The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

Summary Astrocytes extend highly branched processes that form functionally isolated microdomains, facilitating local homeostasis by redistributing ions, removing neurotransmitters and releasing factors to influence blood flow and neuronal activity. Microdomains exhibit spontaneous increases in calcium (Ca2+), but the mechanisms and functional significance of this localized signaling are unknown. By developing conditional, membrane anchored GCaMP3 mice, we found that microdomain activity that occurs in the absence of IP3-dependent release from endoplasmic reticulum arises through Ca2+ efflux from mitochondria during brief openings of the mitochondrial permeability transition pore. These microdomain Ca2+ transients were facilitated by the production of reactive oxygen species during oxidative phosphorylation and enhanced by expression of a mutant form of superoxide dismutase 1 (SOD1 G93A) that causes astrocyte dysfunction and neurodegeneration in amyotrophic lateral sclerosis (ALS). By localizing mitochondria to microdomains, astrocytes ensure local metabolic support for energetically demanding processes and enable coupling between metabolic demand and Ca2+ signaling events.

Cell migration through 3D extracellular matrices is critical to the normal development of tissues and organs and in disease processes, yet adequate analytical tools to characterize 3D migration are lacking. Here, we quantified the migration patterns of individual fibrosarcoma cells on 2D substrates and in 3D collagen matrices and found that 3D migration does not follow a random walk. Both 2D and 3D migration features a non-Gaussian, exponential mean cell velocity distribution, which we show is primarily a result of cellto-cell variations. Unlike in the 2D case, 3D cell migration is anisotropic: velocity profiles display different speed and selfcorrelation processes in different directions, rendering the classical persistent random walk (PRW) model of cell migration inadequate. By incorporating cell heterogeneity and local anisotropy to the PRW model, we predict 3D cell motility over a wide range of matrix densities, which identifies density-independent emerging migratory properties. This analysis also reveals the unexpected robust relation between cell speed and persistence of migration over a wide range of matrix densities.theory | 3D motility | cancer R andom walks are ubiquitous in biology (1). In particular, the motility of bacteria and eukaryotic cells in the absence of symmetry-breaking gradients has long been described in terms of random walk statistics. Eukaryotic cell migration is a complex process that is a tightly regulated and critical to the normal development of organs and tissues (2-4). Cell migration is activated in a wide range of human diseases, including cancer metastasis (5, 6), immunological responses (7), and wound healing (8). Most of what we know about eukaryotic cell migration at a mechanistic molecular level has stemmed from well-controlled studies of cell migration on flat dishes (i.e., 2D environment). However, cell migration in vivo often forces cells to remodel, exert pulling forces on, and move through a 3D collagen I-rich matrix. Recent work has demonstrated that mechanisms of 3D migration are often different from their 2D counterparts (9-15). Migration on 2D dishes, which induces a basal-apical polarization of the cell, is driven by actomyosin contractility of stress fibers between large focal adhesions and the formation of a wide lamellipodium terminated by thin filopodial protrusions at the leading cellular edge (4, 16). The same cells in collagen-rich 3D matrix do not display a lamellipodium or filopodia. Instead, they display highly dendritic pseudopodial protrusions controlled by distinct proteins that rely both on acto-myosin contractility and microtubule assembly/disassembly dynamics (11, 17). 3D cell migration depends on the expression of metalloproteinases (MMPs), which are dispensable in 2D migration, and physical properties of the 3D matrix (18), such as pore size (6). Recent work has also shown how cancer cells in 3D can alternate between a mesenchymal and an amoeboid migratory phenotype depending on the physical properties of the matrix (19,20) and MMP inhibition (17...

Secreted proteins dictate a range of cellular functions in human health and disease. Due to the high degree of cellular heterogeneity and, more importantly, polyfunctionality of individual cells, there is an unmet need to simultaneously measure an array of proteins from single cells and to rapidly assay a large number of single cells (more than 1000) in parallel. We describe a simple bioanalytical assay platform consisting of a large array of sub-nanoliter microchambers integrated with high-density antibody barcode microarrays for highly multiplexed protein detection from over a thousand single cells in parallel. This platform has been tested for both cell lines and complex biological samples such as primary cells from patients. We observed distinct heterogeneity among the single cell secretomic signatures that, for the first time, can be directly correlated to the cells’ physical behavior such as migration. Compared to the state-of-the-art protein secretion assay such as ELISpot and emerging microtechnology-enabled assays, our approach offers both high throughput and high multiplicity. It also has a number of clinician-friendly features such as ease of operation, low sample consumption and standardized data analysis, representing a potentially transformative tool for informative monitoring of cellular function and immunity in patients.

Multiple attributes of the three-dimensional (3D) extracellular matrix (ECM) have been independently implicated as regulators of cell motility, including pore size, crosslink density, structural organization, and stiffness. However, these parameters cannot be independently varied within a complex 3D ECM protein network. We present an integrated, quantitative study of these parameters across a broad range of complex matrix configurations using self-assembling 3D collagen and show how each parameter relates to the others and to cell motility. Increasing collagen density resulted in a decrease and then an increase in both pore size and fiber alignment, which both correlated significantly with cell motility but not bulk matrix stiffness within the range tested. However, using the crosslinking enzyme Transglutaminase II to alter microstructure independently of density revealed that motility is most significantly predicted by fiber alignment. Cellular protrusion rate, protrusion orientation, speed of migration, and invasion distance showed coupled biphasic responses to increasing collagen density not predicted by 2D models or by stiffness, but instead by fiber alignment. The requirement of matrix metalloproteinase (MMP) activity was also observed to depend on microstructure, and a threshold of MMP utility was identified. Our results suggest that fiber topography guides protrusions and thereby MMP activity and motility.

Following uncontrolled proliferation, a subset of primary tumour cells acquires additional traits/mutations to trigger phenotypic changes that enhance migration and are hypothesized to be the initiators of metastasis. This study reveals an adaptive mechanism that harnesses synergistic paracrine signalling via IL-6/8, which is amplified by cell proliferation and cell density, to directly promote cell migration. This effect occurs in metastatic human sarcoma and carcinoma cells– but not in normal or non-metastatic cancer cells-, and likely involves the downstream signalling of WASF3 and Arp2/3. The transcriptional phenotype of high-density cells that emerges due to proliferation resembles that of low-density cells treated with a combination of IL-6/8. Simultaneous inhibition of IL-6/8 receptors decreases the expression of WASF3 and Arp2/3 in a mouse xenograft model and reduces metastasis. This study reveals a potential mechanism that promotes tumour cell migration and infers a strategy to decrease metastatic capacity of tumour cells.

SUMMARYCells adopt distinct signaling pathways to optimize cell locomotion in different physical microenvironments. However, the underlying mechanism that enables cells to sense and respond to physical confinement is unknown. Using microfabricated devices and substrate-printing methods along with FRET-based biosensors, we report that, as cells transition from unconfined to confined spaces, intracellular Ca2+ level is increased, leading to phosphodiesterase 1 (PDE1)-dependent suppression of PKA activity. This Ca2+ elevation requires Piezo1, a stretch-activated cation channel. Moreover, differential regulation of PKA and cell stiffness in unconfined versus confined cells is abrogated by dual, but not individual, inhibition of Piezo1 and myosin II, indicating that these proteins can independently mediate confinement sensing. Signals activated by Piezo1 and myosin II in response to confinement both feed into a signaling circuit that optimizes cell motility. This study provides a mechanism by which confinement-induced signaling enables cells to sense and adapt to different physical microenvironments.

Cell migration through three-dimensional (3D) extracellular matrices is critical to the normal development of tissues and organs and in disease processes, yet adequate analytical tools to characterize 3D migration are lacking. The motility of eukaryotic cells on 2D substrates in the absence of gradients has long been described using persistent random walks (PRW). Recent work shows that 3D migration is anisotropic and features an exponential mean cell velocity distribution, rendering the PRW model invalid. Here we present a protocol for the analysis of 3D cell motility using the anisotropic persistent random walk model. The software, implemented in MATLAB, enables statistical profiling of experimentally observed 2D and 3D cell trajectories and extracts the persistence and speed of cells along primary and non-primary directions and an anisotropic index of migration. Basic computer skills and experience with MATLAB software are recommended for successful use of the protocol. This protocol is highly automated and fast, taking less than 30 minutes to analyze trajectory data per biological condition.