Touch, Grasp, Deliver and Control: Functional Cross-Talk Between Microtubules and Cell Adhesions

Cross-talk between microtubule networks and sites of cell–matrix and cell–cell adhesion has profound impact on these structures and is essential for proper cell organization, polarization and motility. Components of adhesion sites can interact directly with microtubules or with proteins that specifically associate with microtubule plus ends and minus ends and in this way capture, stabilize or destabilize microtubules. In their turn, microtubules can serve as routes for delivery of structural and regulatory factors that control adhesion site turnover. In addition, the microtubule lattice or growing microtubule plus ends can serve as diffusional sinks that accumulate and scaffold regulatory molecules, thereby affecting their activity in the vicinity of adhesions. Combination of these mechanisms underlies the functional co-operation between microtubules and adhesion sites and defines their dynamic behavior.     

The basic framework of VE-cadherin junctions revealed by cryo-EM

Artificial adherens junctions were reconstituted in vitro by assembly of cadherin fragments at the surfaces of liposomes. The architecture of the adherens junctions was revealed by cryo-electron microscopy (cryo-EM). The formation of these artificial adherens junctions was shown to result from the two-dimensional (2D) self-assembly of cadherin fragments at membrane surfaces. The molecular architecture of the junctions was resolved by combining information from several cryo-EM views. This study concludes to the 2D ordered nature of the cadherin assembly and shows that the minimal information required to build up an adherens junction is contained within the extracellular moiety of cadherin molecules.     

Crossroads of integrins and cadherins in epithelia and stroma remodeling

Adhesion events mediated by cadherin and integrin adhesion receptors have fundamental roles in the maintenance of the physiological balance of epithelial tissues, and it is well established that perturbations in their normal functional activity and/or changes in their expression are associated with tumorigenesis. Over the last decades, increasing evidence of a dynamic collaborative interaction between these complexes through their shared interactions with cytoskeletal proteins and common signaling pathways has emerged not only as an important regulator of several aspects of epithelial cell behavior, but also as a coordinated adhesion module that senses and transmits signals from and to the epithelia surrounding microenvironment. The tight regulation of their crosstalk is particularly important during epithelial remodeling events that normally take place during morphogenesis and tissue repair, and when defective it leads to cell transformation and aggravated responses of the tumor microenvironment that contribute to tumorigenesis. In this review we highlight some of the interactions that regulate their crosstalk and how this could be implicated in regulating signals across epithelial tissues to sustain homeostasis.     

机械力及力学信号转导影响干细胞命运的研究进展

干细胞作为一种未分化的祖细胞,目前已被广泛应用于开展组织损伤修复、再生以及干细胞特异谱系分化的研究.大量研究表明,干细胞所处的微环境对调控干细胞的生长和分化具有重要作用,多种溶液介质、细胞外基质和信号通路等参与了干细胞命运的调控.尽管已有大量研究证明,溶液介质(如激素和生长因子)在干细胞的生长和分化中发挥重要作用,但近年来越来越多的研究表明,机械力及力学信号转导同样在干细胞自我更新、分化、衰老和凋亡等细胞生理过程中起到重要的作用.本文将对机械应力响应的细胞基础、生物力学及力学信号调控干细胞自我更新和分化,以及生物力学调控干细胞命运可能的作用机制几个方面加以综述.     

Crossroads of integrins and cadherins in epithelia and stroma remodeling

Adhesion events mediated by cadherin and integrin adhesion receptors have fundamental roles in the maintenance of the physiological balance of epithelial tissues, and it is well established that perturbations in their normal functional activity and/or changes in their expression are associated with tumorigenesis. Over the last decades, increasing evidence of a dynamic collaborative interaction between these complexes through their shared interactions with cytoskeletal proteins and common signaling pathways has emerged not only as an important regulator of several aspects of epithelial cell behavior, but also as a coordinated adhesion module that senses and transmits signals from and to the epithelia surrounding microenvironment. The tight regulation of their crosstalk is particularly important during epithelial remodeling events that normally take place during morphogenesis and tissue repair, and when defective it leads to cell transformation and aggravated responses of the tumor microenvironment that contribute to tumorigenesis. In this review we highlight some of the interactions that regulate their crosstalk and how this could be implicated in regulating signals across epithelial tissues to sustain homeostasis.     

Regulation of Cadherin Trafficking

Cadherins are a large family of cell–cell adhesion molecules that tether cytoskeletal networks of actin and intermediate filaments to the plasma membrane. This function of cadherins promotes tissue organization and integrity, as demonstrated by numerous disease states that are characterized by the loss of cadherin-based adhesion. However, plasticity in cell adhesion is often required in cellular processes such as tissue patterning during development and epithelial migration during wound healing. Recent work has revealed a pivotal role for various membrane trafficking pathways in regulating cellular transitions between quiescent adhesive states and more dynamic phenotypes. The regulation of cadherins by membrane trafficking is emerging as a key player in this balancing act, and studies are beginning to reveal how this process goes awry in the context of disease. This review summarizes the current understanding of how cadherins are routed and how the interface between cadherins and membrane trafficking pathways regulates cell surface adhesive potential. Particular emphasis is placed on the regulation of cadherin trafficking by catenins and the interplay between growth factor signaling pathways and cadherin endocytosis.     

A primer to traction force microscopy

Traction force microscopy (TFM) has emerged as a versatile technique for the measurement of single-cell-generated forces. TFM has gained wide use among mechanobiology laboratories, and several variants of the original methodology have been proposed. However, issues related to the experimental setup and, most importantly, data analysis of cell traction datasets may restrain the adoption of TFM by a wider community. In this review, we summarize the state of the art in TFM-related research, with a focus on the analytical methods underlying data analysis. We aim to provide the reader with a friendly compendium underlying the potential of TFM and emphasizing the methodological framework required for a thorough understanding of experimental data. We also compile a list of data analytics tools freely available to the scientific community for the furtherance of knowledge on this powerful technique.     

Atomic Force Microscopy in Mechanoimmunology Analysis: A New Perspective for Cancer Immunotherapy

Immunotherapy has remarkable success outcomes against hematological malignancies with high rates of complete remission. To date, many studies have been conducted to increase its effectiveness in other types of cancer. However, it still yields unsatisfying results in solid tumor therapy. This limitation is partly attributed to the lack of understanding of how immunotherapy works in cancer from other perspectives. The traditional studies focus on the biological and chemical perspectives to determine which molecular substrates are involved in the immune system that can eradicate cancer cells. In the last decades, accumulating evidence has shown that physical properties also play important roles in the immune system to combat cancer, which is studied in mechanoimmunology. Mechanoimmunology analysis requires special tools; and herein, atomic force microscopy (AFM) appears as a versatile tool to determine and quantify the mechanical properties of a sample in nanometer precisions. Owing to its multifunctional capabilities, AFM can be used to explore immune system function from the physical perspective. This review paper explains the mechanoimmunology of how immune systems work through AFM, which includes mechanosignaling, mechanosensing, and mechanotransduction, with the aim to deepen the understanding of the mechanistic role of immunotherapy for further development in cancer treatment.     

Traction Force Measurements of Human Aortic Smooth Muscle Cells Reveal a Motor-Clutch Behavior

The contractile behavior of smooth muscle cells (SMCs) in the aorta is an important determinant of growth, remodeling, and homeostasis. However, quantitative values of SMC basal tone have never been characterized precisely on individual SMCs. Therefore, to address this lack, we developed an in vitro technique based on Traction Force Microscopy (TFM). Aortic SMCs from a human lineage at low passages (4-7) were cultured 2 days in conditions promoting the development of their contractile apparatus and seeded on hydrogels of varying elastic modulus (1, 4, 12 and 25 kPa) with embedded fluorescent microspheres. After complete adhesion, SMCs were artificially detached from the gel by trypsin treatment. The microbeads movement was tracked and the deformation fields were processed with a mechanical model, assuming linear elasticity, isotropic material, plane strain, to extract the traction forces formerly applied by individual SMCs on the gel. Two major interesting and original observations about SMC traction forces were deduced from the obtained results: 1. they are variable but driven by cell dynamics and show an exponential distribution, with 40% to 80% of traction forces in the range 0-10 μN. 2. They depend on the substrate stiffness: the fraction of adhesion forces below 10 μN tend to decrease when the substrate stiffness increases, whereas the fraction of higher adhesion forces increases. As these two aspects of cell adhesion (variability and stiffness dependence) and the distribution of their traction forces can be predicted by the probabilistic motor-clutch model, we conclude that this model could be applied to SMCs. Further studies will consider stimulated contractility and primary culture of cells extracted from aneurysmal human aortic tissue.     

Colon cancer cell adhesion in response to Src kinase activation and actin-cytoskeleton by non-laminar shear stress

Malignant cells shed from tumors during surgical resection or spontaneous metastasis experience physical forces such as shear stress and turbulence within the peritoneal cavity during irrigation, laparoscopic air insufflation, or surgical manipulation, and within the venous or lymphatic system. Since physical forces can activate intracellular signals that modulate the biology of various cell types in vitro, we hypothesized that shear stress and turbulence might increase colon cancer cell adhesion to extracellular matrix, potentiating metastatic implantation. Primary human malignant colon cancer cells isolated from resected tumors and SW620 were subjected to shear stress and turbulence by stirring cells in suspension at 600 rpm for 10 min. Shear stress for 10 min increased subsequent SW620 colon cancer cell adhesion by 40.0 +/- 3.0% (n = 3; P < 0.001) and primary cancer cells by 41.0 +/- 3.0% to collagen I when compared to control cells. In vitro kinase assay (1.5 +/- 0.13 fold) and Western analysis (1.34 +/- 0.04 fold) demonstrated a significant increase in Src kinase activity in cells exposed shear stress. Src kinase inhibitors PP1 (0.1 microM), PP2 (20 microM), and actin-cytoskeleton stabilizer phalloidin (10 microM) prevented the shear stress stimulated cell adhesion to collagen I. Furthermore, PP2 inhibited basal (50.0 +/- 2.8%) and prevented shear stress induced src activation but phalloidin pretreatment did not. These results raise the possibility that shear stress and turbulence may stimulate the adhesion of malignant cells shed from colon cancers by a mechanism that requires both actin-cytoskeletal reorganization an independent physical force activation of Src kinase. Blocking this pathway might reduce tumor metastasis during surgical resection.     

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts. Traction forces are generated through myosin-actin interactions and play an important role in our physiology. During development, they enable cells to move from one location to the next in order to form the early structures of tissue. Traction forces help in the healing processes. They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body. These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor. The most common method by which to study cells in vitro has been to use a glass or polystyrene dish. However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces. Our lab has developed a technique to overcome these limitations. The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process. The pillars we use are 3 μm in diameter, 10 μm tall, and are configured in a regular array with 9 μm center-to-center spacing. But these physical dimensions can be readily varied to accommodate a variety of studies. We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS. We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections. We call these substrates microfabricated post-array-detectors, or mPADs. Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.     

A helping hand: How vinculin contributes to cell-matrix and cell-cell force transfer

Vinculin helps cells regulate and respond to mechanical forces. It is a scaffolding protein that tightly regulates its interactions with potential binding partners within adhesive structures—including focal adhesions that link the cell to the extracellular matrix and adherens junctions that link cells to each other—that physically connect the force-generating actin cytoskeleton (CSK) with the extracellular environment. This tight control of binding partner interaction—mediated by vinculin''s autoinhibitory head–tail interaction—allows vinculin to rapidly interact and detach in response to changes in the dynamic forces applied through the cell. In doing so, vinculin modulates the structural composition of focal adhesions and the cell''s ability to generate traction forces and adhesion strength. Recent evidence suggests that vinculin plays a similar role in regulating the fate and function of cell–cell junctions, further underscoring the importance of this protein. Using our lab''s recent work as a starting point, this commentary explores several outstanding questions regarding the nature of vinculin activation and its function within focal adhesions and adherens junctions.     

A helping hand: How vinculin contributes to cell-matrix and cell-cell force transfer

Vinculin helps cells regulate and respond to mechanical forces. It is a scaffolding protein that tightly regulates its interactions with potential binding partners within adhesive structures—including focal adhesions that link the cell to the extracellular matrix and adherens junctions that link cells to each other—that physically connect the force-generating actin cytoskeleton (CSK) with the extracellular environment. This tight control of binding partner interaction—mediated by vinculin's autoinhibitory head–tail interaction—allows vinculin to rapidly interact and detach in response to changes in the dynamic forces applied through the cell. In doing so, vinculin modulates the structural composition of focal adhesions and the cell's ability to generate traction forces and adhesion strength. Recent evidence suggests that vinculin plays a similar role in regulating the fate and function of cell–cell junctions, further underscoring the importance of this protein. Using our lab's recent work as a starting point, this commentary explores several outstanding questions regarding the nature of vinculin activation and its function within focal adhesions and adherens junctions.     

Dynamics of ligand-induced, Rac1-dependent anchoring of cadherins to the actin cytoskeleton

Cadherin receptors are key morphoregulatory molecules during development. To dissect their mode of action, we developed an approach based on the use of myogenic C2 cells and beads coated with an Ncad-Fc ligand, allowing us to mimic cadherin-mediated adhesion. We used optical tweezers and video microscopy to investigate the dynamics of N-cadherin anchoring within the very first seconds of bead-cell contact. The analysis of the bead movement by single-particle tracking indicated that N-cadherin molecules were freely diffusive in the first few seconds after bead binding. The beads rapidly became diffusion-restricted and underwent an oriented rearward movement as a result of N-cadherin anchoring to the actin cytoskeleton. The kinetics of anchoring were dependent on ligand density, suggesting that it was an inducible process triggered by active cadherin recruitment. This anchoring was inhibited by the dominant negative form of Rac1, but not that of Cdc42. The Rac1 mutant had no effect on cell contact formation or cadherin-catenin complex recruitment, but did inhibit actin recruitment. Our results suggest that cadherin anchoring to the actin cytoskeleton is an adhesion-triggered, Rac1-regulated process enabling the transduction of mechanical forces across the cell membrane; they uncover novel aspects of the action of cadherins in cell sorting, cell migration, and growth cone navigation.     

原子力显微技术在细胞生物学中的应用

对近年来原子力显微技术(AFM)在细胞生物学中的应用大致归纳为几个方面进行了简单介绍,还指出了细胞表面结构难于识别、细胞内部结构难以原位观察等AFM应用于细胞生物学中的难题,并提出了“形状探针”的概念以及超薄切片的思路以解决这些难题。AFM在细胞生物学中的应用研究还远远不足,需要更多的科学工作者加入其中。     

Direct Force Measurement of the Interaction Between Liposome and the C2A Domain of Synaptotagmin I using Atomic Force Microscopy

The binding force between a liposome and the C2A domain of synaptotagmin I was determined by an atomic force microscopy (AFM). Liposomes were immobilized on the surface of the L1 sensor chip and the C2A domains, which recognize phosphatidylserine, were chemically conjugated onto a gold-coated cantilever tip. The average interaction force between the C2A domain and the liposome was 306 (±57) pN while the force between untreated cantilever and the liposome was 58 (±16) pN. This work helps understand the physicochemical interactions between proteins and lipid vesicles for the design of high affinity protein probes against the apoptotic cell surface. Revisions requested 13 December 2005; Revisions received 9 January 2006     

Sanguinarine Decreases Cell Stiffness and Traction Force and Inhibits the Reactivity of Airway Smooth Muscle Cells in Culture

Airway hyperresponsiveness (AHR) is the cardinal character of asthma, which involves the biomechanical properties such as cell stiffness and traction force of airway smooth muscle cells (ASMCs). Therefore, these biomechanical properties comprise logical targets of therapy. β2-adrenergic agonist is currently the mainstream drug to target ASMCs in clinical practice for treating asthma. However, this drug is known for side effects such as desensitization and non-responsiveness in some patients. Therefore, it is desirable to search for new drug agents to be alternative of β2-adrenergic agonist. In this context, sanguinarine, a natural product derived from plants such as bloodroots, that has been reported to relax gut smooth muscle emerges as a potential candidate. So far, it is unknown whether sanguinarine can regulate the biomechanical properties of ASMCs and reactivity of ASMCs to irritants. Thus, we tested the hypothesis that sanguinarine reduce the contractile potentials of ASMCs in culture. To do so, the primary cultured rat ASMCs were first treated with different concentration of sanguinarine. Then, cell stiffness, traction force, fiber distribution, and calcium signaling of the ASMCs were evaluated by optical magnetic twisting cytometry, Fourier transform traction microscopy, atomic force microscopy, and Fluo-4/AM based fluorescence confocal scanning microscopy, respectively. The results indicated that sanguinarine (0.05 and 0.5 μmol/L) significantly decreased cell stiffness and traction force, inhibited reactivity of ASMCs to histamine, and disrupted the fiber structures in ASMCs in dose-dependent manner. These findings establish that sanguinarine can indeed change the biomechanical properties of ASMCs and may be used to treat AHR in asthma.     

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.