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Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

measurement-liver-stiffness-using-atomic-force-microscopy-coupled

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell ...

Institute of Molecular Genetics of the Czech Academy of Sciences

Lecithin:retinol acyltransferase (LRAT) is the major enzyme responsible for retinol esterification in the mammalian body. LRAT exhibits specific activity in the cells with active retinol metabolism where it converts retinols into retinyl esters, which represents the major storage form of retinol. Besides hepatic stellate cells in the liver, LRAT appears to have a key physiologic role in several other tissues. In this study, we generated a transgenic reporter mouse expressing green fluorescence protein (EGFP) under the control of region containing -1166 bps from promoter upstream from the putative transcriptional start site and 262 bps downstream of this start. Transgenic reporter mice exhibited specific expression in eyes and testes. In eyes, expression of EGFP-reporter is found in lens and lens epithelium and fibers from embryo to adulthood. In testes, LRAT-EGFP reporter is expressed both in Sertoli and in spermatocytes marking initiation of spermatogenesis in prepubertal mice. Our data show that the examined LRAT regulatory region is sufficient to achieve strong and selective expression in the eye and testes but not in liver and other organs. 

Transgenic reporter mice with promoter region of murine LRAT specifically marks lens and meiosis spermatocytes.

The complex architecture of the liver biliary network represents a structural prerequisite for the formation and secretion of bile as well as excretion of toxic substances through bile ducts. Disorders of the biliary tract affect a significant portion of the worldwide population, often leading to cholestatic liver diseases. Cholestatic liver disease is a condition that results from an impairment of bile formation or bile flow to the gallbladder and duodenum. Cholestasis leads to dramatic changes in biliary tree architecture, worsening liver disease and systemic illness. Recent studies show that the prevalence of cholestatic liver diseases is increasing. The availability of well characterized animal models, as well as development of visualization approaches constitutes a critical asset to develop novel pathogenetic concepts and new treatment strategies.

Biliary system architecture: experimental models and visualization techniques.

Plectin, a highly versatile cytolinker protein, controls intermediate filament cytoarchitecture and cellular stress response. In the present study, we investigate the role of plectin in the liver under basal conditions and in experimental cholestasis.

Plectin controls biliary tree architecture and stability in cholestasis.

Intermediate filaments constitute the third component of the cellular skeleton. Unlike actin and microtubule cytoskeletons, the intermediate filaments are composed of a wide variety of structurally related proteins showing distinct expression patterns in tissues and cell types. Changes in the expression patterns of intermediate filaments are often associated with cancer progression; in particular with phenotypes leading to increased cellular migration and invasion. In this review we will describe the role of vimentin intermediate filaments in cancer cell migration, cell adhesion structures, and metastasis formation. The potential for targeting vimentin in cancer treatment and the development of drugs targeting vimentin will be reviewed.

Vimentin Intermediate Filaments as Potential Target for Cancer Treatment.

Autoinflammatory diseases are characterized by dysregulation of the innate immune system, leading to spontaneous inflammation.  mouse strain is a well-characterized model of this class of disorders. Because of the mutation leading to the lack of adaptor protein PSTPIP2, these animals suffer from autoinflammatory chronic multifocal osteomyelitis similar to several human syndromes. Current evidence suggests that it is driven by hyperproduction of IL-1β by neutrophil granulocytes. In this study, we show that in addition to IL-1β, PSTPIP2 also negatively regulates pathways governing reactive oxygen species generation by neutrophil NOX2 NADPH oxidase.  neutrophils display highly elevated superoxide production in response to a range of stimuli. Inactivation of NOX2 NADPH oxidase in  mice did not affect IL-1β levels, and the autoinflammatory process was initiated with similar kinetics. However, the bone destruction was almost completely alleviated, suggesting that dysregulated NADPH oxidase activity is a key factor promoting autoinflammatory bone damage in  mice.

Dysregulated NADPH Oxidase Promotes Bone Damage in Murine Model of Autoinflammatory Osteomyelitis.

Plectin, a highly versatile cytolinker protein, provides tissues with mechanical stability through the integration of intermediate filaments (IFs) with cell junctions. Here, we hypothesize that plectin-controlled cytoarchitecture is a critical determinant of the intestinal barrier function and homeostasis. Mice lacking plectin in an intestinal epithelial cell (IEC; Ple) spontaneously developed colitis characterized by extensive detachment of IECs from the basement membrane (BM), increased intestinal permeability, and inflammatory lesions. Moreover, plectin expression was reduced in the colons of ulcerative colitis (UC) patients and negatively correlated with the severity of colitis. Mechanistically, plectin deficiency in IECs led to aberrant keratin filament (KF) network organization and the formation of dysfunctional hemidesmosomes (HDs) and intercellular junctions. In addition, the hemidesmosomal α6β4 integrin (Itg) receptor showed attenuated association with KFs, and protein profiling revealed prominent downregulation of junctional constituents. Consistent with the effects of plectin loss in the intestinal epithelium, plectin-deficient IECs exhibited remarkably reduced mechanical stability and limited adhesion capacity in vitro. Feeding mice with a low-residue liquid diet that reduced mechanical stress and antibiotic treatment successfully mitigated epithelial damage in the Ple colon.

Plectin ensures intestinal epithelial integrity and protects colon against colitis.

ADAM10 and ADAM17 are proteases that affect multiple signalling pathways by releasing molecules from the cell surface. As their substrate specificities partially overlaps, we investigated their concurrent role in liver regeneration and fibrosis, using three liver-specific deficient mouse lines: ADAM10- and ADAM17-deficient lines, and a line deficient for both proteases. In the model of partial hepatectomy, double deficient mice exhibited decreased AKT phosphorylation, decreased release of EGFR activating factors and lower shedding of HGF receptor c-Met. Thus, simultaneous ablation of ADAM10 and ADAM17 resulted in inhibited EGFR signalling, while HGF/c-Met signalling pathway was enhanced. In contrast, antagonistic effects of ADAM10 and ADAM17 were observed in the model of chronic CCl intoxication. While ADAM10-deficient mice develop more severe fibrosis manifested by high ALT, AST, ALP and higher collagen deposition, combined deficiency of ADAM10 and ADAM17 surprisingly results in comparable degree of liver damage as in control littermates. Therefore, ADAM17 deficiency is not protective in fibrosis development per se, but can ameliorate the damaging effect of ADAM10 deficiency on liver fibrosis development. Furthermore, we show that while ablation of ADAM17 resulted in decreased shedding of TNF RI, ADAM10 deficiency leads to increased levels of soluble TNF RI in serum. In conclusion, hepatocyte-derived ADAM10 and ADAM17 are important regulators of growth receptor signalling and TNF RI release, and pathological roles of these proteases are dependent on the cellular context.

ADAM10 and ADAM17 regulate EGFR, c-Met and TNF RI signalling in liver regeneration and fibrosis.

Cellular force generation and force transmission are of fundamental importance for numerous biological processes and can be studied with the methods of Traction Force Microscopy (TFM) and Monolayer Stress Microscopy. Traction Force Microscopy and Monolayer Stress Microscopy solve the inverse problem of reconstructing cell-matrix tractions and inter- and intra-cellular stresses from the measured cell force-induced deformations of an adhesive substrate with known elasticity. Although several laboratories have developed software for Traction Force Microscopy and Monolayer Stress Microscopy computations, there is currently no software package available that allows non-expert users to perform a full evaluation of such experiments. Here we present pyTFM, a tool to perform Traction Force Microscopy and Monolayer Stress Microscopy on cell patches and cell layers grown in a 2-dimensional environment. pyTFM was optimized for ease-of-use; it is open-source and well documented (hosted at https://pytfm.readthedocs.io/) including usage examples and explanations of the theoretical background. pyTFM can be used as a standalone Python package or as an add-on to the image annotation tool ClickPoints. In combination with the ClickPoints environment, pyTFM allows the user to set all necessary analysis parameters, select regions of interest, examine the input data and intermediary results, and calculate a wide range of parameters describing forces, stresses, and their distribution. In this work, we also thoroughly analyze the accuracy and performance of the Traction Force Microscopy and Monolayer Stress Microscopy algorithms of pyTFM using synthetic and experimental data from epithelial cell patches.

pyTFM: A tool for traction force and monolayer stress microscopy.

Correction: Plectin ensures intestinal epithelial integrity and protects colon against colitis.

Fibrogenic processes instigate fatal chronic diseases leading to organ failure and death. Underlying biological processes involve induced massive deposition of extracellular matrix (ECM) by aberrant fibroblasts. We subjected diseased primary human lung fibroblasts to an advanced three-dimensional phenotypic high-content assay and screened a repurposing drug library of small molecules for inhibiting ECM deposition. Fibrotic Pattern Detection by Artificial Intelligence identified tranilast as an effective inhibitor. Structure-activity relationship studies confirmed -(2-butoxyphenyl)-3-(phenyl)acrylamides (N23Ps) as a novel and highly potent compound class. N23Ps suppressed myofibroblast transdifferentiation, ECM deposition, cellular contractility, and altered cell shapes, thus advocating a unique mode of action. Mechanistically, transcriptomics identified SMURF2 as a potential therapeutic target network. Antifibrotic activity of N23Ps was verified by proteomics in a human ex vivo tissue fibrosis disease model, suppressing profibrotic markers SERPINE1 and CXCL8. Conclusively, N23Ps are a novel class of highly potent compounds inhibiting organ fibrosis in patients.

Phenotypic drug screening in a human fibrosis model identified a novel class of antifibrotic therapeutics.

The coordinated interplay of cytoskeletal networks critically determines tissue biomechanics and structural integrity. Here, we show that plectin, a major intermediate filament-based cytolinker protein, orchestrates cortical cytoskeletal networks in epithelial sheets to support intercellular junctions. By combining CRISPR/Cas9-based gene editing and pharmacological inhibition, we demonstrate that in an F-actin-dependent context, plectin is essential for the formation of the circumferential keratin rim, organization of radial keratin spokes, and desmosomal patterning. In the absence of plectin-mediated cytoskeletal cross-linking, the aberrant keratin-desmosome (DSM)-network feeds back to the actin cytoskeleton, which results in elevated actomyosin contractility. Also, by complementing a predictive mechanical model with Förster resonance energy transfer-based tension sensors, we provide evidence that in the absence of cytoskeletal cross-linking, major intercellular junctions (adherens junctions and DSMs) are under intrinsically generated tensile stress. Defective cytoarchitecture and tensional disequilibrium result in reduced intercellular cohesion, associated with general destabilization of plectin-deficient sheets upon mechanical stress.

Plectin-mediated cytoskeletal crosstalk controls cell tension and cohesion in epithelial sheets.

Correction to: Plectin ensures intestinal epithelial integrity and protects colon against colitis.

Cells in the lungs, the heart, and numerous other organs, are constantly exposed to dynamic forces and deformations. To mimic these dynamic mechanical loading conditions and to study the resulting cellular responses such as morphological changes or the activation of biochemical signaling pathways, cells are typically seeded on flexible 2D substrates that are uniaxially or biaxially stretched. Here, we present an open-source cell stretcher built from parts of an Anet A8 3D printer. The cell stretcher is controlled by a fully programmable open-source software using GCode and Python. Up to six flexible optically clear substrates can be stretched simultaneously, allowing for comparative multi-batch biological studies including microscopic image analysis. The cell yield from the cell culture area of 4 cm per substrate is sufficient for Western-blot protein analysis. As a proof-of-concept, we study the activation of the Yes-associated protein (YAP) mechanotransduction pathway in response to increased cytoskeletal tension induced by uniaxial stretching of epithelial cells. Our data support the previously observed activation of the YAP transcription pathway by stretch-induced increase in cytoskeletal tension and demonstrate the suitability of the cell stretcher to study complex mechano-biological processes.

Martin Gregor

Laboratory of Integrative Biology

Institute of Molecular Genetics of the Czech Academy of Sciences

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Martin Gregor

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Integrative Biology

Institute of Molecular Genetics of the Czech Academy of Sciences

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Laboratory of Integrative Biology