Biology subjects

Arias-Garcia, M.

Publications and source records attributed to Arias-Garcia, M..

6 recordsLinked to original sources

bioRxiv2026

Multi-Site Reproducibility Study of 3D High-Content Analysis with Dual-View Oblique Plane Microscopy

High content imaging is being applied to achieve quantitative fluorescence readouts in increasingly complex 3-dimensional (3D) cell culture models such as spheroids and organoids. Compared to conventional 2D assays, 3D assays better represent biological heterogeneity but require more complex sample preparation, 3D imaging and 3D image analysis that can affect the accuracy and precision of such assays. We used spheroids formed from the NRAS-activated melanoma cell line 19161 modified to express an ERK kinase translocation reporter (KTR) as an exemplar 3D phenotypic assay carried out in 96-well plates. The spheroids were treated with the ERK activator TPA and a range of concentrations of the MEK inhibitor Binimetinib. 3D live-cell imaging with sub-cellular spatial resolution was performed using a dual-view oblique plane microscope (dOPM) - a form of single-objective light-sheet microscope - and the experiment was performed separately at 4 different institutes. The results were analysed using an identical 3D analysis pipeline and parameters. We assessed the variation in assay readout using a linear mixed effects model. Random variance at the well level was negligible (SD = 0.0048 relative to range of KTR biosensor readout at reference site of 0.17), indicating low technical noise. Treatment effects were dose-dependent and highly statistically significant compared to DMSO control across all sites (Dunnett-corrected p < 0.001). The range in KTR readout between the minimum (3.5 M Binimetinib) and maximum (100 nM TPA) treatments varied between 59 to 96% relative to the reference site. Measured bias in KTR readout between sites was between 6 and 12% of the range of the reference site. This study quantifies the reproducibility of a 3D live spheroid-based assay employing a fluorescence biosensor requiring readout out at the per-cell level using the dOPM platform and discusses areas where experimental protocol could be improved in the future to further improve reproducibility.

bioengineering↗

bioRxiv2022

Characterization of proteome-size scaling by integrative omics reveals mechanisms of proliferation control in cancer

Almost all living cells maintain size uniformity through successive divisions. Proteins that sub- or super-scale with size act as rheostats which regulate cell progression. A comprehensive atlas of these proteins is lacking; particularly in cancer cells where both mitogen and growth signalling are dysregulated. Utilising a multi-omic strategy, that integrates quantitative single cell imaging, phosphoproteomic and transcriptomic datasets, we leverage the inherent size heterogeneity of melanoma cells to investigate how peptides, post-translational modifications, and mRNAs scale with cell size to regulate proliferation. We find melanoma cells have different mean sizes, but all retain uniformity. Across the proteome, we identify proteins and phosphorylation events that sub and super scale with cell size. In particular, G2/M, biosynthetic, and cytoskeletal regulators sub- and super-scale with size. In small cells growth and proliferation processes are tightly coupled by translation which promotes CCND1 accumulation and anabolic increases in mass. Counter intuitively, anabolic growth pathways and translational process are low in large cells, which throttles the expression of factors such as CCND1 and thereby coupling proliferation from anabolic growth. Strikingly, these cells exhibit increased growth and comparable proliferation rates. Mathematical modelling suggests that decoupling growth and proliferative signalling fosters proliferation under mitogenic inhibition. As factors which promote adhesion and actin reorganization super-scale with size or are enriched in large cells, we suggest that growth/proliferation in these cells may be decoupled by cell spreading and mechanics. This study provides one of the first demonstrations of size-scaling phenomena in cancer and how morphology determines the chemistry of the cell.

systems biology↗

bioRxiv2021

Membrane voltage fluctuations in human breast cancer cells

Cancer cells feature a resting membrane potential (Vm) that is depolarized compared to normal cells, and express active ionic conductances, which factor directly in their pathophysiological behavior. Despite similarities to excitable tissues, relatively little is known about cancer cell Vm dynamics. With high-throughput, cellular-resolution Vm imaging, we characterized Vm fluctuations of hundreds of human triple-negative breast cancer MDA-MB-231 cells and compared to non-cancerous breast epithelial MCF-10A cells. By quantifying their Dynamic Electrical Signatures (DESs) through an unsupervised machine-learning protocol, we identified four classes ranging from "noisy" to "blinking/waving". The Vm of MDA-MB-231 cells exhibited spontaneous, transient hyperpolarizations that were inhibited by the voltage-gated sodium channel blocker tetrodotoxin. The Vm of MCF-10A cells was comparatively static, but fluctuations increased following treatment with transforming growth factor-{beta}1, a canonical inducer of the epithelial-to-mesenchymal transition. These data suggest that the ability to generate Vm fluctuations is acquired during transformation and may participate in oncogenesis.

systems biology↗

bioRxiv2021

PERK-dependent reciprocal crosstalk between ER and non-centrosomal microtubules coordinates ER architecture and cell shape

The architecture of the endoplasmic reticulum (ER) is tightly controlled as a key determinant of its function. Its dynamics are linked to those of the cytoskeleton, but our understanding of how this coordination occurs and what its functional relevance is, is limited. We found the Unfolded Protein Response (UPR) transducer EIF2AK3/PERK is essential for acute stress-induced peripheral redistribution and remodeling of the ER, through eIF2a phosphorylation and translation initiation shutdown. PERK-mediated eIF2a phosphorylation can be bypassed by blocking ribosome activity; by depleting microtubule-anchoring ER proteins such as REEP4, p180/RRBP1 and Climp63/CKAP4; or by disrupting the microtubule cytoskeleton. Notably, specific disruption of non-centrosomal microtubules, but not centrosome depletion, relieved blockade of ER redistribution in PERK-deficient cells. Conversely, PERK deficiency stabilized non-centrosomal microtubules, promoting polarized protrusiveness in epithelial cells and neuroblasts. We propose that PERK coordinates ER architecture and homeostasis with cell morphogenesis by coupling ER remodeling and non-centrosomal MT dynamics.

cell biology↗

bioRxiv2020

DOCK5 couples CDC42-GSK3beta activation with focal adhesion morphogenesis to reinforce the establishment of polarity and promote YAP/TAZ activation during migration.

YAP and TAZ are transcriptional co-activators that are often constitutively active in triple negative breast cancer (TNBC) cells driving proliferation, invasion, and drug resistance. Through multiplexed quantitative genetic screens for YAP/TAZ localisation and cell shape, we found that the RhoGEF DOCK5 is essential for YAP/TAZ activation in metastatic cells and is required for the maintenance of polarity during migration. DOCK5 regulates cell shape and thus YAP/TAZ through different genetic interactions with CDC42, RAC, and RHOA GTPases. DOCK5 regulates focal adhesion (FA) morphogenesis in RAC-dependent fashions that promote RHOA mediated actomyosin engagement of FA. Using unbiased systems-level quantification of protein levels by mass spectrometry we show that DOCK5 maintains polarity by stabilising protein levels of the CDC42 effector GSK3{beta}. We conclude DOCK5 acts as a coincidence detector to promote leading edge persistence in subcellular locations where there is both RAC and RHOA dependent FA morphogenesis and active CDC42 mediated cell polarisation.

cell biology↗

bioRxiv2019

Microtubule-mediated nuclear deformation drives the Epithelial-to-Mesenchymal Transition and breast cancer.

Morphology dictates how cells sense physical and soluble cues in their environment; thus contributing to fate decisions. The differentiation of epithelial cells into mesenchymal forms, or epithelial-mesenchymal plasticity (EMP), is essential for metazoan development and homeostasis. Here we show that the decision to engage EMP is coupled to cell morphology by cell-cell adhesions by microtubule and nuclear organization (MTNO). Using an integrative omic approach we identify Junctional Adhesion Molecule 3 (JAM3) as a new tumour suppressor in breast cancer patients. JAM3 depletion in epithelial cells alters MTNO and causes differentiation into mesenchymal forms. Soluble TGF{beta} also changes MTNO, and synergizes with JAM3 depletion to promote mesenchymal morphogenesis. Through systematic proteomic analysis we show that changes in MTNO lead to the upregulation of an inflammatory signalling network where YAP/TAZ, FOXO, IKK-NFKB, and JNK pathways are active; but where insulin signalling is suppressed. The actions of the MT-motor Kinesin-1 serve to both change MTNO and promote the upregulation of the core EMP network. Critically, the upregulation of the EMP network predicts the mesenchymal state across cancers. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=137 HEIGHT=200 SRC="FIGDIR/small/689737v3_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@6ab6d1org.highwire.dtl.DTLVardef@8787borg.highwire.dtl.DTLVardef@1dff5dorg.highwire.dtl.DTLVardef@16856a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗