Assembly of a chondrocyte-like pericellular matrix on non-chondrogenic cells. Role of the cell surface hyaluronan receptors in the assembly of a pericellular matrix

1991 ◽  
Vol 99 (2) ◽  
pp. 227-235 ◽  
Author(s):  
W. Knudson ◽  
C.B. Knudson
Keyword(s):  
Cell Surface ◽  
Plasma Membranes ◽  
Cell Types ◽  
Competitive Inhibitor ◽  
Live Cells ◽  
Pericellular Matrix ◽  
The Matrix ◽  
Express Cell ◽  
Hyaluronan Receptors

In this study, we have examined the capacity of various cell types, which express cell surface hyaluronan receptors, to organize a chondrocyte-like pericellular matrix when given chondrocyte-derived extracellular matrix macromolecules exogenously. The assembly of a pericellular matrix was visualized by a particle exclusion assay. Without the addition of exogenous macromolecular components, none of the cell types studied exhibited significant pericellular matrices extending from their plasma membranes. However, upon the addition of high molecular weight hyaluronan in combination with aggregating cartilage proteoglycan monomers, large pericellular matrices were formed within two hours of incubation. No pericellular matrices were formed if these macromolecular components were added separately at equivalent concentrations or if the components were added in the presence of hyaluronan hexasaccharide, a competitive inhibitor of hyaluronan interaction with cell surface hyaluronan receptors. Fully assembled pericellular matrices could also be displaced by the subsequent addition of hyaluronan hexasaccharides. Nonliving, glutaraldehyde-fixed cells, which retained functional hyaluronan receptors, maintained the capacity to assembly pericellular matrices with exogenous components, in serum-containing or serum-free medium. Cells that were incubated with exogenous matrix macromolecules for 24 h, followed by a chase incubation in medium minus the exogenous macromolecules, continued to maintain the matrix for up to 6 h on live cells and more than 24 h on glutaraldehyde-fixed cells. Cell types that did not express hyaluronan receptors were not capable of organizing such pericellular matrices when incubated with these exogenous components. These findings suggest that cells expressing hyaluronan receptors have a significant capacity to organize their immediate extracellular environment via hyaluronan-hyaluronan receptor interactions. Possible physiological functions for this type of matrix organizing capacity are discussed.

scholarly journals Generation of a Quantitative Luciferase Reporter for Sox9 SUMOylation

2020 ◽  
Vol 21 (4) ◽  
pp. 1274
Author(s):  
Hideka Saotome ◽  
Atsumi Ito ◽  
Atsushi Kubo ◽  
Masafumi Inui
Keyword(s):  
Cell Types ◽  
Live Cells ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Post Translational Modifications ◽  
Extracellular Signal ◽  
Multiple Cell ◽  
Sox9 Activity ◽  
Reporter Signal

Sox9 is a master transcription factor for chondrogenesis, which is essential for chondrocyte proliferation, differentiation, and maintenance. Sox9 activity is regulated by multiple layers, including post-translational modifications, such as SUMOylation. A detection method for visualizing the SUMOylation in live cells is required to fully understand the role of Sox9 SUMOylation. In this study, we generated a quantitative reporter for Sox9 SUMOylation that is based on the NanoBiT system. The simultaneous expression of Sox9 and SUMO1 constructs that are conjugated with NanoBiT fragments in HEK293T cells induced luciferase activity in SUMOylation target residue of Sox9-dependent manner. Furthermore, the reporter signal could be detected from both cell lysates and live cells. The signal level of our reporter responded to the co-expression of SUMOylation or deSUMOylation enzymes by several fold, showing dynamic potency of the reporter. The reporter was active in multiple cell types, including ATDC5 cells, which have chondrogenic potential. Finally, using this reporter, we revealed a extracellular signal conditions that can increase the amount of SUMOylated Sox9. In summary, we generated a novel reporter that was capable of quantitatively visualizing the Sox9-SUMOylation level in live cells. This reporter will be useful for understanding the dynamism of Sox9 regulation during chondrogenesis.

scholarly journals Hyaluronan as an Immune Regulator in Human Diseases

2011 ◽  
Vol 91 (1) ◽  
pp. 221-264 ◽  
Author(s):  
Dianhua Jiang ◽  
Jiurong Liang ◽  
Paul W. Noble
Keyword(s):  
Cell Surface ◽  
Tissue Injury ◽  
Inflammatory Cells ◽  
Cell Types ◽  
Surface Proteins ◽  
Human Diseases ◽  
Inflammatory Genes ◽  
Extracellular Matrix Components ◽  
Injury And Repair

Accumulation and turnover of extracellular matrix components are the hallmarks of tissue injury. Fragmented hyaluronan stimulates the expression of inflammatory genes by a variety of immune cells at the injury site. Hyaluronan binds to a number of cell surface proteins on various cell types. Hyaluronan fragments signal through both Toll-like receptor (TLR) 4 and TLR2 as well as CD44 to stimulate inflammatory genes in inflammatory cells. Hyaluronan is also present on the cell surface of epithelial cells and provides protection against tissue damage from the environment by interacting with TLR2 and TLR4. Hyaluronan and hyaluronan-binding proteins regulate inflammation, tissue injury, and repair through regulating inflammatory cell recruitment, release of inflammatory cytokines, and cell migration. This review focuses on the role of hyaluronan as an immune regulator in human diseases.

scholarly journals Endothelial Cell Inflammation and Barriers Are Regulated by the Rab26-Mediated Balance between β2-AR and TLR4 in Pulmonary Microvessel Endothelial Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Huaping Chen ◽  
Ming Yuan ◽  
Chunji Huang ◽  
Zhi Xu ◽  
Mingchun Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Membrane ◽  
Cell Surface ◽  
Cell Types ◽  
Tlr4 Expression ◽  
Barrier Dysfunction ◽  
Surface Receptors ◽  
G Protein Coupled

Rab26 GTPase modulates the trafficking of cell surface receptors, such as G protein-coupled receptors including α2-adrenergic receptors in some cell types. However, the effect of Rab26 on β2-adrenergic receptor (β2-AR) trafficking or/and Toll-like receptor 4 (TLR4) expression in human pulmonary microvascular endothelial cells (HPMECs) is still unclear. Here, we investigated the role of Rab26 in regulating the expression of β2-ARs and TLR4 in HPMECs and the effect of these receptors’ imbalance on endothelial cell barrier function. The results showed that there was unbalance expression in these receptors, where β2-AR expression was remarkably reduced, and TLR4 was increased on the cell membrane after lipopolysaccharide (LPS) treatment. Furthermore, we found that Rab26 overexpression not only upregulated β2-ARs but also downregulated TLR4 expression on the cell membrane. Subsequently, the TLR4-related inflammatory response was greatly attenuated, and the hyperpermeability of HPMECs also was partially relived. Taken together, these data suggest that basal Rab26 maintains the balance between β2-ARs and TLR4 on the cell surface, and it might be a potential therapeutic target for diseases involving endothelial barrier dysfunction.

Neutral sphingomyelinase inhibitor scyphostatin prevents and ceramide mimics mechanotransduction in vascular endothelium

2004 ◽  
Vol 287 (3) ◽  
pp. H1344-H1352 ◽  
Author(s):  
Malgorzata Czarny ◽  
Jan E. Schnitzer
Keyword(s):  
Endothelial Cell ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Fluid Shear Stress ◽  
Experimental Models ◽  
Surface Proteins ◽  
Rat Lung ◽  
Neutral Sphingomyelinase

Recently, we showed that neutral sphingomyelinase (N-SMase) is concentrated at the endothelial cell surface in caveolae and is activated to produce ceramide in an acute and transient manner by increase in flow rate and pressure in rat lung vasculature (Czarny M, Liu J, Oh P, and Schnitzer JE, J Biol Chem 278: 4424–4430, 2003). Here, we report further on our investigations of this new acute mechanotransduction pathway. We employed three experimental models to explore the role of N-SMase and ceramides in mechanosignaling: 1) a cell-free, in vitro model using isolated luminal plasma membranes of rat lung endothelium; 2) a fluid shear stress model using monolayers of intact bovine aorta endothelial cell in culture; and 3) an in situ model using controlled perfusion of the rat lung vasculature. Scyphostatin, which specifically inhibited N-SMase but not acid SMase activity, prevented mechanoactivation of N-SMase as well as downstream tyrosine and mitogen-activated protein kinases. Cell-permeable ceramide analogs ( N-acetylsphingosine, C2-ceramide, and N-hexanoylsphingosine, C6-ceramide) but not the inactive dihydroderivatives D2-ceramide and D6-ceramide ( N-acetylsphinganine and N-hexanoylsphinganine, respectively) mimic rapid mechano-induced tyrosine phosphorylation of cell surface proteins as well as mechanoactivation of Src-like kinases and the extracellular regulated kinase pathway. The responses common to ceramide and mechanical stress were inhibited by genistein, herbamycin A, and PP2, but not PP3, which suggests an obligate role of Src-like kinases in ceramide-mediated mechanotransduction. Ceramides also induced serine/threonine phosphorylation to activate the Akt/endothelial nitric oxide synthase pathway. Thus N-SMase at the plasma membrane in caveolae may be an upstream initiating mechanosensor, which acutely triggers mechanotransduction by generation of the lipid second messenger ceramide.

scholarly journals Amino acid transporter B0AT1 influence on ADAM17 interactions with SARS-CoV-2 receptor ACE2 putatively expressed in intestine, kidney, and cardiomyocytes

2020 ◽  
Author(s):  
Jacob T. Andring ◽  
Robert McKenna ◽  
Bruce R. Stevens
Keyword(s):  
Amino Acid ◽  
Plasma Membranes ◽  
Renin Angiotensin System ◽  
Neck Region ◽  
Cell Types ◽  
Amino Acid Transporter ◽  
Common Denominator ◽  
Organ Systems ◽  
Acid Transporter

ABSTRACTSARS-CoV-2 exhibits significant experimental and clinical gastrointestinal, renal, and cardiac muscle tropisms responsible for local tissue-specific and systemic pathophysiology capriciously occurring in about half of COVID-19 patients. The underlying COVID-19 mechanisms engaged by these extra-pulmonary organ systems are largely unknown. We approached this knowledge gap by recognizing that neutral amino acid transporter B0AT1 (alternately called NBB, B, B0 in the literature) is a common denominator expressed nearly exclusively by three particular cell types: intestinal epithelia, renal proximal tubule epithelium, and cardiomyocytes. B0AT1 provides uptake of glutamine and tryptophan. The gut is the main depot expressing over 90% of the body’s entire pool of SARS-CoV-2 receptor angiotensin converting enzyme-2 (ACE2) and B0AT1. Recent cryo-EM studies established that ACE2 forms a thermodynamically favored dimer-of-heterodimers complex with B0AT1 assembled in the form of a dimer of two ACE2:B0AT1 heterodimers anchored in plasma membranes. Prior epithelial cell studies demonstrated ACE2 chaperone trafficking of B0AT1. This contrasts with monomeric expression of ACE2 in lung pneumocytes, in which B0AT1 is undetectable. The cell types in question also express a disintegrin and metalloproteinase-17 (ADAM17) known to cleave and shed the ectodomain of monomeric ACE2 from the cell surface, thereby relinquishing protection against unchecked renin-angiotensin-system (RAS) events of COVID-19. The present study employed molecular docking modeling to examine the interplaying assemblage of ACE2, ADAM17 and B0AT1. We report that in the monomer form of ACE2, neck region residues R652-N718 provide unimpeded access to ADAM17 active site pocket, but notably R708 and S709 remained >10-15 Å distant. In contrast, interference of ADAM17 docking to ACE2 in a dimer-of-heterodimers arrangement was directly correlated with the presence of a neighboring B0AT1 subunit complexed to the partnering ACE2 subunit of the 2ACE2:2B0AT1] dimer of heterodimers, representing the expression pattern putatively exclusive to intestinal, renal and cardiomyocyte cell types. The monomer and dimer-of-heterodimers docking models were not influenced by the presence of SARS-CoV-2 receptor binding domain (RBD) complexed to ACE2. The results collectively provide the underpinnings for understanding the role of B0AT1 involvement in COVID-19 and the role of ADAM17 steering ACE2 events in intestinal and renal epithelial cells and cardiomyocytes, with implications useful for consideration in pandemic public hygiene policy and drug development.

scholarly journals Automated analysis of invadopodia dynamics in live cells

2014 ◽  
Author(s):  
Matthew E Berginski ◽  
Sarah J Creed ◽  
Shelly Cochran ◽  
David W Roadcap ◽  
James E Bear ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Metastatic Cancer ◽  
Automated Analysis ◽  
Time Lapse ◽  
Cell Types ◽  
Live Cells ◽  
Imaging Data ◽  
The Matrix ◽  
Invadopodia Formation

Multiple cell types form specialized protein complexes, podosomes or invadopodia and collectively referred to as invadosomes, which are used by the cell to actively degrade the surrounding extracellular matrix. Due to their potential importance in both healthy physiology as well as in pathological conditions such as cancer, the characterization of these structures has been of increasing interest. Following early descriptions of invadopodia, assays were developed which labelled the matrix underneath metastatic cancer cells allowing for the assessment of invadopodia activity in motile cells. However, characterization of invadopodia using these methods has traditionally been done manually with time-consuming and potentially biased quantification methods, limiting the number of experiments and the quantity of data that can be analysed. We have developed a system to automate the segmentation, tracking and quantification of invadopodia in time-lapse fluorescence image sets at both the single invadopodia level and whole cell level. We rigorously tested the ability of the method to detect changes in invadopodia formation and dynamics through the use of well-characterized small molecule inhibitors, with known effects on invadopodia. Our results demonstrate the ability of this analysis method to quantify changes in invadopodia formation from live cell imaging data in a high throughput, automated manner.

scholarly journals Integrins alpha v beta 3 and alpha v beta 5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface.

1991 ◽  
Vol 113 (4) ◽  
pp. 919-929 ◽  
Author(s):  
E A Wayner ◽  
R A Orlando ◽  
D A Cheresh
Keyword(s):  
Cell Surface ◽  
Lung Carcinoma ◽  
Cell Attachment ◽  
Cell Types ◽  
Carcinoma Cells ◽  
Lung Carcinoma Cells ◽  
Distinct Cell ◽  
Contact Distribution ◽  
Alpha V Beta 3

We investigated the role of the integrins alpha v beta 3 and alpha v beta 5 in mediating vitronectin adhesion of three phenotypically distinct cell types. M21 human melanoma cells and H2981 lung carcinoma cells use both alpha v-containing integrins in adhering to vitronectin while UCLA-P3 lung carcinoma cells adhere exclusively with alpha v beta 5. Specifically, monoclonal antibodies directed to functional epitopes on both receptors were required to block adhesion of M21 or H2981 cells while adhesion of UCLA-P3 cells to vitronectin could be blocked with a monoclonal antibody to alpha v beta 5. Although both receptors are involved in M21 and H2981 cell adhesion to vitronectin, only alpha v beta 3 can be detected in focal contacts, colocalizing with vinculin, talin, and the ends of actin filaments, while alpha v beta 5 shows a distinct, nonfocal contact, distribution on the cell surface. These results provide the first evidence that two homologous integrins that recognize the same ligand distribute differentially on the cell surface.

scholarly journals Immunohistochemical localization of cellCAM 105 in rat tissues: appearance in epithelia, platelets, and granulocytes.

1988 ◽  
Vol 36 (7) ◽  
pp. 729-739 ◽  
Author(s):  
P Odin ◽  
M Asplund ◽  
C Busch ◽  
B Obrink
Keyword(s):  
Cell Surface ◽  
Plasma Membranes ◽  
Surface Membrane ◽  
Cell Types ◽  
Cell Contact ◽  
Rat Tissues ◽  
Common Denominator ◽  
Surface Distribution ◽  
Organ Systems ◽  
Cell Cell

CellCAM 105 is an integral membrane glycoprotein, with apparent Mr 105,000, which has been purified from rat liver plasma membranes. It consists of two structurally similar, highly glycosylated polypeptide chains and is involved in cell-cell adhesion of adult rat hepatocytes in vitro. In this communication we report on the distribution and cell surface location of cellCAM 105 in rat tissues, obtained by using highly sensitive immunodetection systems based on complex formation between biotinylated antibodies, biotinylated peroxidase and avidin, or on antibodies coupled to alkaline phosphatase. CellCAM was found in many organs and organ systems, including liver, kidney, blood, blood vessels, glands, respiratory system, and gastrointestinal tract. It was mainly localized to epithelial structures but showed a varying cell surface distribution. In some cell types it was predominantly localized to cell-cell contact areas. In other cell types the highest concentrations were seen in brush-border areas containing densely packed microvilli. In addition to epithelial structures, cellCAM 105 was found in rat platelets, where it became strongly expressed on the cell surfaces after activation with ADP or collagen, suggesting that it might be involved in platelet adhesion and/or aggregation mechanisms. Granulocytes also contained cellCAM 105. By SDS-PAGE/immunoblotting, significant differences were found in the apparent Mr values of cellCAM 105 in different tissues. The collected data suggest that cellCAM 105 participates in several different cell surface membrane interactions, of which the common denominator might be membrane-membrane binding.

scholarly journals Linkage of extracellular plasminogen activator to the fibroblast cytoskeleton: colocalization of cell surface urokinase with vinculin.

1988 ◽  
Vol 106 (4) ◽  
pp. 1241-1247 ◽  
Author(s):  
C A Hébert ◽  
J B Baker
Keyword(s):  
Cell Surface ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Human Fibroblasts ◽  
Cell Types ◽  
Actin Binding ◽  
Double Labeling ◽  
Amino Acid Peptide ◽  
Adhesion Sites ◽  
Fibrosarcoma Cells

Several cell types display binding sites for [125I]urokinase (Vassalli, J.-D., D. Baccino, D. Belin. 1985. J. Cell Biol. 100:86-92) which in certain cases are occupied with endogenous urokinase. These sites appear to focus urokinase at cell surfaces and hence may participate in tissue matrix destruction and cell invasion. Recently Pöllänen et al. (1987) demonstrated that the cell surface urokinase of human fibroblasts and fibrosarcoma cells is deposited underneath the cells in strands, apparently at sites of cell-to-substratum contact. Here, using immunofluorescence double labeling, we show that the urokinase strands present on human foreskin fibroblasts are colocalized with strands of vinculin, an intracellular actin-binding protein that is deposited at cell-to-substratum focal adhesion sites. Thus, this indicates linkage of the plasminogen/plasmin system both to sites of cell adhesion and to the cytoskeleton. The urokinase strands on HT 1080 fibrosarcoma cells are more numerous and have shapes that are more tortuous than those on normal fibroblasts. In intact HT 1080 cells, colocalized vinculin strands are obscured by an intense background of soluble vinculin but are apparent on isolated ventral plasma membranes. Certain properties of the urokinase strands suggest that they are related to the [125I]urokinase-binding sites that have been described by several groups: (a) incubating fibroblasts with dexamethasone for 48 h or at pH 3 at 5 degrees C for 10 min greatly decreases the number and intensity of the urokinase strands; (b) strands reappear when glucocorticoid-treated cells are incubated with exogenous 54-kD (but not 35-kD) urokinase, and this process is inhibited by a previously described 16-amino acid peptide that blocks [125I]urokinase binding to the cells.

scholarly journals Aquaporin-4 Dynamics in Orthogonal Arrays in Live Cells Visualized by Quantum Dot Single Particle Tracking

2008 ◽  
Vol 19 (8) ◽  
pp. 3369-3378 ◽  
Author(s):  
Jonathan M. Crane ◽  
Alfred N. Van Hoek ◽  
William R. Skach ◽  
A. S. Verkman
Keyword(s):  
Plasma Membranes ◽  
Freeze Fracture ◽  
Orthogonal Arrays ◽  
Cell Types ◽  
Single Particle Tracking ◽  
Aquaporin 4 ◽  
Live Cells ◽  
Restoring Force ◽  
Orthogonal Arrays Of Particles ◽  
Freeze Fracture Electron Microscopy

Freeze-fracture electron microscopy (FFEM) indicates that aquaporin-4 (AQP4) water channels can assemble in cell plasma membranes in orthogonal arrays of particles (OAPs). We investigated the determinants and dynamics of AQP4 assembly in OAPs by tracking single AQP4 molecules labeled with quantum dots at an engineered external epitope. In several transfected cell types, including primary astrocyte cultures, the long N-terminal “M1” form of AQP4 diffused freely, with diffusion coefficient ∼5 × 10−10 cm2/s, covering ∼5 μm in 5 min. The short N-terminal “M23” form of AQP4, which by FFEM was found to form OAPs, was relatively immobile, moving only ∼0.4 μm in 5 min. Actin modulation by latrunculin or jasplakinolide did not affect AQP4-M23 diffusion, but deletion of its C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding domain increased its range by approximately twofold over minutes. Biophysical analysis of short-range AQP4-M23 diffusion within OAPs indicated a spring-like potential, with a restoring force of ∼6.5 pN/μm. These and additional experiments indicated that 1) AQP4-M1 and AQP4-M23 isoforms do not coassociate in OAPs; 2) OAPs can be imaged directly by total internal reflection fluorescence microscopy; and 3) OAPs are relatively fixed, noninterconvertible assemblies that do not require cytoskeletal or PDZ-mediated interactions for formation. Our measurements are the first to visualize OAPs in live cells.

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