Biology subjects

Marcotti, S.

Publications and source records attributed to Marcotti, S..

16 recordsLinked to original sources

bioRxiv2026

Advanced in High-Resolution Cryo Volume Electron Microscopy (cvEM) Imaging for Unicellular and Multicellular Organisms

Cryo-Focused Ion Beam Scanning Electron Microscopy (cryoFIB-SEM) using samples fixed by high-pressure freezing uniquely enables high resolution cryo-volume Electron Microscope (cvEM) images of cell ultrastructure to be obtained from whole cells and complex tissues in their near native state. As the freezing process also preserves fluorescence, the link between three-dimensional (3D) ultrastructure and biological process is also enabled by targeted cryo-Correlative Light and Electron Microscopy (CLEM). However, the overall viability of cvEM is challenged by sample preparation, charge balance during imaging, sample sensitivity to beam damage, contamination, and very long acquisition times. Here we detail new experimental workflows to significantly reduce each of these effects and demonstrate the improvement in resolution possible with results from the nematode Caenorhabditis elegans and the ciliated protozoon Paramecium bursaria containing many endosymbiotic algae. These results demonstrate the versatility and potential wide-ranging utility of cvEM for 3D ultrastructural imaging of whole multicellular and unicellular organisms.

biophysics↗

bioRxiv2025

Spatially distinct chromatin compaction states predict neoadjuvant chemotherapy resistance in Triple Negative Breast Cancer

Organisation and dynamics of chromatin play a key role in regulation of cell state and function. In cancer, chromatin plasticity is known to be important in control of drug resistance, but the relationship between chromatin compaction and chemotherapy response within complex tissue settings remains unclear. Here, we measured single nuclei chromatin compaction using fluorescence lifetime imaging microscopy (FLIM) in situ in whole biopsies from 53 pre treatment and 14 post surgery tissue samples of human triple-negative breast cancer (TNBC) patients to determine whether single nuclei spatial chromatin compaction state can predict resistance to neoadjuvant chemotherapy (NACT). Bulk chromatin compaction across 53 pre-treatment core biopsies did not predict patient outcome. However, machine learning analysis revealed that a subset of patients exhibited distinct distributions of single nuclei with more open chromatin states, which was predictive of NACT-resistance. Graph neural network analysis established that the spatial arrangement of chromatin compaction contributed to prediction of NACT resistance and that chromatin compaction signatures were preserved in tissue state transitions from pre- to 14 post-NACT samples. Our findings shed new light on spatial control of chromatin structure and relationship to therapeutic resistance and establish a foundation for further molecular analysis of chromatin states in complex biological tissues.

systems biology↗

bioRxiv2025

Inhibiting epithelial cell extrusion and inflammation synergistically reverses asthmatic airway remodelling

Airway smooth muscle (ASM) hyperresponsiveness is the defining feature of asthma, yet neither its origins nor the processes that establish and maintain it are understood. Here, using a mouse model, we show that hyperresponsiveness arises from ASM realignment into dense, parallel bundles reinforced by excess extracellular matrix, a fundamental remodelling rather than mere amplification of existing contractile ASM. This remodelling is initiated and sustained by a mechanical feedforward cycle. Allergen treatment initiates a contraction-relaxation cycle that realigns ASM into a bronchoconstrictive state. As bronchoconstriction triggers crowding and excess airway epithelial cell extrusion 1, the wounding then locks ASM into a hypercontractile architecture that drives further constriction. Gadolinium, a mechanosensitive channel blocker that inhibits extrusion, prevents this remodelling and hyperresponsiveness--unlike the corticosteroid budesonide. Critically, in mice with fully established disease, gadolinium reverses all asthmatic features and restores normal airway function, while budesonide alone cannot. Budesonide, however, synergises with gadolinium to normalise protein and gene expression. These findings reframe asthma pathogenesis as a mechanically driven, pharmacologically reversible process -- suggesting that asthma may be curable rather than merely treatable.

cell biology↗

bioRxiv2025

SMART: A Spatio-Molecular Atlas of Response Trajectories in Triple-Negative Breast Cancer

A major challenge in treating Triple-Negative Breast Cancer (TNBC) lies in its molecular, morphological and clinical heterogeneity, which hampers accurate prediction of responses to neoadjuvant treatment. To address this, we introduce SMART: Spatio-Molecular Atlas of Response Trajectories, a comprehensive, multimodal resource compiled from 129 TNBC samples across 89 patients, obtained before, during, and after neoadjuvant chemotherapy (NACT). SMART comprises of 5,096 high quality manually selected spatial transcriptomic profiles enriched for epithelial, immune, or stromal compartments; paralleled with histological annotations, imagebased network analysis and protein expression. Seven novel spatial epithelial archetypes (EAs), seven tumour-immune microenvironments (TIMEs) and their co-localisation patterns were defined, revealing an opposing prevalence of functionally divergent EAs between response groups and the prognostic significance of B-cell enriched TIMEs, in particular those surrounding histologically normal epithelium adjacent to the tumour. The SMART dataset and analytical tools are publicly available via the PharosAI platform, providing the research community with the most comprehensive, manually annotated spatio-molecular transcriptomics atlas of NACT-treated TNBC to date.

cancer biology↗

bioRxiv2025

Proteomic Analysis Links Truncated Tau to Lysosome Motility, Autophagy and Endo-lysosomal Dysfunction

Tauopathies are characterized by the progressive accumulation of abnormal tau species, which disrupt the autophagy-lysosomal pathway (ALP), a critical system for degrading intracellular macromolecules and aggregated proteins, causing toxicity and cell death. This study investigates the impact of the N-terminally truncated Tau35 protein overexpression on proteolytic pathways, including effects on autophagy and endo-lysosomal processes. Using a Tau35 mouse model and SH-SY5Y cell lines stably expressing either the Tau35 fragment or full-length tau, we employed western blotting, proteomic analysis of lysosome-enriched brain fractions, proteolysis/endocytosis assays, and live-cell imaging with the lysotracker reporter to assess protein degradation and lysosomal function. Our findings identify early pathological changes in endo-lysosomal processes, including increased endocytosis, proteolytic dysfunction and lysosomal motility abnormalities, associated with Tau35-induced toxicity. This work extends previous research by providing new insights into the mechanisms of Tau35-induced neurotoxicity, offering a foundation for developing targeted therapeutic strategies to address tauopathies.

neuroscience↗

bioRxiv2025

Enhancing Reproducibility Through Bioimage Analysis: The Significance of Effect Sizes and Controls

Bioimage analysis is a powerful tool for investigating complex biological processes, but its robustness depends on technical precision and rigorous experimental design. In particular, the use of appropriate controls and experimental repetition is critical for drawing meaningful conclusions. However, both are often used inadequately or overlooked, with "statistical significance" often prioritised, frequently obtained through the misuse or misinterpretation of statistical tests. In this study, we reanalyse publicly available image datasets to highlight the crucial role of robust experimental design in interpreting results. Our findings underscore the importance of focusing on effect sizes and biological relevance over arbitrary statistical thresholds. We also discuss the diminishing returns of increased data collection once statistical stability has been achieved. By refining control usage and emphasising effect sizes, this work aims to enhance the reproducibility and robustness of research findings. We provide open-access code to allow researchers to engage with the dataset, promoting better practices in experimental design and data interpretation.

cell biology↗

bioRxiv2024

Derivation and simulation of a computational model of active cell populations: How overlap avoidance, deformability, cell-cell junctions and cytoskeletal forces affect alignment

Collective alignment of cell populations is a commonly observed phenomena in biology. An important example are aligning fibroblasts in healthy or scar tissue. In this work we derive and simulate a mechanistic agent-based model of the collective behaviour of actively moving and interacting cells, with a focus on understanding collective alignment. The derivation strategy is based on energy minimisation. The model ingredients are motivated by data on the behaviour of different populations of aligning fibroblasts and include: Self-propulsion, overlap avoidance, deformability, cell-cell junctions and cytoskeletal forces. We find that there is an optimal ratio of self-propulsion speed and overlap avoidance that maximises collective alignment. Further we find that deformability aids alignment, and that cell-cell junctions by themselves hinder alignment. However, if cytoskeletal forces are transmitted via cell-cell junctions we observe strong collective alignment over large spatial scales.

cell biology↗

bioRxiv2023

Characterisation and correction of polarisation effects in fluorescently labelled fibres

Many biological structures take the form of fibres and filaments, and quantitative analysis of fibre organisation is important for understanding their functions in both normal physiological conditions and disease. In order to visualise these structures, fibres can be fluorescently labelled and imaged, with specialised image analysis methods available for quantifying the degree and strength of fibre alignment. Here we show that fluorescently labelled fibres can display polarised emission, with the strength of this effect varying depending on structure and fluorophore identity. This can bias automated analysis of fibre alignment and mask the true underlying structural organisation. We present a method for quantifying and correcting these polarisation effects without requiring polarisation resolved microscopy, and demonstrate its efficacy when applied to images of fluorescently labelled collagen gels, allowing for more reliable characterisation of fibre microarchitecture.

cell biology↗

bioRxiv2023

Bridging Imaging Users to Imaging Analysis - A community survey

The "Bridging Imaging Users to Imaging Analysis" survey was conducted in 2022 by the Center for Open Bioimage Analysis (COBA), Bioimaging North America (BINA), and the Royal Microscopical Society Data Analysis in Imaging Section (RMS DAIM) to understand the needs of the imaging community. Through multi-choice and open-ended questions, the survey inquired about demographics, image analysis experiences, future needs, and suggestions on the role of tool developers and users. Participants of the survey were from diverse roles and domains of the life and physical sciences. To our knowledge, this is the first attempt to survey cross-community to bridge knowledge gaps between physical and life sciences imaging. Survey results indicate that respondents overarching needs are documentation, detailed tutorials on the usage of image analysis tools, user-friendly intuitive software, and better solutions for segmentation, ideally in a format tailored to their specific use cases. The tool creators suggested the users familiarize themselves with the fundamentals of image analysis, provide constant feedback, and report the issues faced during image analysis while the users would like more documentation and an emphasis on tool friendliness. Regardless of the computational experience, there is a strong preference for written tutorials to acquire knowledge on image analysis. We also observed that the interest in having office hours to get an expert opinion on their image analysis methods has increased over the years. In addition, the community suggests the need for a common repository for the available image analysis tools and their applications. The opinions and suggestions of the community, released here in full, will help the image analysis tool creation and education communities to design and deliver the resources accordingly.

scientific communication and education↗

bioRxiv2022

GliaMorph: A modular image analysis toolkit to quantify Müller glial cell morphology

Cell morphology is critical for all cell functions. This is particularly true for glial cells as they rely on their complex shape to contact and support neurons. However, methods to quantify complex glial cell shape accurately and reproducibly are lacking. To address this gap in quantification approaches, we developed an analysis pipeline called "GliaMorph". GliaMorph is a modular image analysis toolkit developed to perform (i) image pre-processing, (ii) semi-automatic region-of-interest (ROI) selection, (iii) apicobasal texture analysis, (iv) glia segmentation, and (v) cell feature quantification. Muller Glia (MG) are the principal retinal glial cell type with a stereotypic shape linked to their maturation and physiological status. We here characterized MG on three levels, including (a) global image-level, (b) apicobasal texture, and (c) apicobasal vertical-to-horizontal alignment. Using GliaMorph, we show structural changes occurring in the developing retina. Additionally, we study the loss of cadherin2 in the zebrafish retina, as well as a glaucoma mouse disease model. The GliaMorph toolkit enables an in-depth understanding of MG morphology in the developing and diseased retina. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=144 HEIGHT=200 SRC="FIGDIR/small/490765v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@198fca6org.highwire.dtl.DTLVardef@f319fcorg.highwire.dtl.DTLVardef@2f0fadorg.highwire.dtl.DTLVardef@1ae4ce7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGlial morphology is complex, making it challenging to accurately quantify 3D cell shape. C_LIO_LIWe developed the GliaMorph toolkit for image pre-processing, glial segmentation, and quantification of Muller glial cells. C_LIO_LIMuller glia elaborate their morphology and rearrange subcellular features during embryonic development. C_LIO_LIGliaMorph accurately identifies subcellular changes in models with disrupted glia cells, including zebrafish cadherin2 loss of function and a mouse glaucoma model. C_LI

developmental biology↗

bioRxiv2022

Extracellular matrix assembly stress drives Drosophila central nervous system morphogenesis

The forces controlling tissue morphogenesis are attributed to cellular-driven activities and any role for extracellular matrix (ECM) is assumed to be passive. However, all polymer networks, including ECM, can theoretically develop autonomous stresses during their assembly. Here we examine the morphogenetic function of an ECM prior to reaching homeostatic equilibrium by analyzing de novo ECM assembly during Drosophila ventral nerve cord (VNC) condensation. Asymmetric VNC shortening and a rapid decrease in surface area correlate with exponential assembly of Collagen-IV (Col4) surrounding the tissue. Concomitantly, a transient developmentally-induced Col4 gradient leads to coherent long-range flow of ECM, which equilibrates the Col4 network. Finite element analysis and perturbation of Col4 network formation through the generation of dominant Col4-truncations that affect assembly, reveals that VNC morphodynamics is driven by a sudden increase in ECM-driven surface tension. These data highlight that ECM assembly stress and associated network instabilities can actively participate in tissue morphogenesis.

developmental biology↗

bioRxiv2021

A Workflow for Rapid Unbiased Quantification of Fibrillar Feature Alignment in Biological Images

Measuring the organisation of the cellular cytoskeleton and the surrounding extracellular matrix (ECM) is currently of wide interest as changes in both local and global alignment can highlight alterations in cellular functions and material properties of the extracellular environment. Different approaches have been developed to quantify these structures, typically based on fibre segmentation or on matrix representation and transformation of the image, each with its own advantages and disadvantages. Here we present AFT - Alignment by Fourier Transform, a workflow to quantify the alignment of fibrillar features in microscopy images exploiting 2D Fast Fourier Transforms (FFT). Using pre-existing datasets of cell and ECM images, we demonstrate our approach and compare and contrast this workflow with two other well-known ImageJ algorithms to quantify image feature alignment. These comparisons reveal that AFT has a number of advantages due to its grid-based FFT approach. 1) Flexibility in defining the window and neighbourhood sizes allows for performing a parameter search to determine an optimal length scale to carry out alignment metrics. This approach can thus easily accommodate different image resolutions and biological systems. 2) The length scale of decay in alignment can be extracted by comparing neighbourhood sizes, revealing the overall distance that features remain anisotropic. 3) The approach is ambivalent to the signal source, thus making it applicable for a wide range of imaging modalities and is dependent on fewer input parameters than segmentation methods. 4) Finally, compared to segmentation methods, this algorithm is computationally inexpensive, as high-resolution images can be evaluated in less than a second on a standard desktop computer. This makes it feasible to screen numerous experimental perturbations or examine large images over long length scales. Implementation is made available in both MATLAB and Python for wider accessibility, with example datasets for single images and batch processing. Additionally, we include an approach to automatically search parameters for optimum window and neighbourhood sizes, as well as to measure the decay in alignment over progressively increasing length scales.

biophysics↗

bioRxiv2021

The glycocalyx affects force loading-dependent mechanotransductive topography sensing at the nanoscale

The cell/microenvironment interface is the starting point of integrin-mediated mechanotransduction, but many details of mechanotransductive signal integration remain elusive due to the complexity of the involved (extra)cellular structures, such as the glycocalyx. We used nano-bio-interfaces reproducing the complex nanotopographical features of the extracellular matrix to analyse the glycocalyx impact on PC12 cell mechanosensing at the nanoscale (e.g., by force spectroscopy with functionalised probes). Our data demonstrates that the glycocalyx configuration affects spatio-temporal nanotopography-sensitive mechanotransductive events at the cell/microenvironment interface. Opposing effects of glycocalyx removal were observed, when comparing flat and specific nanotopographical conditions. The excessive retrograde actin flow speed and force loading are strongly reduced on certain nanotopographies upon removal of the native glycocalyx, while on the flat substrate we observe the opposite trend. Our results highlight the importance of the glycocalyx configuration in a molecular clutch force loading-dependent cellular mechanism for mechanosensing of microenvironmental nanotopographical features.

biophysics↗

bioRxiv2020

Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration.

Cell migration is important for development and its aberrant regulation contributes to many diseases. The Scar/WAVE complex is essential for Arp2/3 mediated lamellipodia formation during mesenchymal cell migration and several coinciding signals activate it. However, so far, no direct negative regulators are known. We have identified Nance-Horan Syndrome-like 1 protein (NHSL1) as a novel, direct binding partner of the Scar/WAVE complex, which co-localise at protruding lamellipodia. This interaction is mediated by the Abi SH3 domain and two binding sites in NHSL1. Furthermore, active Rac binds to NHSL1 at two regions that mediate leading edge targeting of NHSL1. Surprisingly, NHSL1 inhibits cell migration through its interaction with the Scar/WAVE complex. Mechanistically, NHSL1 may reduce cell migration efficiency by impeding Arp2/3 activity, as measured in cells using a novel Arp2/3 FRET-FLIM biosensor, resulting in reduced F-actin density of lamellipodia, and consequently impairing the stability of lamellipodia protrusions.

cell biology↗

bioRxiv2020

Comparative study of contact repulsion in control and mutant macrophages using a novel interaction detection

This paper compares the contact-repulsion movement of mutant and wild-type macrophages using a novel interaction detection mechanism. The migrating macrophages are observed in Drosophila embryos. The study is carried out by a framework called macrosight, which analyses the movement and interaction of migrating macrophages. The framework incorporates a segmentation and tracking algorithm into analysing motion characteristics of cells after contact. In this particular study, the interactions between cells is characterised in the case of control embryos and Shot3 mutants, where the cells have been altered to suppress a specific protein, looking to understand what drives the movement. Statistical significance between control and mutant cells was found when comparing the direction of motion after contact in specific conditions. Such discoveries provide insights for future developments in combining biological experiments to computational analysis. Cell Segmentation, Cell Tracking, Macrophages, Cell Shape, Contact Analysis

cell biology↗

bioRxiv2019

A multi-structural finite element model to simulate atomic force microscopy nanoindentation of single cells

Single cell mechanical properties represent an increasingly studied descriptor for health and disease. Atomic force microscopy (AFM) has been widely used to measure single cell stiffness, despite its experimental limitations. The development of a computational framework to simulate AFM nanoindentation experiments could be a valuable tool to complement experimental findings. A single cell multi-structural finite element model was designed to this aim by using confocal images of bone cells, comprised of the cell nucleus, cytoplasm and actin cytoskeleton. The computational cell stiffness values were in the range of experimental values acquired on the same cells for nanoindentation of the cell nucleus and periphery, despite showing higher stiffness for the nucleus than for the periphery, oppositely to the average experimental findings. These results suggest it would be of interest to model different single cells with known experimental effective moduli to evaluate the ability of the computational models to replicate experimental results.

bioengineering↗