Effect of Substrate Stiffness and Formin on Fibrillar Force Generation by Fibroblasts

Fibroblasts in connective tissues often interact with a fibrillar extra-cellular matrix (ECM) that restricts their shape along one-dimension (1D, along the fiber). At the same time, the fibroblast responds to and affects the mechanical nature of its microenvironment which consists of the inter-woven fibrillary ECM, other matrix components and cells. The determinants of force generation by fibroblasts, which is necessary to understand normal physiology and disease, is however unclear. In order to construct the 1D geometry of fibroblasts, we plated NIH 3T3 fibroblasts on micropatterned 1.5 μm-wide fibronectin lines on polyacrylamide gels with stiffness of 13 or 45 kPa. We used traction force microscopy to quantify the cellular traction force exerted and the associated strain energy stored in the substrate. We found that strain energy or maximum traction stress is not a function of cell length. Even though cell length depends on substrate stiffness, the strain energy and the maximum traction forces exerted were independent of substrate stiffness. Besides, we found that fibroblasts in a 1D morphology have prominent linear actin structures and inhibition of a family of actin nucleators (formin) significantly reduced linear actin level. Importantly, we found that the fibrillar force exerted by fibroblasts also strongly decreased, implicating formin in fibrillar fibroblast force exertion.https://digitalcommons.odu.edu/engineering_batten/1010/thumbnail.jp

Biophysical Tools to Study Cellular Mechanotransduction

The cell membrane is the interface that volumetrically isolates cellular components from the cell’s environment. Proteins embedded within and on the membrane have varied biological functions: reception of external biochemical signals, as membrane channels, amplification and regulation of chemical signals through secondary messenger molecules, controlled exocytosis, endocytosis, phagocytosis, organized recruitment and sequestration of cytosolic complex proteins, cell division processes, organization of the cytoskeleton and more. The membrane’s bioelectrical role is enabled by the physiologically controlled release and accumulation of electrochemical potential modulating molecules across the membrane through specialized ion channels (e.g., Na+, Ca2+, K+ channels). The membrane’s biomechanical functions include sensing external forces and/or the rigidity of the external environment through force transmission, specific conformational changes and/or signaling through mechanoreceptors (e.g., platelet endothelial cell adhesion molecule (PECAM), vascular endothelial (VE)-cadherin, epithelial (E)-cadherin, integrin) embedded in the membrane. Certain mechanical stimulations through specific receptor complexes induce electrical and/or chemical impulses in cells and propagate across cells and tissues. These biomechanical sensory and biochemical responses have profound implications in normal physiology and disease. Here, we discuss the tools that facilitate the understanding of mechanosensitive adhesion receptors. This article is structured to provide a broad biochemical and mechanobiology background to introduce a freshman mechano-biologist to the field of mechanotransduction, with deeper study enabled by many of the references cited herein

Essential Role of the \u3ci\u3eCrk\u3c/i\u3e Family-Dosage in DiGeorge-Like Anomaly and Metabolic Homeostasis

CRK and CRKL (CRK-like) encode adapter proteins with similar biochemical properties. Here, we show that a 50% reduction of the family-combined dosage generates developmental defects, including aspects of DiGeorge/del22q11 syndrome in mice. Like the mouse homologs of two 22q11.21 genes CRKL and TBX1, Crk and Tbx1 also genetically interact, thus suggesting that pathways shared by the three genes participate in organogenesis affected in the syndrome. We also show that Crk and Crkl are required during mesoderm development, and Crk/Crkl deficiency results in small cell size and abnormal mesenchyme behavior in primary embryonic fibroblasts. Our systems-wide analyses reveal impaired glycolysis, associated with low Hif1a protein levels as well as reduced histone H3K27 acetylation in several key glycolysis genes. Furthermore, Crk/Crkl deficiency sensitizes MEFs to 2deoxy-D-glucose, a competitive inhibitor of glycolysis, to induce cell blebbing. Activated Rapgef1, a Crk/Crkl-downstream effector, rescues several aspects of the cell phenotype, including proliferation, cell size, focal adhesions, and phosphorylation of p70 S6k1 and ribosomal protein S6. Our investigations demonstrate that Crk/Crkl-shared pathways orchestrate metabolic homeostasis and cell behavior through widespread epigenetic controls

Molecular Mechanisms of Cell Adhesion

151 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.NCAM forms a complex between its terminal domains Ig1 and Ig2. When NCAM of cell A and cell B connect to each other through complexes Ig12(A)/Ig12(B), the relative mobility of cells A and B and membrane tension exerts a force on the Ig12(A)/Ig12(B) complex. Here we investigate the response of the complex to force, using steered molecular dynamics. Starting from the structure of the complex from the Ig1-Ig2-Ig3 fragment, we first equilibrate the complex in solvent and show that its actual end-to-end length is markedly larger than in the crystal structure. We then show that the Ig12/Ig12 complex can behave as a molecular spring of spring constant ∼0.03 N/m in response to forces of tens of pico-Newton. Such tertiary structure elasticity can be expected to be pervasive considering the large number of multi-modular CAMs. Finally, we rupture the complex using higher forces to identify E16, F19, K98, and L175 as key residues stabilizing the complex.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Quantifying Cell Adhesion Strength with a Novel Flow Assay

Quantifying the level of adhesion of cells to the extracellular matrix (ECM) is vital in efforts to understand complex biological functions. Cell adhesion to the ECM involves multiple cell surface proteins binding to components of the ECM. The ECM consists of collagen fibers, proteoglycans, and other matrix components, all of which are produced by cells themselves. Fluid flow in microfluidic channels has previously been used to quantify the fluid shear forces required to disrupt cell-ECM adhesion. Enzymatic digestion of cell-ECM adhesion components by the enzyme trypsin has also been used qualitatively. Here, we combine elements of these two methods to develop an accessible alternative. We flowed 0.25% trypsin through a microfluidic channel to rupture the adhesion between single Madin-Darby Canine Kidney (MDCK) cells and collagen I. The fraction of cells that stayed adherent to the collagen surface precipitously dropped at higher flow rates. The channel allowed cell adhesion rupture events to be spatially resolved via time lapse imaging. Our setup enables the use of different extracellular matrix properties as well as cells in different biochemical states to model cell adhesive states relevant to healthy and diseased tissues. Our approach has a large dynamic range – i.e., the assay can quantify the adhesion strength of very weak as well as very strong cell adhesive contacts. Thus, it is of relevance to the study of cell physiology in a multitude of normal as well as diseased states like cancer

In situ determination of exerted forces in magnetic pulling cytometry

Localized application of exogenous forces on soft biomaterials and cells is often essential for the study of their response to external mechanical stimuli. Magnetic means of applying forces, particularly those based on permanent magnets and magnetic beads coupled to substrates or cells provide an accessible means of exerting forces of appropriate magnitude. The amount of force exerted, however, is often inferred from calibration performed ex situ, with typically similar but different magnetic beads. Here, we construct a simple magnetic tweezer by coupling a pencil-shaped stainless-steel probe to permanent neodymium magnets using a 3D printed adapter. We then demonstrate the in situ determination of magnetic bead pulling forces on a super-paramagnetic micro-bead coupled to a soft substrate using traction force microscopy. We determine the force exerted on the magnetic bead by the magnet probe – and thus exerted by the magnetic bead on the soft polyacrylamide substrate – as a function of the distance between the probe tip and the magnetic bead. We also show that we can determine the force exerted on a magnetic bead coupled to a cell by the changes in the traction force exerted by the cell on the soft substrate beneath. We thus demonstrate that forces of nanonewton magnitude can be locally exerted on soft substrates or cells and simultaneously determined using traction force microscopy. Application of this method for the in situ measurement of localized exogenous forces exerted on cells can also enable dissection of cellular force transmission pathways

High-Force Magnetic Pulling Cytometer for Probing Cellular Mechanotransduction Pathways

Cellular domains accrue mechanical fluctuations as a means of communication. The process by which these mechanical forces are transmitted into cellular signals is termed, mechanotransduction. To explore these mechanotransduction pathways we developed a magnetic pulling cytometer for applying localized exogenous forces to a target receptor. We coated a micron-sized superparamagnetic bead such that it will bind to the cell’s integrins, the primary receptors responsible for cell adhesion to an extracellular matrix. Using the magnetic pulling cytometer a physiologically relevant force on the order of a few nanonewtons was applied to the bead and thus the cell. In order to determine the forces applied to the cell in situ, we utilized traction force microscopy. Cellular responses to the applied force such as the distribution of forces across the cell and changes in the cytoskeletal network or focal adhesions may now be probed further

Electrokinetic Phenomena in Pencil Lead-Based Microfluidics

Fabrication of microchannels and associated electrodes to generate electrokinetic phenomena often involves costly materials and considerable effort. In this study, we used graphite pencil-leads as low cost, disposable 3D electrodes to investigate various electrokinetic phenomena in straight cylindrical microchannels, which were themselves fabricated by using a graphite rod as the microchannel mold. Individual pencil-leads were employed as the micro-electrodes arranged along the side walls of the microchannel. Efficient electrokinetic phenomena provided by the 3D electrodes, including alternating current electroosmosis (ACEO), induced-charge electroosmosis (ICEO), and dielectrophoresis (DEP), were demonstrated by the introduced pencil-lead based microfluidic devices. The electrokinetic phenomena were characterized by micro-particle image velocimetry (micro-PIV) measurements and microscopy imaging. Highly efficient electrokinetic phenomena using 3D pencil-lead electrodes showed the affordability and ease of this technique to fabricate microfluidic devices embedded with electrodes for electrokinetic fluid and particle manipulations