification Technique for Structural and Morphological Analysis of Super-Resolution Microscopy Data

We introduce a new automated machine learning analysis pipeline to precisely classify cellular structures captured through single molecule localization microscopy, which we call ECLiPSE (Enhanced Classification of Localized Pointclouds by Shape Extraction). ECLiPSE leverages 67 comprehensive shape descriptors encompassing geometric, boundary, skeleton and other properties, the majority of which are directly extracted from the localizations to accurately characterize individual structures. We validate ECLiPSE through unsupervised and supervised classification on a dataset featuring five distinct cellular structures, achieving exceptionally high classification accuracies nearing 100%. Moreover, we demonstrate the versatility of our approach by applying it to two novel biological applications: quantifying the clearance of tau protein aggregates, a critical marker for neurodegenerative diseases, and differentiating between two distinct morphological features (morphotypes) of TAR DNA-binding protein 43 proteinopathy, potentially associated to different TDP-43 strains, each exhibiting unique seeding and spreading properties. We anticipate that this versatile approach will significantly enhance the way we study cellular structures across various biological contexts, elucidating their roles in disease development and progression.

biophysics↗

bioRxiv2023

Active Transcription and Epigenetic Reactions Synergistically RegulateMeso-Scale Genomic Organization

In interphase nuclei, chromatin is organized into interspersed dense domains with characteristic sizes, both in the nuclear interior and periphery. However, the quantitative impact of transcription and histone modifications on the size and distribution of these domains remains unclear. Here, we introduce a mesoscale theoretical model that investigates the relationship between heterochromatic domain sizes and loop extrusion rates from these domains. The model considers chromatin-chromatin and chromatin-lamina interactions, methylation and acetylation kinetics, and diffusion of epigenetic marks and nucleoplasm. Our model generates testable predictions that help reveal the biophysics underlying chromatin organization in the presence of transcription-driven loop extrusion. This process is kinetically captured through the conversion of heterochromatin to euchromatin in response to RNAPII activity. We discovered that a balance between diffusive and reactive fluxes governs the steady-state sizes of heterochromatin domains. Using theory and simulations, we predicted that a loss of transcription results in increased chromatin compaction and larger heterochromatin domain sizes. To validate our predictions, we employed complementary super-resolution and nano-imaging techniques on five different cell lines with impaired transcription. We quantitatively assessed how domain sizes scale with loop extrusion rates at the hetero-euchromatin interfaces. Our analysis of previously obtained super-resolution images of nuclei revealed that excessive loop extrusion leads to smaller heterochromatin domains. The model successfully recapitulated these observations, explaining how transcription loss can counteract the effects of cohesin overloading. As the general biophysical mechanisms regulating heterochromatin domain sizes are independent of cell type, our findings have significant implications for understanding the role of transcription in global genome organization.

biophysics↗

bioRxiv2022

Detyrosination enrichment on microtubule subsets is established by the interplay between a stochastically-acting enzyme and microtubule stability

Microtubules in cells consist of functionally diverse subpopulations carrying distinct post-translational modifications (PTMs). Akin to the histone code, the tubulin code regulates a myriad of microtubule functions ranging from intracellular transport to chromosome segregation. Yet, how individual PTMs only occur on subsets of microtubules to contribute to microtubule specialization is not well understood. In particular, microtubule detyrosination, which is the removal of the C-terminal tyrosine on -tubulin subunits, marks the stable population of microtubules and modifies how microtubules interact with other microtubule-associated proteins to regulate a wide range of cellular processes. Previously, we found that, in certain cell types, only a small subpopulation of microtubules is highly enriched with the detyrosination mark ([~]30%) and that detyrosination spans most of the length of a microtubule, often adjacent to a completely tyrosinated microtubule. How the activity of a cytosolic detyrosinase, Vasohibin (VASH) leads to only a small subpopulation of highly detyrosinated microtubules is unclear. Here, using quantitative super-resolution microscopy, we visualized nascent microtubule detyrosination events in cells consisting of 1-3 detyrosinated -tubulin subunits after Nocodazole washout. Microtubule detyrosination accumulates slowly and in a disperse pattern across the microtubule length. By visualizing single molecules of VASH in live cells, we found that VASH engages with microtubules stochastically on a short time scale suggesting limited removal of tyrosine per interaction, consistent with the super-resolution results. Combining these quantitative imaging results with simulations incorporating parameters from our experiments, we propose a stochastic model for cells to establish a subset of detyrosinated microtubules via a detyrosination-stabilization feedback mechanism.

cell biology↗

bioRxiv2021

Chemo-Mechanical Cues Modulate Nano-Scale Chromatin Organization in Healthy and Diseased Connective Tissue Cells

Microscale changes in tissue environment are translated to changes in cell behavior and phenotype, yet the mechanisms behind how these phenotypic changes occur are poorly understood. Here, we describe and model chromatin, which stores genetic information within the cell nucleus, as a dynamic nanomaterial whose configuration is modulated by chemo-mechanical cues in the microenvironment. Our findings indicate that physiologic chemo-mechanical cues can directly regulate chromatin architecture in progenitor cell populations. Via direct experimental observation and modeling that incorporates phase transitions and histone methylation kinetics, we demonstrate that soft environmental cues drive chromatin relocalization to the nuclear boundary and compaction. Conversely, dynamic stiffening attenuates these changes. Interestingly, in diseased human fibrous tissue cells, this link between mechanical inputs and chromatin nano-scale remodeling is abrogated. These data indicate that chromatin dynamics and plasticity may be hallmarks of disease progression and targets for therapeutic intervention.

bioengineering↗