Identification of phenotype-specific networks from paired gene expression-cell shape imaging data.

The morphology of breast cancer cells is often used as an indicator of tumor severity and prognosis. Additionally, morphology can be used to identify more fine-grained, molecular developments within a cancer cell, such as transcriptomic changes and signaling pathway activity. Delineating the interface between morphology and signaling is important to understand the mechanical cues that a cell processes in order to undergo epithelial-to-mesenchymal transition and consequently metastasize. However, the exact regulatory systems that define these changes remain poorly characterized. In this study, we used a network-systems approach to integrate imaging data and RNA-seq expression data. Our workflow allowed the discovery of unbiased and context-specific gene expression signatures and cell signaling subnetworks relevant to the regulation of cell shape, rather than focusing on the identification of previously known, but not always representative, pathways. By constructing a cell-shape signaling network from shape-correlated gene expression modules and their upstream regulators, we found central roles for developmental pathways such as WNT and Notch, as well as evidence for the fine control of NF-kB signaling by numerous kinase and transcriptional regulators. Further analysis of our network implicates a gene expression module enriched in the RAP1 signaling pathway as a mediator between the sensing of mechanical stimuli and regulation of NF-kB activity, with specific relevance to cell shape in breast cancer.

Nanoneedle-Based Materials for Intracellular Studies.

Nanoneedles, defined as high aspect ratio structures with tip diameters of 5 to approximately 500 nm, are uniquely able to interface with the interior of living cells. Their nanoscale dimensions mean that they are able to penetrate the plasma membrane with minimal disruption of normal cellular functions, allowing researchers to probe the intracellular space and deliver or extract material from individual cells. In the last decade, a variety of strategies have been developed using nanoneedles, either singly or as arrays, to investigate the biology of cancer cells in vitro and in vivo. These include hollow nanoneedles for soluble probe delivery, nanocapillaries for single-cell biopsy, nano-AFM for direct physical measurements of cytosolic proteins, and a wide range of fluorescent and electrochemical nanosensors for analyte detection. Nanofabrication has improved to the point that nanobiosensors can detect individual vesicles inside the cytoplasm, delineate tumor margins based on intracellular enzyme activity, and measure changes in cell metabolism almost in real time. While most of these applications are currently in the proof-of-concept stage, nanoneedle technology is poised to offer cancer biologists a powerful new set of tools for probing cells with unprecedented spatial and temporal resolution.

Cell shape and the microenvironment regulate nuclear translocation of NF-κB in breast epithelial and tumor cells.

Although a great deal is known about the signaling events that promote nuclear translocation of NF-κB, how cellular biophysics and the microenvironment might regulate the dynamics of this pathway is poorly understood. In this study, we used high-content image analysis and Bayesian network modeling to ask whether cell shape and context features influence NF-κB activation using the inherent variability present in unperturbed populations of breast tumor and non-tumor cell lines. Cell­cell contact, cell and nuclear area, and protrusiveness all contributed to variability in NF-κB localization in the absence and presence of TNFα. Higher levels of nuclear NF-κB were associated with mesenchymal-like versus epithelial-like morphologies, and RhoA-ROCK-myosin II signaling was critical for mediating shape-based differences in NF-κB localization and oscillations. Thus, mechanical factors such as cell shape and the microenvironment can influence NF-κB signaling and may in part explain how different phenotypic outcomes can arise from the same chemical cues.

How cells explore shape space: a quantitative statistical perspective of cellular morphogenesis.

Through statistical analysis of datasets describing single cell shape following systematic gene depletion, we have found that the morphological landscapes explored by cells are composed of a small number of attractor states. We propose that the topology of these landscapes is in large part determined by cell-intrinsic factors, such as biophysical constraints on cytoskeletal organization, and reflects different stable signaling and/or transcriptional states. Cell-extrinsic factors act to determine how cells explore these landscapes, and the topology of the landscapes themselves. Informational stimuli primarily drive transitions between stable states by engaging signaling networks, while mechanical stimuli tune, or even radically alter, the topology of these landscapes. As environments fluctuate, the topology of morphological landscapes explored by cells dynamically adapts to these fluctuations. Finally we hypothesize how complex cellular and tissue morphologies can be generated from a limited number of simple cell shapes.

A role for Lin28 in primordial germ-cell development and germ-cell malignancy.

The rarity and inaccessibility of the earliest primordial germ cells (PGCs) in the mouse embryo thwart efforts to investigate molecular mechanisms of germ-cell specification. stella (also called Dppa3) marks the rare founder population of the germ lineage. Here we differentiate mouse embryonic stem cells carrying a stella transgenic reporter into putative PGCs in vitro. The Stella(+) cells possess a transcriptional profile similar to embryo-derived PGCs, and like their counterparts in vivo, lose imprints in a time-dependent manner. Using inhibitory RNAs to screen candidate genes for effects on the development of Stella(+) cells in vitro, we discovered that Lin28, a negative regulator of let-7 microRNA processing, is essential for proper PGC development. Furthermore, we show that Blimp1 (also called Prdm1), a let-7 target and a master regulator of PGC specification, can rescue the effect of Lin28 deficiency during PGC development, thereby establishing a mechanism of action for Lin28 during PGC specification. Overexpression of Lin28 promotes formation of Stella(+) cells in vitro and PGCs in chimaeric embryos, and is associated with human germ-cell tumours. The differentiation of putative PGCs from embryonic stem cells in vitro recapitulates the early stages of gamete development in vivo, and provides an accessible system for discovering novel genes involved in germ-cell development and malignancy.

Actin networks modulate heterogenous NF-κB dynamics in response to TNFα.

The canonical NF-κB transcription factor RELA is a master regulator of immune and stress responses and is upregulated in PDAC tumours. In this study, we characterised previously unexplored endogenous RELA-GFP dynamics in PDAC cell lines through live single cell imaging. Our observations revealed that TNFα stimulation induces rapid, sustained, and non-oscillatory nuclear translocation of RELA. Through Bayesian analysis of single cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. RNA-seq analysis identified distinct clusters of RELA-regulated gene expression in PDAC cells, including TNFα-induced RELA upregulation of the actin regulators NUAK2 and ARHGAP31. Further, siRNA-mediated depletion of ARHGAP31 and NUAK2 altered TNFα-stimulated nuclear RELA dynamics in PDAC cells, establishing a novel negative feedback loop that regulates RELA activation by TNFα. Additionally, we characterised the NF-κB pathway in PDAC cells, identifying how NF-κB/IκB proteins genetically and physically interact with RELA in the absence or presence of TNFα. Taken together, we provide computational and experimental support for interdependence between the F-actin network and the NF-κB pathway with RELA translocation dynamics in PDAC.

YAP activation is robust to dilution.

The concentration of many transcription factors exhibits high cell-to-cell variability due to differences in synthesis, degradation, and cell size. Whether the functions of these factors are robust to fluctuations in concentration, and how this may be achieved, is poorly understood. Across two independent panels of breast cancer cells, we show that the average whole cell concentration of YAP decreases as a function of cell area. However, the nuclear concentration distribution remains constant across cells grouped by size, across a 4-8 fold size range, implying unperturbed nuclear translocation despite the falling cell wide concentration. Both the whole cell and nuclear concentration was higher in cells with more DNA and CycA/PCNA expression suggesting periodic synthesis of YAP across the cell cycle offsets dilution due to cell growth and/or cell spreading. The cell area - YAP scaling relationship extended to melanoma and RPE cells. Integrative analysis of imaging and phospho-proteomic data showed the average nuclear YAP concentration across cell lines was predicted by differences in RAS/MAPK signalling, focal adhesion maturation, and nuclear transport processes. Validating the idea that RAS/MAPK and cell cycle regulate YAP translocation, chemical inhibition of MEK or CDK4/6 increased the average nuclear YAP concentration. Together, this study provides an example case, where cytoplasmic dilution of a protein, for example through cell growth, does not limit a cognate cellular function. Here, that same proteins translocation into the nucleus.

Epigallocatechin Gallate Modulates Microglia Phenotype to Suppress Pro-inflammatory Signalling Cues and Inhibit Phagocytosis.

Microglia are crucial players in the pathogenesis of late-onset Alzheimer's disease (AD), with evidence for both deleterious and beneficial effects. Identifying interventions to modulate microglial responsiveness, promote amyloid ß (Aß) clearance, disrupt plaque formation, or dampen excessive inflammation has therapeutic potential. Bioavailable flavonoids, such as the flavan 3-ols, are of interest due to their antioxidant, metal chelating, signalling, and anti-inflammatory potential. Primary microglia were treated with a series of structurally related flavanol 3-ols to assess effects on phagocytosis, cytokine release, and transcriptional responses by RNA sequencing. Data indicated that the extent of hydroxylation and the presence of the galloyl moiety were strong determinants of flavan 3-ol activity. Epigallocatechin gallate (EGCG) was the most effective flavan-3-ol tested and strongly inhibited phagocytosis of Aß independent of any metal chelating properties, suggesting a more direct modulation of microglia responsiveness. EGCG was broadly anti-inflammatory, reducing cytokine release and downregulating transcription, particularly of components of the microglia extracellular matrix such as MMP3 and SerpinB2. Collectively, this brings new insight into the actions of flavonoids on microglial responsiveness with potential implications for the therapeutic use of EGCG and structurally related flavanol-3-ols in AD.

High-Aspect-Ratio Nanostructured Surfaces as Biological Metamaterials.

Materials patterned with high-aspect-ratio nanostructures have features on similar length scales to cellular components. These surfaces are an extreme topography on the cellular level and have become useful tools for perturbing and sensing the cellular environment. Motivation comes from the ability of high-aspect-ratio nanostructures to deliver cargoes into cells and tissues, access the intracellular environment, and control cell behavior. These structures directly perturb cells' ability to sense and respond to external forces, influencing cell fate, and enabling new mechanistic studies. Through careful design of their nanoscale structure, these systems act as biological metamaterials, eliciting unusual biological responses. While predominantly used to interface eukaryotic cells, there is growing interest in nonanimal and prokaryotic cell interfacing. Both experimental and theoretical studies have attempted to develop a mechanistic understanding for the observed behaviors, predominantly focusing on the cell-nanostructure interface. This review considers how high-aspect-ratio nanostructured surfaces are used to both stimulate and sense biological systems.

Size-Tunable Nanoneedle Arrays for Influencing Stem Cell Morphology, Gene Expression, and Nuclear Membrane Curvature.

High-aspect-ratio nanostructures have emerged as versatile platforms for intracellular sensing and biomolecule delivery. Here, we present a microfabrication approach in which a combination of reactive ion etching protocols were used to produce high-aspect-ratio, nondegradable silicon nanoneedle arrays with tip diameters that could be finely tuned between 20 and 700 nm. We used these arrays to guide the long-term culture of human mesenchymal stem cells (hMSCs). Notably, we used changes in the nanoneedle tip diameter to control the morphology, nuclear size, and F-actin alignment of interfaced hMSCs and to regulate the expression of nuclear lamina genes, Yes-associated protein (YAP) target genes, and focal adhesion genes. These topography-driven changes were attributed to signaling by Rho-family GTPase pathways, differences in the effective stiffness of the nanoneedle arrays, and the degree of nuclear membrane impingement, with the latter clearly visualized using focused ion beam scanning electron microscopy (FIB-SEM). Our approach to design high-aspect-ratio nanostructures will be broadly applicable to design biomaterials and biomedical devices used for long-term cell stimulation and monitoring.

Nanoscale Molecular Quantification of Stem Cell-Hydrogel Interactions.

A common approach to tailoring synthetic hydrogels for regenerative medicine applications involves incorporating RGD cell adhesion peptides, yet assessing the cellular response to engineered microenvironments at the nanoscale remains challenging. To date, no study has demonstrated how RGD concentration in hydrogels affects the presentation of individual cell surface receptors. Here we studied the interaction between human mesenchymal stem cells (hMSCs) and RGD-functionalized poly(ethylene glycol) hydrogels, by correlating macro- and nanoscale single-cell interfacial quantification techniques. We quantified RGD unbinding forces on a synthetic hydrogel using single cell atomic force spectroscopy, revealing that short-term binding of hMSCs was sensitive to RGD concentration. We also performed direct stochastic optical reconstruction microscopy (dSTORM) to quantify the molecular interactions between integrin α5ß1 and a biomaterial, unexpectedly revealing that increased integrin clustering at the hydrogel-cell interface correlated with fewer available RGD binding sites. Our complementary, quantitative approach uncovered mechanistic insights into specific stem cell-hydrogel interactions, where dSTORM provides nanoscale sensitivity to RGD-dependent differences in cell surface localization of integrin α5ß1. Our findings reveal that it is possible to precisely determine how peptide-functionalized hydrogels interact with cells at the molecular scale, thus providing a basis to fine-tune the spatial presentation of bioactive ligands.

Multiparametric Analysis of Cell Shape Demonstrates that ß-PIX Directly Couples YAP Activation to Extracellular Matrix Adhesion.

Mechanical signals from the extracellular matrix (ECM) and cellular geometry regulate the nuclear translocation of transcriptional regulators such as Yes-associated protein (YAP). Elucidating how physical signals control the activity of mechanosensitive proteins poses a technical challenge, because perturbations that affect cell shape may also affect protein localization indirectly. Here, we present an approach that mitigates confounding effects of cell-shape changes, allowing us to identify direct regulators of YAP localization. This method uses single-cell image analysis and statistical models that exploit the naturally occurring heterogeneity of cellular populations. Through systematic depletion of all human kinases, Rho family GTPases, GEFs, and GTPase activating proteins (GAPs), together with targeted chemical perturbations, we found that ß-PIX, a Rac1/Ccd42 GEF, and PAK2, a Rac1/Cdc42 effector, drive both YAP activation and cell-ECM adhesion turnover during cell spreading. Our observations suggest that coupling YAP to adhesion dynamics acts as a mechano-timer, allowing cells to rapidly tune gene expression in response to physical signals.

RNAi screens for Rho GTPase regulators of cell shape and YAP/TAZ localisation in triple negative breast cancer.

In order to metastasise, triple negative breast cancer (TNBC) must make dynamic changes in cell shape. The shape of all eukaryotic cells is regulated by Rho Guanine Nucleotide Exchange Factors (RhoGEFs), which activate Rho-family GTPases in response to mechanical and informational cues. In contrast, Rho GTPase-activating proteins (RhoGAPs) inhibit Rho GTPases. However, which RhoGEFs and RhoGAPS couple TNBC cell shape to changes in their environment is very poorly understood. Moreover, whether the activity of particular RhoGEFs and RhoGAPs become dysregulated as cells evolve the ability to metastasise is not clear. Towards the ultimate goal of identifying RhoGEFs and RhoGAPs that are essential for TNBC metastasis, we performed an RNAi screen to isolate RhoGEFs and RhoGAPs that contribute to the morphogenesis of the highly metastatic TNBC cell line LM2, and its less-metastatic parental cell line MDA-MB-231. For ~6 million cells from each cell line, we measured 127 different features following the depletion of 142 genes. Using a linear classifier scheme we also describe the morphological heterogeneity of each gene-depleted population.

Visualizing cellular imaging data using PhenoPlot.

Visualization is essential for data interpretation, hypothesis formulation and communication of results. However, there is a paucity of visualization methods for image-derived data sets generated by high-content analysis in which complex cellular phenotypes are described as high-dimensional vectors of features. Here we present a visualization tool, PhenoPlot, which represents quantitative high-content imaging data as easily interpretable glyphs, and we illustrate how PhenoPlot can be used to improve the exploration and interpretation of complex breast cancer cell phenotypes.

Differential RNAi screening provides insights into the rewiring of signalling networks during oxidative stress.

Reactive Oxygen Species (ROS) are a natural by-product of cellular growth and proliferation, and are required for fundamental processes such as protein-folding and signal transduction. However, ROS accumulation, and the onset of oxidative stress, can negatively impact cellular and genomic integrity. Signalling networks have evolved to respond to oxidative stress by engaging diverse enzymatic and non-enzymatic antioxidant mechanisms to restore redox homeostasis. The architecture of oxidative stress response networks during periods of normal growth, and how increased ROS levels dynamically reconfigure these networks are largely unknown. In order to gain insight into the structure of signalling networks that promote redox homeostasis we first performed genome-scale RNAi screens to identify novel suppressors of superoxide accumulation. We then infer relationships between redox regulators by hierarchical clustering of phenotypic signatures describing how gene inhibition affects superoxide levels, cellular viability, and morphology across different genetic backgrounds. Genes that cluster together are likely to act in the same signalling pathway/complex and thus make "functional interactions". Moreover we also calculate differential phenotypic signatures describing the difference in cellular phenotypes following RNAi between untreated cells and cells submitted to oxidative stress. Using both phenotypic signatures and differential signatures we construct a network model of functional interactions that occur between components of the redox homeostasis network, and how such interactions become rewired in the presence of oxidative stress. This network model predicts a functional interaction between the transcription factor Jun and the IRE1 kinase, which we validate in an orthogonal assay. We thus demonstrate the ability of systems-biology approaches to identify novel signalling events.

Paxillin controls directional cell motility in response to physical cues.

Physical cues from the extracellular environment that influence cell shape and directional migration are transduced into changes in cytoskeletal organization and biochemistry through integrin-based cell adhesions to extracellular matrix (ECM). Paxillin is a focal adhesion (FA) scaffold protein that mediates integrin anchorage to the cytoskeleton, and has been implicated in regulation of FA assembly and cell migration. To determine whether paxillin is involved in coupling mechanical distortion with directional movement, cell shape was physically constrained by culturing cells on square-shaped fibronectin-coated adhesive islands surrounded by non-adhesive barrier regions that were created with a microcontact printing technique. Square-shaped cells preferentially formed FAs and extended lamellipodia from their corner regions when stimulated with PDGF, and loss of paxillin resulted in loss of this polarized response. Selective expression of the N- and C-terminal domains of paxillin produced opposite, but complementary, effects on suppressing or promoting lamellipodia formation in different regions of square cells, which corresponded to directional motility defects in vitro. Paxillin loss or mutation was also shown to affect the formation of circular dorsal ruffles, and this corresponded to changes in cell invasive behavior in 3D. This commentary addresses the implications of these findings in terms of how a multifunctional FA scaffold protein can link physical cues to cell adhesion, protrusion and membrane trafficking so as to control directional migration in 2D and 3D. We also discuss how microengineered ECM islands and in vivo model systems can be used to further elucidate the functions of paxillin in directional migration.

Paxillin mediates sensing of physical cues and regulates directional cell motility by controlling lamellipodia positioning.

Physical interactions between cells and the extracellular matrix (ECM) guide directional migration by spatially controlling where cells form focal adhesions (FAs), which in turn regulate the extension of motile processes. Here we show that physical control of directional migration requires the FA scaffold protein paxillin. Using single-cell sized ECM islands to constrain cell shape, we found that fibroblasts cultured on square islands preferentially activated Rac and extended lamellipodia from corner, rather than side regions after 30 min stimulation with PDGF, but that cells lacking paxillin failed to restrict Rac activity to corners and formed small lamellipodia along their entire peripheries. This spatial preference was preceded by non-spatially constrained formation of both dorsal and lateral membrane ruffles from 5-10 min. Expression of paxillin N-terminal (paxN) or C-terminal (paxC) truncation mutants produced opposite, but complementary, effects on lamellipodia formation. Surprisingly, pax-/- and paxN cells also formed more circular dorsal ruffles (CDRs) than pax+ cells, while paxC cells formed fewer CDRs and extended larger lamellipodia even in the absence of PDGF. In a two-dimensional (2D) wound assay, pax-/- cells migrated at similar speeds to controls but lost directional persistence. Directional motility was rescued by expressing full-length paxillin or the N-terminus alone, but paxN cells migrated more slowly. In contrast, pax-/- and paxN cells exhibited increased migration in a three-dimensional (3D) invasion assay, with paxN cells invading Matrigel even in the absence of PDGF. These studies indicate that paxillin integrates physical and chemical motility signals by spatially constraining where cells will form motile processes, and thereby regulates directional migration both in 2D and 3D. These findings also suggest that CDRs may correspond to invasive protrusions that drive cell migration through 3D extracellular matrices.

Methods for studying mechanical control of angiogenesis by the cytoskeleton and extracellular matrix.

Mechanical forces that capillary endothelial cells generate in their cytoskeleton and exert on their extracellular matrix adhesions feed back to modulate cell sensitivity to soluble angiogenic factors, and thereby control vascular development. Here we describe various genetic, biochemical, and engineering methods that can be used to study, manipulate, and probe this physical mechanism of developmental control. These techniques are useful as in vitro angiogenesis models and for analyzing the molecular and biophysical basis of vascular control.

Tools to study cell mechanics and mechanotransduction.

Analysis of how cells sense and respond to mechanical stress has been limited by the availability of techniques that can apply controlled mechanical forces to living cells while simultaneously measuring changes in cell and molecular distortion, as well as alterations of intracellular biochemistry. We have confronted this challenge by developing new engineering methods to measure and manipulate the mechanical properties of cells and their internal cytoskeletal and nuclear frameworks, and by combining them with molecular cell biological techniques that rely on microscopic analysis and real-time optical readouts of biochemical signaling. In this chapter, we describe techniques like microcontact printing, magnetic twisting cytometry, and magnetic pulling cytometry that can be systematically used to study the molecular basis of cellular mechanotransduction.