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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The role of E2F4 in adipogenesis is independent of its cell cycle regulatory activity.

The E2F and pocket protein families are known to play an important role in the regulation of both cellular proliferation and terminal differentiation. In this study, we have used compound E2F and pocket protein mutant mouse embryonic fibroblasts to dissect the role of these proteins in adipogenesis. This analysis shows that loss of E2F4 allows cells to undergo spontaneous differentiation. The ability of E2F4 to prevent adipogenesis seems to be quite distinct from the known properties of E2F. First, it can be separated from any change in either E2F-responsive gene expression or cell cycle regulation. Second, it is a specific property of E2F4, and not other E2Fs, and it occurs independently of E2F4's ability to interact with pocket proteins. In addition, E2F4 loss does not override the differentiation defect resulting from pRB loss even though it completely suppresses the proliferation defect of Rb(-/-) mouse embryonic fibroblasts. This finding definitively separates the known, positive role of pRB in adipogenesis from its cell cycle function and shows that this pocket protein is required to act downstream of E2F4 in the differentiation process.