THE SECRETED LEUKOCYTE PROTEIN HETERODIMER S100A8/A9 DISSOCIATES PANCREATIC CELL-CELL CONTACTS IN VIVO

AbstractIn the homeostatic liver, ductal cells intermingle with a microenvironment of endothelial and mesenchymal cells to form the functional unit of the portal tract. Ductal cells proliferate rarely in homeostasis but do so transiently after tissue injury to replenish any lost epithelium. We have shown that liver ductal cells can be expanded as liver organoids that recapitulate several of the cell-autonomous mechanisms of regeneration, but lack the stromal cell milieu of the biliary tract in vivo. Here, we describe a subpopulation of SCA1+ periportal mesenchymal cells that closely surrounds ductal cells in vivo and exerts a dual control on their proliferative capacity. Mesenchymal-secreted mitogens support liver organoid formation and expansion from differentiated ductal cells. However, direct mesenchymal-to-ductal cell-cell contact, established following a microfluidic co-encapsulation that enables the cells to self-organize into chimeric organoid structures, abolishes ductal cell proliferation in a mesenchyme-dose dependent manner. We found that it is the ratio between mesenchymal and epithelial cell contacts that determines the net outcome of ductal cell proliferation both in vitro, and in vivo, during damage-regeneration. SCA1+ mesenchymal cells control ductal cell proliferation dynamics by a mechanism involving, at least in part, Notch signalling activation. Our findings underscore how the relative abundance of cell-cell contacts between the epithelium and its mesenchymal microenvironment are key regulatory cues involved in the control of tissue regeneration.SummaryIn the homeostatic liver, the ductal epithelium intermingles with a microenvironment of stromal cells to form the functional unit of the portal tract. Ductal cells proliferate rarely in homeostasis but do so transiently after tissue injury. We have shown that these cells can be expanded as liver organoids that recapitulate several of the cell-autonomous mechanisms of regeneration, but lack the stromal cell milieu of the portal tract in vivo. Here, we describe a subpopulation of SCA1+ periportal mesenchymal niche cells that closely surrounds ductal cells in vivo and exerts a dual control on their proliferative capacity. Mesenchymal-secreted mitogens support liver organoid formation and expansion from differentiated ductal cells. However, direct mesenchymal-to-ductal cell-cell contact, established through a microfluidic co-encapsulation method that enables the cells to self-organize into chimeric organoid structures, abolishes ductal cell proliferation in a mesenchyme-dose dependent manner. We found that it is the ratio between mesenchymal and epithelial cell contacts that determines the net outcome of ductal cell proliferation both in vitro, and in vivo, during damage-regeneration. SCA1+ mesenchymal cells control ductal cell proliferation dynamics by a mechanism involving, at least in part, Notch signalling activation. Our findings re-evaluate the concept of the cellular niche, whereby the proportions of cell-cell contacts between the epithelium and its mesenchymal niche, and not the absolute cell numbers, are the key regulatory cues involved in the control of tissue regeneration.

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Direct laser manipulation reveals the mechanics of cell contacts in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1418732112 ◽

2015 ◽

Vol 112 (5) ◽

pp. 1416-1421 ◽

Cited By ~ 123

Author(s):

Kapil Bambardekar ◽

Raphaël Clément ◽

Olivier Blanc ◽

Claire Chardès ◽

Pierre-François Lenne

Keyword(s):

Cell Shape ◽

Constitutive Law ◽

Cell Junctions ◽

Scale Model ◽

Tissue Morphogenesis ◽

Cell Contacts ◽

Cell Shape Changes ◽

Shape Changes ◽

Cell Cell

Cell-generated forces produce a variety of tissue movements and tissue shape changes. The cytoskeletal elements that underlie these dynamics act at cell–cell and cell–ECM contacts to apply local forces on adhesive structures. In epithelia, force imbalance at cell contacts induces cell shape changes, such as apical constriction or polarized junction remodeling, driving tissue morphogenesis. The dynamics of these processes are well-characterized; however, the mechanical basis of cell shape changes is largely unknown because of a lack of mechanical measurements in vivo. We have developed an approach combining optical tweezers with light-sheet microscopy to probe the mechanical properties of epithelial cell junctions in the early Drosophila embryo. We show that optical trapping can efficiently deform cell–cell interfaces and measure tension at cell junctions, which is on the order of 100 pN. We show that tension at cell junctions equilibrates over a few seconds, a short timescale compared with the contractile events that drive morphogenetic movements. We also show that tension increases along cell interfaces during early tissue morphogenesis and becomes anisotropic as cells intercalate during germ-band extension. By performing pull-and-release experiments, we identify time-dependent properties of junctional mechanics consistent with a simple viscoelastic model. Integrating this constitutive law into a tissue-scale model, we predict quantitatively how local deformations propagate throughout the tissue.

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Vinculin Is Part of the Cadherin–Catenin Junctional Complex: Complex Formation between α-Catenin and Vinculin

The Journal of Cell Biology ◽

10.1083/jcb.141.3.755 ◽

1998 ◽

Vol 141 (3) ◽

pp. 755-764 ◽

Cited By ~ 180

Author(s):

Elisabeth E. Weiss ◽

Martina Kroemker ◽

Angelika-H. Rüdiger ◽

Brigitte M. Jockusch ◽

Manfred Rüdiger

Keyword(s):

Complex Formation ◽

Transmembrane Protein ◽

Adherens Junction ◽

Focal Adhesions ◽

Junctional Complex ◽

Cell Contacts ◽

Junction Protein ◽

Cell Cell

In epithelial cells, α-, β-, and γ-catenin are involved in linking the peripheral microfilament belt to the transmembrane protein E-cadherin. α-Catenin exhibits sequence homologies over three regions to vinculin, another adherens junction protein. While vinculin is found in cell–matrix and cell–cell contacts, α-catenin is restricted to the latter. To elucidate, whether vinculin is part of the cell–cell junctional complex, we investigated complex formation and intracellular targeting of vinculin and α-catenin. We show that α-catenin colocalizes at cell–cell contacts with endogenous vinculin and also with the transfected vinculin head domain forming immunoprecipitable complexes. In vitro, the vinculin NH2-terminal head binds to α-catenin, as seen by immunoprecipitation, dot overlay, cosedimentation, and surface plasmon resonance measurements. The Kd of the complex was determined to 2–4 × 10−7 M. As seen by overlays and affinity mass spectrometry, the COOH-terminal region of α-catenin is involved in this interaction. Complex formation of vinculin and α-catenin was challenged in transfected cells. In PtK2 cells, intact α-catenin and α-catenin1-670, harboring the β-catenin– binding site, were directed to cell–cell contacts. In contrast, α-catenin697–906 fragments were recruited to cell–cell contacts, focal adhesions, and stress fibers. Our results imply that in vivo α-catenin, like vinculin, is tightly regulated in its ligand binding activity.

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Cell based strain stiffening of a non-fibrous matrix as organizing principle for morphogenesis

10.1101/816496 ◽

2019 ◽

Author(s):

Daniel Rüdiger ◽

Kerstin Kick ◽

Andriy Goychuk ◽

Angelika M. Vollmar ◽

Erwin Frey ◽

...

Keyword(s):

Extracellular Matrix ◽

Pattern Formation ◽

Basement Membrane ◽

Self Assembly ◽

Matrix Material ◽

Cell Contacts ◽

Cell Contractility ◽

Cell Cell

AbstractEndothelial tube formation on a reconstituted extracellular matrix (Matrigel) is a well-established in vitro model for studying the processes of angiogenesis and vasculogenesis. However, to date, the organizing principles that underlie the morphogenesis of this network, and that shape the initial process of cell-cell finding remain elusive. Furthermore, it is unclear how in vitro results extrapolate to in vivo morphogenesis. Here, we identify a mechanism that allows cells to form networks by mechanically reorganizing and stiffening their extracellular matrix, independent of chemical guidance cues. Interestingly, we find that this cellular self-organization strongly depends on the connectivity and topology of the surrounding matrix, as well as on cell contractility and cell density. Cells rearrange the matrix, and form bridges of matrix material that are stiffer than their surroundings, thus creating a durotactic track for the initiation of cell-cell contacts. This contractility-based communication via strain stiffening and matrix rearrangement might be a general organizing principle during tissue development or regeneration.Significance StatementIn addition to chemotactic gradients, biomechanical cues are important for guiding biological pattern formation. Self-assembly of cells has often been ascribed to reorganization of collagen fibres in the extracellular matrix. However, the basement membrane surrounding vascular cells, is per se non-fibrous. Here, we find that this difference in matrix topology can crucially influence cell behaviour and pattern formation. In a homogeneously elastic environment like the basement membrane, endothelial cells rearrange extracellular matrix proteins by contractile force, forming stiff intercellular bridges as tracks for cell-cell contacts. Our findings shine some light why there is a lot of merit in having multiple approaches to matrix elasticity (like continuum theories or dilated network approaches). Our observations might help to understand why vascular nets look different in different tissues and after rearrangement of the extracellular matrix during disease.

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Dismantling Cell–Cell Contacts during Apoptosis Is Coupled to a Caspase-dependent Proteolytic Cleavage of β-Catenin

The Journal of Cell Biology ◽

10.1083/jcb.139.3.759 ◽

1997 ◽

Vol 139 (3) ◽

pp. 759-771 ◽

Cited By ~ 156

Author(s):

Claudio Brancolini ◽

Dean Lazarevic ◽

Joe Rodriguez ◽

Claudio Schneider

Keyword(s):

Cell Death ◽

Cell Adhesion ◽

Actin Cytoskeleton ◽

Caspase 3 ◽

Cell Types ◽

Cell Contacts ◽

Carboxy Terminal ◽

Cell Cell

Cell death by apoptosis is a tightly regulated process that requires coordinated modification in cellular architecture. The caspase protease family has been shown to play a key role in apoptosis. Here we report that specific and ordered changes in the actin cytoskeleton take place during apoptosis. In this context, we have dissected one of the first hallmarks in cell death, represented by the severing of contacts among neighboring cells. More specifically, we provide demonstration for the mechanism that could contribute to the disassembly of cytoskeletal organization at cell–cell adhesion. In fact, β-catenin, a known regulator of cell–cell adhesion, is proteolytically processed in different cell types after induction of apoptosis. Caspase-3 (cpp32/apopain/yama) cleaves in vitro translated β-catenin into a form which is similar in size to that observed in cells undergoing apoptosis. β-Catenin cleavage, during apoptosis in vivo and after caspase-3 treatment in vitro, removes the amino- and carboxy-terminal regions of the protein. The resulting β-catenin product is unable to bind α-catenin that is responsible for actin filament binding and organization. This evidence indicates that connection with actin filaments organized at cell–cell contacts could be dismantled during apoptosis. Our observations suggest that caspases orchestrate the specific and sequential changes in the actin cytoskeleton occurring during cell death via cleavage of different regulators of the microfilament system.

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RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes

Molecular Biology of the Cell ◽

10.1091/mbc.e09-10-0859 ◽

2011 ◽

Vol 22 (5) ◽

pp. 593-605 ◽

Cited By ~ 87

Author(s):

Ben Jackson ◽

Karine Peyrollier ◽

Esben Pedersen ◽

Astrid Basse ◽

Richard Karlsson ◽

...

Keyword(s):

Normal Skin ◽

Cell Spreading ◽

Fiber Formation ◽

Cell Contraction ◽

Skin Development ◽

Cell Contacts ◽

Directed Migration ◽

Cell Cell

RhoA is a small guanosine-5’-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell–cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell–cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC–mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.

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Generation of Bioactive Nanostructured Interfaces to Mimic Cellular Microenvironments

MRS Proceedings ◽

10.1557/proc-1062-nn03-04 ◽

2007 ◽

Vol 1062 ◽

Author(s):

Tobias Wolfram ◽

Ferdinand Belz ◽

Tobias Schoen ◽

Daniel Aydin ◽

Joachim P Spatz

Keyword(s):

Cell Adhesion ◽

Neuronal Cell ◽

Target Cells ◽

Length Scale ◽

Primary Fibroblast ◽

Cell Contacts ◽

Nanometer Length Scale ◽

Cellular Microenvironments ◽

Cell Cell

ABSTRACTThere is currently great interest in material science to mimic cellular microenvironments with controlled bioactive features and biomolecule presentation at the nanometer length scale. Target cells exposed to those 2- or 3D-substrates are stimulated to manifest a variety of cellular functions like cell adhesion, migration and differentiation.Based on micellar nanolithography, we developed an experimental setup to present single biomolecules on a 2D-nanoarray over large areas on cell-adhesive or cell-repellent substrates. The spacing between biomolecules can be adjusted to 30-250 nm. To mimic a more complex cellular micro-environment with cell-cell contacts as well as cell-extracellular matrix (ECM) contacts, we used biomolecules from different cell adhesion molecule families, like cadherins, immunglobulin-superfamily, integrins and laminins. Via a Ni2+- nitrilo triacetic acid (NTA) system, the amount and the orientation of proteins is chemically controlled such that it resembles the native in vivo settings in the cellular microenvironment. By using poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG) molecules, we generated 2D-protein-nanoarrays with a cell repellent background matrix. After modification of this matrix we incorporated small bioactive peptides to create a microenvironment which allows cell adhesion. These approaches resulted in substrates with specific presentation of biomolecules to mimic cell-cell contacts and cell-ECM interactions simultaneously.With these substrates we investigated primary fibroblast and neuronal cell adhesion mediated by different biomolecules and with different nanometer spacing. We quantified neurite outgrowth of dorsal root ganglion (DRG) neurons as a differentiation paradigm on nanostructured substrates. Gold nanoparticles were functionalized with peptides, containing the arginine-glycine-aspartic acid (RGD) motif, an integrin activation sequence. Cell adhesion as well as differentiation showed a dependency on the nanometer length scale and on the presented biomolecules.

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Ordered Assembly of the Adhesive and Electrochemical Connections within Newly Formed Intercalated Disks in Primary Cultures of Adult Rat Cardiomyocytes

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/624719 ◽

2010 ◽

Vol 2010 ◽

pp. 1-14 ◽

Cited By ~ 26

Author(s):

Sarah B. Geisler ◽

Kathleen J. Green ◽

Lori L. Isom ◽

Sasha Meshinchi ◽

Jeffrey R. Martens ◽

...

Keyword(s):

Cardiac Myocytes ◽

Heart Development ◽

Complex Structure ◽

Primary Cultures ◽

Rat Cardiomyocytes ◽

Fully Integrated ◽

Cell Contacts ◽

Adult Rat ◽

Cell Cell

The intercalated disk (ID) is a complex structure that electromechanically couples adjoining cardiac myocytes into a functional syncitium. The integrity of the disk is essential for normal cardiac function, but how the diverse elements are assembled into a fully integrated structure is not well understood. In this study, we examined the assembly of new IDs in primary cultures of adult rat cardiac myocytes. From 2 to 5 days after dissociation, the cells flatten and spread, establishing new cell-cell contacts in a manner that recapitulates the in vivo processes that occur during heart development and myocardial remodeling. As cells make contact with their neighbors, transmembrane adhesion proteins localize along the line of apposition, concentrating at the sites of membrane attachment of the terminal sarcomeres. Cx43 gap junctions and ankyrin-G, an essential cytoskeletal component of voltage gated sodium channel complexes, were secondarily recruited to membrane domains involved in cell-cell contacts. The consistent order of the assembly process suggests that there are specific scaffolding requirements for integration of the mechanical and electrochemical elements of the disk. Defining the relationships that are the foundation of disk assembly has important implications for understanding the mechanical dysfunction and cardiac arrhythmias that accompany alterations of ID architecture.

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Non-junctional Cadherin3 regulates cell migration and contact inhibition of locomotion via domain-dependent opposing regulations of Rac1

10.1101/750752 ◽

2019 ◽

Author(s):

Takehiko Ichikawa ◽

Carsten Stuckenholz ◽

Lance A. Davidson

Keyword(s):

Cell Migration ◽

Contact Inhibition ◽

Cell Contact ◽

Collective Cell Migration ◽

Cell Contacts ◽

Rac1 Activity ◽

Classical Cadherins ◽

The Stability ◽

Cell Cell

AbstractClassical cadherins are well-known primary adhesion molecules responsible for physically connecting neighboring cells and signaling the cell-cell contact. Recent studies have suggested novel signaling roles for “non-junctional” cadherins (Niessen and Gottardi, 2008; Padmanabhan et al., 2017); however, the function of cadherin signaling independent of cell-cell contacts remains unknown. In this study, we used mesendoderm cells and tissues from gastrula stage Xenopus laevis embryos to demonstrate that extracellular and cytoplasmic cadherin domains regulate Rac1 in opposing directions in the absence of cell-cell contacts. Furthermore, we found that non-junctional cadherins regulate contact inhibition of locomotion (CIL) during gastrulation through alterations in the stability of the cytoskeleton. Live FRET imaging of Rac1 activity illustrated how non-junction cadherin3 (formerly C-cadherin) spatio-temporally regulates CIL. Our study provides novel insights into adhesion-independent signaling by cadherin3 and its role in regulating single and collective cell migration in vivo.

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Monolayer and aggregate cultures of rainbow trout hepatocytes: long-term and stable liver-specific expression in aggregates

Journal of Cell Science ◽

10.1242/jcs.105.2.407 ◽

1993 ◽

Vol 105 (2) ◽

pp. 407-416 ◽

Cited By ~ 1

Author(s):

G. Flouriot ◽

C. Vaillant ◽

G. Salbert ◽

C. Pelissero ◽

J.M. Guiraud ◽

...

Keyword(s):

Estrogen Receptor ◽

Rainbow Trout ◽

Hormonal Response ◽

Specific Expression ◽

Cell Contacts ◽

Vitellogenin Synthesis ◽

Estradiol Stimulation ◽

Cell Cell

An aggregate culture system for rainbow trout hepatocytes was developed to study liver-specific mRNA expression. Maintenance of differentiated functions and morphology of hepatocytes were examined using both monolayer and aggregate systems. The rainbow trout estrogen receptor and vitellogenin genes were induced by estradiol and their mRNAs used as markers of cell differentiation during cell culture. In monolayer culture, rainbow trout hepatocytes established very few cell-cell contacts in minimal media. The use of more complete media promotes cell-cell contacts and cell islet formation. Hepatocyte response to estradiol stimulation was generally lower than in vivo but a correlation between the degree of cellular organization and the intensity of the hormonal response was observed. However, in this system hepatocytes progressively lost their specific hormonal response between 5 and 10 days. In aggregates with DMEM/F12 and Ultroser SF, cell-cell contacts were maximized and stabilized during at least one month. The levels of rainbow trout estrogen receptor and vitellogenin mRNAs induced by estradiol were stable and maintained at a level comparable to in vivo levels; vitellogenin synthesis and secretion remained fully functional for the duration of the culture.

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