Cultured Primary Turtle Hepatocytes: A Cellular Model for The Study of Temperature and Anoxia.

Turtle hepatocytes are a non-excitable model for metabolic depression during low-temperature and/or anoxic overwintering conditions. Cytoskeletal structure and mitochondrial distribution are continuously modified in cells, and we hypothesized that metabolic depression would inhibit such processes as cell attachment and spreading and promote withdrawal of cell protrusions and peripheral mitochondria. After developing a methodology for culturing painted turtle hepatocytes, maintenance of cell attachment after a media change, and 2D area, were used as indicators of structural rearrangement and spreading/volume.

Membrane Ruffling is a Mechanosensor of Extracellular Fluid Viscosity.

Cell behaviour is affected by the physical forces and mechanical properties of the cells and of their microenvironment. The viscosity of extracellular fluid - a component of the cellular microenvironment - can vary by orders of magnitude, but its effect on cell behaviour remains largely unexplored. Using bio-compatible polymers to increase the viscosity of the culture medium, we characterize how viscosity affects cell behaviour.

Force-dependent activation of actin elongation factor mDia1 protects the cytoskeleton from mechanical damage and promotes stress fiber repair.

Plasticity of cell mechanics underlies a wide range of cell and tissue behaviors allowing cells to migrate through narrow spaces, resist shear forces, and safeguard against mechanical damage. Such plasticity depends on spatiotemporal regulation of the actomyosin cytoskeleton, but mechanisms of adaptive change in cell mechanics remain elusive. Here, we report a mechanism of mechanically activated actin polymerization at focal adhesions (FAs), specifically requiring the actin elongation factor mDia1.

Control of Cell Geometry through Infrared Laser Assisted Micropatterning.

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment.

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading.

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out shape. Following the cell-substrate attachment, the cell forms a thin sheet of lamellipodia emanating from the cell body. In the lamellipodia, globular actin (G-actin) monomers polymerize into a dense filamentous actin (F-actin) meshwork that pushes against the plasma membrane, thereby providing the mechanical forces required for the cell to spread.

Quantitative Analysis of Myofibroblast Contraction by Traction Force Microscopy.

Myofibroblasts play important roles in physiological processes such as wound healing and tissue repair. While high contractile forces generated by the actomyosin network enable myofibroblasts to physically contract the wound and bring together injured tissue, prolonged and elevated levels of contraction also drive the progression of fibrosis and cancer. However, quantitative mapping of these forces has been difficult due to their extremely low magnitude ranging from 100 pN/μm to 2 nN/μm.

Mechanosensitive Regulation of Fibrosis.

Cells in the human body experience and integrate a wide variety of environmental cues. A growing interest in tissue mechanics in the past four decades has shown that the mechanical properties of tissue drive key biological processes and facilitate disease development. However, tissue stiffness is not only a potent behavioral cue, but also a product of cellular signaling activity.

Publisher Correction: A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions.

The interrelationship between microtubules and the actin cytoskeleton in mechanoregulation of integrin-mediated adhesions is poorly understood. Here, we show that the effects of microtubules on two major types of cell-matrix adhesion, focal adhesions and podosomes, are mediated by KANK family proteins connecting the adhesion protein talin with microtubule tips. Both total microtubule disruption and microtubule uncoupling from adhesions by manipulations with KANKs trigger a massive assembly of myosin IIA filaments, augmenting focal adhesions and disrupting podosomes.

A Noisy Analog-to-Digital Converter Connects Cytosolic Calcium Bursts to Transcription Factor Nuclear Localization Pulses in Yeast.

Several examples of transcription factors that show stochastic, unsynchronized pulses of nuclear localization have been described. Here we show that under constant calcium stress, nuclear localization pulses of the transcription factor Crz1 follow stochastic variations in cytosolic calcium concentration. We find that the size of the stochastic calcium bursts is positively correlated with the number of subsequent Crz1 pulses.

Coupling of Human Rhodopsin to a Yeast Signaling Pathway Enables Characterization of Mutations Associated with Retinal Disease.

G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in eukaryotes, with multiple GPCR mutations linked to human diseases. With the growing number of sequenced human genomes, determining the pathogenicity of a mutation is challenging, but can be aided by a direct measurement of GPCR-mediated signaling. This is particularly difficult for the visual pigment rhodopsin-a GPCR activated by light-for which hundreds of mutations have been linked to inherited degenerative retinal diseases such as retinitis pigmentosa.

Talin Autoinhibition Regulates Cell-ECM Adhesion Dynamics and Wound Healing In Vivo.

Cells in multicellular organisms are arranged in complex three-dimensional patterns. This requires both transient and stable adhesions with the extracellular matrix (ECM). Integrin adhesion receptors bind ECM ligands outside the cell and then, by binding the protein talin inside the cell, assemble an adhesion complex connecting to the cytoskeleton.

Two Distinct Actin Networks Mediate Traction Oscillations to Confer Focal Adhesion Mechanosensing.

Focal adhesions (FAs) are integrin-based transmembrane assemblies that connect a cell to its extracellular matrix (ECM). They are mechanosensors through which cells exert actin cytoskeleton-mediated traction forces to sense the ECM stiffness. Interestingly, FAs themselves are dynamic structures that adapt their growth in response to mechanical force.

Bridging the gap.

A new study reveals that a protein called talin forms a vital link between microtubules and focal adhesions at the surface of cells.

A lateral signalling pathway coordinates shape volatility during cell migration.

Cell migration is fundamental for both physiological and pathological processes. Migrating cells usually display high dynamics in morphology, which is orchestrated by an integrative array of signalling pathways. Here we identify a novel pathway, we term lateral signalling, comprised of the planar cell polarity (PCP) protein Pk1 and the RhoGAPs, Arhgap21/23.

A ternary complex comprising FAK, PTPα and IP3 receptor 1 functionally engages focal adhesions and the endoplasmic reticulum to mediate IL-1-induced Ca2+ signalling in fibroblasts.

Ca(2+) release is tightly sequestered in eukaryotic cells to enable fine spatio-temporal control of signalling but how Ca(2+) release from the endoplasmic reticulum (ER) is linked to cell adhesions is not defined. We examined the spatial restriction of Ca(2+) release through the inositol 1,4,5-triphosphate receptor 1 (IP3R1) in response to interleukin-1 (IL-1) and the functions of the adhesion-associated proteins, focal adhesion kinase (FAK) and protein tyrosine phosphatase-α (PTPα). In cultured fibroblasts IL-1 treatment promoted co-localization of PTPα and FAK with the ER and increased association of IP3R1 with PTPα and FAK at focal adhesions (FAs).

Inverted formin 2 in focal adhesions promotes dorsal stress fiber and fibrillar adhesion formation to drive extracellular matrix assembly.

Actin filaments and integrin-based focal adhesions (FAs) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator, inverted formin 2 (INF2), localizes specifically to FAs and dorsal stress fibers (SFs) in fibroblasts.

High-resolution traction force microscopy.

Cellular forces generated by the actomyosin cytoskeleton and transmitted to the extracellular matrix (ECM) through discrete, integrin-based protein assemblies, that is, focal adhesions, are critical to developmental morphogenesis and tissue homeostasis, as well as disease progression in cancer. However, quantitative mapping of these forces has been difficult since there has been no experimental technique to visualize nanonewton forces at submicrometer spatial resolution. Here, we provide detailed protocols for measuring cellular forces exerted on two-dimensional elastic substrates with a high-resolution traction force microscopy (TFM) method.

Vinculin-actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth.

In migrating cells, integrin-based focal adhesions (FAs) assemble in protruding lamellipodia in association with rapid filamentous actin (F-actin) assembly and retrograde flow. How dynamic F-actin is coupled to FA is not known. We analyzed the role of vinculin in integrating F-actin and FA dynamics by vinculin gene disruption in primary fibroblasts.

Guiding cell migration by tugging.

The ability of cells to move directionally toward areas of stiffer extracellular matrix (ECM) via a process known as 'durotaxis' is thought to be critical for development and wound healing, but durotaxis can also drive cancer metastasis. Migration is driven by integrin-mediated focal adhesions (FAs), protein assemblies that couple contractile actomyosin bundles to the plasma membrane, transmit force generated by the cytoskeleton to the ECM, and convert the mechanical properties of the microenvironment into biochemical signals. To probe the stiffness of the ECM, motile fibroblasts modulate FA mechanics on the nanoscale and exert forces that are reminiscent of repeated tugging on the ECM.

Force fluctuations within focal adhesions mediate ECM-rigidity sensing to guide directed cell migration.

Cell migration toward areas of higher extracellular matrix (ECM) rigidity via a process called "durotaxis" is thought to contribute to development, immune response, and cancer metastasis. To understand how cells sample ECM rigidity to guide durotaxis, we characterized cell-generated forces on the nanoscale within single mature integrin-based focal adhesions (FAs). We found that individual FAs act autonomously, exhibiting either stable or dynamically fluctuating ("tugging") traction.

Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure.

Second-harmonic generation (SHG) microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. Because of its underlying physical origin, it is highly sensitive to the collagen fibril/fiber structure, and, importantly, to changes that occur in diseases such as cancer, fibrosis and connective tissue disorders. We discuss how SHG can be used to obtain more structural information on the assembly of collagen in tissues than is possible by other microscopy techniques.

Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration.

Migration and proliferation of cardiac fibroblasts (CFs) play an important role in the myocardial remodeling process. While many factors have been identified that regulate CF growth and migration, less is known about the signaling mechanisms involved in these processes. Here, we utilized Cre-LoxP technology to obtain focal adhesion kinase (FAK)-deficient adult mouse CFs and studied how FAK functioned in modulating cell adhesion, proliferation, and migration of these cells.

Measurement of muscle disease by quantitative second-harmonic generation imaging.

Determining the health of muscle cells by in vivo imaging could impact the diagnosis and monitoring of a large number of congenital and acquired muscular or cardiac disorders. However, currently used technologies are hampered by insufficient resolution, lack of specificity, or invasiveness. We have combined intrinsic optical second-harmonic generation from sarcomeric myosin with a novel mathematical treatment of striation pattern analysis, to obtain measures of muscle contractile integrity that correlate strongly with the neuromuscular health of mice suffering from genetic, acquired, and age-related decline in skeletal muscle function.