Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments

Biophysical and biomolecular analysis of EVs and their interaction with target cells

Triple negative breast cancer (TNBC) is one of the most aggressive breast cancer subtype and with a poor prognosis. Nowadays, chemotherapy is the main treatment in both early and advanced stage of the TNBC, but patients without complete response to conventional chemotherapy are approximately 80%. In light of that, clarifying biological mechanisms of the metastatic process is crucial in finding new therapeutic approaches for effective interventions. Metastasis is thought to be easier for more deformable and, therefore, soft cancer cells, which can migrate through narrow pores of extracellular matrix and vessels. Extracellular vesicles derived from triple-negative breast cancer, by sharing oncogenic molecules, have been shown to promote proliferation, drug resistance migration and metastatic capability in target cells proportional to properties of donor ones. Considering all these evidence, we wondered if small-EVs could also transfer information to target cells about biomechanical properties, a key step in metastasis, of the cell from which they originate. Our results showed that small-EVs derived from the MDA-MB-231 cell line (TNBC) can directly modulate biomechanical properties (stiffness/Young\u2019s modulus), cytoskeleton, nuclear morphology and Yap activity of MCF7 cell line (Luminal A) as target cell. Therefore, in this study, we found out a new mechanism through which small-EVs derived from TNBC subtype could be able to contribute to progression and metastatic processes in breast cancer; this new knowledge could be used in diagnostic and therapeutic field

Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments

Correlating confocal microscopy and atomic force indentation reveals metastatic cancer cells stiffen during invasion into collagen I matrices

abstract: Mechanical interactions between cells and their microenvironment dictate cell phenotype and behavior, calling for cell mechanics measurements in three-dimensional (3D) extracellular matrices (ECM). Here we describe a novel technique for quantitative mechanical characterization of soft, heterogeneous samples in 3D. The technique is based on the integration of atomic force microscopy (AFM) based deep indentation, confocal fluorescence microscopy, finite element (FE) simulations and analytical modeling. With this method, the force response of a cell embedded in 3D ECM can be decoupled from that of its surroundings, enabling quantitative determination of the elastic properties of both the cell and the matrix. We applied the technique to the quantification of the elastic properties of metastatic breast adenocarcinoma cells invading into collagen hydrogels. We found that actively invading and fully embedded cells are significantly stiffer than cells remaining on top of the collagen, a clear example of phenotypical change in response to the 3D environment. Treatment with Rho-associated protein kinase (ROCK) inhibitor significantly reduces this stiffening, indicating that actomyosin contractility plays a major role in the initial steps of metastatic invasion.The final version of this article, as published in Scientific Reports, can be viewed online at: https://www.nature.com/articles/srep1968

Mapping the dynamics of force transduction at cell–cell junctions of epithelial clusters

Force transduction at cell-cell adhesions regulates tissue development, maintenance and adaptation. We developed computational and experimental approaches to quantify, with both sub-cellular and multi-cellular resolution, the dynamics of force transmission in cell clusters. Applying this technology to spontaneously-forming adherent epithelial cell clusters, we found that basal force fluctuations were coupled to E-cadherin localization at the level of individual cell-cell junctions. At the multi-cellular scale, cell-cell force exchange depended on the cell position within a cluster, and was adaptive to reconfigurations due to cell divisions or positional rearrangements. Importantly, force transmission through a cell required coordinated modulation of cell-matrix adhesion and actomyosin contractility in the cell and its neighbors. These data provide insights into mechanisms that could control mechanical stress homeostasis in dynamic epithelial tissues, and highlight our methods as a resource for the study of mechanotransduction in cell-cell adhesions. DOI: http://dx.doi.org/10.7554/eLife.03282.00

The role of E-cadherin in the translocation dynamics of Yes-associated Protein

The Yes-associated protein (YAP) transcriptional co-activator is a key mechanosensitive regulator of mammalian growth control. When cells are subjected to mechanical stimuli such as stretching or a change in substrate stiffness, YAP moves from the cytoplasm to the nucleus where it activates pro-growth, cytoskeletal, and stem cell identity genes. Intrinsic and extrinsic physical forces experienced by cells are sensed by mechanosensitive structures such as the cytoskeleton, focal adhesions, and adherens junctions. Past studies have shown that upon application of force, focal adhesions and cell–cell junctions both affect YAP localisation. However, the degree to which each mechanotransduction pathway is required for YAP nuclear localisation upon mechanical stimulation remains unexplored. To examine the relative contributions of focal adhesions and adherens junctions to YAP localisation, I developed mechanical assays monitoring live-YAP localisation in monolayer growth, dissociation (via epithelial-mesenchymal transition, EMT) and stretch. I used CRISPR/Cas9 to generate endogenously tagged YAP epithelial cell lines. By live-imaging these lines, I showed that high levels of the adherens junction component, E-cadherin, limits YAP nuclear localisation during monolayer growth. In contrast, cell scattering during epithelial to mesenchymal transition (EMT), leading to loss of polarized cell morphology and E-cadherin adhesions, triggered YAP nuclear translocation. This switch in localisation is dependent on both the ECM specific integrin engagement and Src signalling. Similarly, stretching monolayers solely by their adherens junction does not induce YAP nuclear localisation. Instead, strain-induced YAP nuclear localisation occurs when the cell-ECM contacts are present. In summary, YAP localisation to the nucleus upon mechanical deformation is mainly promoted by focal adhesion signalling, whereas E-cadherins limit nuclear YAP entry. This study therefore proposes that there is an antagonistic behaviour between cell-cell junctions and cell-ECM attachments which tightly regulates YAP to ensure growth homeostasis

A dynamic-shape-prior guided snake model with application in visually tracking dense cell populations

This paper proposes a dynamic-shape-prior guided snake (DSP G-snake) model that is designed to improve the overall stability of the point-based snake model. The dynamic shape prior is first proposed for snakes, that efficiently unifies different types of high-level priors into a new force term. To be specific, a global-topology regularity is first introduced that settles the inherent self-intersection problem with snakes. The problem that a snake’s snaxels tend to unevenly distribute along the contour is also handled, leading to good parameterization. Unlike existing methods that employ learning templates or commonly enforce hard priors, the dynamic-template scheme strongly respects the deformation flexibility of the model, while retaining a decent global topology for the snake. It is verified by experiments that the proposed algorithm can effectively prevent snakes from selfcrossing, or automatically untie an already self-intersected contour. In addition, the proposed model is combined with existing forces and applied to the very challenging task of tracking dense biological cell populations. The DSP G-snake model has enabled an improvement of up to 30% in tracking accuracy with respect to regular model-based approaches. Through experiments on real cellular datasets, with highly dense populations and relatively large displacements, it is confirmed that the proposed approach has enabled superior performance, in comparison to modern active-contour competitors as well as the state-of-the-art cell tracking frameworks

Single-cell analysis of cell competition using quantitative microscopy and machine learning

Cell competition is a widely conserved, fundamental biological quality control mechanism. The cell competition assay of MDCK wild-type versus mutant MDCK Scribble-knockdown (ScribKD) relies on a mechanical mechanism of competition, which posits that the emergence of compressing stresses within the tissue at high confluency drive the competitive outcome. According to this mechanism, proliferating wild-type cells out-compete mutant ScribKD cells, resulting in their apoptosis and apical extrusion. Previous studies show that there is an increased division rate of wild-type cells in neighbourhoods with high numbers of ScribKD cells, but what still remains a mystery is whether this is a cause or consequence of increased apoptosis in the “loser” cell population. This project also interrogated the competitive assay of wild-type versus RasV12 , which is hypothesized to operate on a biochemical mechanism and results in the apical extrusion (but not apoptosis) of the loser RasV12 population. For both these mechanisms of competition it is still unknown which population of cells are driving the winner/loser outcome. Is the winner cell proliferation prompting the loser cell demise? Or is an autonomous loser elimination prompting a subsequent winner cell proliferation? In my research, I have employed multi-modal, time-lapse microscopy to image competition assays continuously for several days. These data were then segmented into wild-type or mutant instances using a Convolutional Neural Network (CNN) that can differentiate between the cell types, after which they were tracked across cellular generations using a Bayesian multi-object tracker. A conjugate analysis of fluorescent cell-cycle indicator probes was then utilised to automatically identify key time points of cellular fate commitment using deep-learning image classification. A spatio-temporal analysis was then conducted in order to quantify any correlation between wild-type proliferation and mutant cell demise. For the case of wild-type versus ScribKD , there was no clear evidence for the wild-type cells mitoses directly impacting upon the ScribKD cell apoptotic elimination. Instead, a subsequent analysis found that a more subtle mechanism of pre-emptive, local density increases around the apoptosis site appeared to be determining the eventual ScribKD fate. On the other hand, there was clear evidence of a direct impact of wild-type mitoses on the subsequent apical extrusion and competitive elimination of RasV12 cells. Both of these conclusions agree with the prevailing classification of cell competition types: mechanical interactions are more diffuse and occur over a larger spatio-temporal domain, whereas biochemical interactions are constrained to nearest neighbour cells. The hypothesized density-dependency of ScribKD elimination was further quantified on a single-cell scale by these analyses, as well as a potential new understanding of RasV12 extrusion. Most interestingly, it appears that there is a clear biophysical mechanism to the elimination in the biochemical RasV12 cell competition. This suggests that perhaps a new semantic approach is needed in the field of cell competition in order to accurately classify different mechanisms of elimination

Jamming and Unjamming in Cancer Cells

Jamming' ist ein faszinierender, nicht vollständig verstandener Prozess in der Physik der weichen Materie. Zelluläres Jamming' tritt auch in biologischem Gewebe auf und muss sich im Fall von Krebszellen im Tumorgewebe aufgrund der dichten Packung der Zellen über der dichtesten Kugelpackung anders verhalten als die bekannten 'Jamming' Systeme. In meiner Dissertation skizziere ich wesentliche Ergebnisse zum Verständnis dieses neuen physikalischen Phänomens. Meine Erkenntnisse tragen dazu bei die Dichotomie zwischen den Theorien der dichteinduzierten und der forminduzierten 'Zelljamming' aufzulösen. Die gewonnenen Erkenntnisse weisen auf die Möglichkeit hin Krebszellformen und deren Zellkernformen als Tumormarker für die Metastasierung zu verwenden. Ich fand ein kritisches Skalierungsverhalten für die Dynamik der Neuanordnung von Zellen in der Nähe des Jamming-Übergangs, abhängig von der Zellform der Nachbarschaft. Dies ist der bisher stärkste Beweis dafür, dass die Zellformen als Kontrollparameter für das 'Zelljamming' fungieren können. Die Zellanzahldichte beeinflusst ebenfalls das 'Jamming', ihr Einfluss kann jedoch als eine Verlangsamung der Eigengeschwindigkeit der Zellen beschrieben werden. Eine hohe Zellanzahldichte allein würde also nur die Viskosität des Gewebes erhöhen und es nicht verfestigen. Darüber hinaus habe ich gezeigt, dass es in dicht gepackten dreidimensionalen Zellsphäroiden sowie in Primärtumorstücken einen mit der Zellform verbundenen 'Jamming'-Übergang gibt. Ich verbinde das 'Unjamming' von Zellen mit dem Fortschreiten des Krebses, indem ich zeigte, dass die Herunterregulierung des Adhäsionsmoleküls E-Cadherin, die ein typischer Schritt während der Krebentwicklung ist, einen 'Unjamming'-Übergang verursacht. Bei diesem 'Unjamming'-Übergang kommt es zu einem ausgeprägten Verlust der Kohäsion und einem reduzierten Volumenanteil der Zellen, was zeigt, dass das 'Zelljamming' einen hohen Volumenanteil erfordert