Cell Biology

The composition and mechanical properties of the cellular microenvironment along with the resulting distribution of cellular devolved forces can affect cellular function and behavior. Traction Force Microscopy (TFM) provides a method to measure the forces applied to a surface by adherent cells. Numerous TFM systems have been described in literature. Broadly, these involve culturing cells on a flexible substrate with embedded fluorescent markers which are imaged before and after relaxion of cell forces. From these images, a displacement field is calculated, and from the displacement field, a traction field. Here we describe a TFM system using polyacrylamide substrates and a microarray spotter to fabricate arrays of multicellular islands on various combinations of extra cellular matrix (ECM) proteins or other biomolecules. A microscope with an automated stage is used to image each of the cellular islands before and after lysing cells with a detergent. These images are analyzed in a semi-automated fashion using a series of MATLAB scripts which produce the displacement and traction fields, and summary data. By combining microarrays with a semi-automated implementation of TFM analysis, this protocol enables evaluation of the impact of substrate stiffness, matrix composition, and tissue geometry on cellular mechanical behavior in high throughput.

Microcracks in materials reflect their mechanical properties. The quantification of the number or orientation of such cracks is thus essential in many fields, including engineering and geology. In biology, cracks in soft tissues can reflect adhesion defects, and the analysis of their pattern can help to deduce the magnitude and orientation of tensions in organs and tissues. Here, we describe a semi-automatic method amenable to analyze cell separations occurring in the epidermis of Arabidopsis thaliana seedlings. Our protocol is applicable to any image exhibiting small cracks, and thus also adapted to the analysis of emerging cracks in animal tissues and materials.

Estimation of stomatal aperture using low viscosity silicone-base impression material has the advantage of working with the whole leaf. The developmental stage and the environment strongly affect the stomatal aperture. Therefore, it is mandatory to have accurate estimations of the stomatal aperture of intact leaves under different situations. With this technique, it is possible to get the real picture at any moment. The outputs of the data include studies on cell area and morphology, epidermis cell and stomata lineages, among others. This protocol is useful for the accurate estimation of stomatal aperture in many samples of intact leaves in Arabidopsis thaliana.

The cortical actomyosin cytoskeleton is found in all non-muscle cells where a key function is to control mechanical force (Salbreux et al., 2012). When coupled to E-cadherin cell-cell adhesion, cortical actomyosin generates junctional tension that influences many aspects of tissue function, organization and morphogenesis (Lecuit and Yap, 2015). Uncovering the molecular mechanisms underlying the generation of junctional tension requires tools for measuring it in live cells with a high spatio-temporal resolution. For this, we have set up a technique of laser ablation, in which we use the high power output of a two-photon laser to physically cut the actin cortex at the sites of cell-cell adhesion labeled with E-cadherin-GFP. Tension, thus is visualized as the outwards recoil of the vertices that define a junction after this was ablated/cut. Analysis of recoil versus time allows extracting parameters related to the amount of contractile force that is applied to the junction before ablation (initial recoil) and the ratio between elasticity of the junction and viscosity of the media (cytoplasm) in which the junctional cortex is immersed. Using this approach we have discovered how Src protein-tyrosine kinase (Gomez et al., 2015); actin-binding proteins such as tropomyosins (Caldwell et al., 2014) and N-WASP (Wu et al., 2014); Myosin II (Priya et al., 2015) and coronin-1B (Michael et al., 2016) contribute to the molecular apparatus responsible for generating tension at the cell-cell junctions. This protocol describes the experimental procedure for setting up laser ablation experiments and how to optimize ablation and acquisition conditions for optimal measurements of junctional tension. It also provides a full description, step by step, of the post-acquisition analysis required to evaluate changes in contractile force as well as cell elasticity and/or cytoplasm viscosity.

Non-muscle myosin II (NMII) form bipolar filaments, which bind F-actin to exert cellular contractility during physiological processes (Vicente-Manzanares et al., 2009). Using a combinatorial approach to fluorescently label both N- and C-termini of the NMII heavy chain, recent works have demonstrated the ability to visualize NMII bipolar filaments at various subcellular localizations (Ebrahim et al., 2013; Beach et al., 2014). At the zonula adherens (ZA) of epithelia, NMII minifilaments bind the circumferential actin bundles in a pseudo-sarcomeric manner (Ebrahim et al., 2013), a conformation required to maintain junctional tension and tissue integrity (Ratheesh et al., 2012). By expressing green fluorescent protein (GFP)-NMIIA heavy chain and immunolabel it using a NMIIA C-terminus specific antibody, we were able to visualize the NMII minifilaments bound to F-actin bundles in Caco-2 cells (Michael et al., 2016), as previously reported (Ebrahim et al., 2013; Beach et al., 2014). In addition, we designed an FIJI/MATLAB analysis module to quantify the size, distance and alignment of these minifilaments with respect to junctional F-actin at the ZA. Measurements of the dispersion of minifilaments angles were proven to be a useful parameter that closely correlated to the extent of contractility at junctions (Michael et al., 2016).

Microtubules (MTs) support an astonishing set of versatile cellular functions ranging from cell division, vesicle transport, and cell and tissue morphogenesis in various organisms. This versatility is in large mediated by MT-associated proteins (MAPs). The neuronal MAP Tau, for example, is stabilizing MTs in axons of the vertebrate nervous system and thus provides the basis for enduring axonal transport and the long life span of neurons (Mandelkow et al., 1994). Tau has been shown to bind to MTs directly in vitro and also to promote their nucleation from α-/β-tubulin subunits (Goode et al., 1994). Recently, we identified a plant-specific protein family called “companion of cellulose synthase” (CC), which was shown to bind MTs and enhance dynamics of the cortical MT array in plant cells under salt stress (Endler et al., 2015). The CCs were therefore hypothesized to help plant cells cope with stress conditions and thereby maintain biomass production under adverse growth conditions. Here, we provide detailed experimental information on in vitro MT binding assays, which allow assessing whether a protein of interest is binding to MTs. The assay utilizes the high molecular weight of MTs in a spin down approach and enables the determination of the dissociation constant K_d, a measure for the protein’s binding strength to MTs.

A technique of atomic force microscopy (AFM) called PeakForce quantitative nanomechanical mapping (PeakForce QNM) is an efficient tool for the quantitative mechanobiological imaging of fibrillar aggregate, human epidermal cell and woody plant cell wall topography (Sweers et al., 2011; Heu et al., 2012; Ďurkovič et al., 2012; Ďurkovič et al., 2013). Here, we describe a detailed protocol for the measurement of nanomechanical properties of primary xylem cell walls in woody plants, for the determination of reduced Young’s modulus of elasticity (MOE), adhesion, deformation, and energy dissipation (Figure 1). This new technique provides direct control of the maximum loading force and the deformation depth in cell wall samples keeping indentations small, while at the same time eliminating damaging lateral forces in order to preserve both the AFM tip and plant sample. High-resolution and non-destructive imaging shed new quantitative mechanistic insights into the structural biology of woody plant cell walls. This procedure can also be adapted for other biological samples with a varying range of stiffness.

Vibrio cholerae (V. cholerae) colonizes the intestinal epithelium and secretes cholera toxin (CT), a potent enterotoxin that causes severe fluid loss characteristic of the disease cholera. V. cholerae is a non-invasive Gram-negative bacterium that adheres to intestinal cells as well as a variety of different cell types. A protocol for adherence of V. cholerae to various cell lines is described. The adhered bacteria can be used to examine expression of genes that are differentially expressed between adhered and unadhered bacteria or other purposes (Dey et al., 2013).

Moraxella catarrhalis is a human-restricted pathogen that is responsible for respiratory tract infections such as childhood otitis media (OM) and exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Successful colonization and infection by M. catarrhalis depends on its ability to attach to the respiratory tract mucosal epithelium. This protocol describes a method to measure adherence of M. catarrhalis to epithelial cell lines in vitro.

Dozens of Mycoplasma species bind to solid surfaces and glide in the direction of the membrane protrusion at a pole. In gliding, Mycoplasma legs catch, pull and release sialylated oligosaccharides fixed on a solid surface. The analyses of inhibitory effects of sialylated compounds on gliding of Mycoplasma can determine the target structure of Mycoplasma for gliding and adhesion.