scholarly journals Intermediate filaments and the initiation of desmosome assembly.

1985 ◽  
Vol 101 (2) ◽  
pp. 506-517 ◽  
Author(s):  
J C Jones ◽  
R D Goldman
Keyword(s):  
Cell Surface ◽  
Intermediate Filaments ◽  
Ultrastructural Localization ◽  
Free Cell ◽  
Cell Protein ◽  
Epidermal Keratinocytes ◽  
Cell Contact ◽  
Cell Surfaces ◽  
Primary Mouse ◽  
And Function

The desmosome junction is an important component in the cohesion of epithelial cells, especially epidermal keratinocytes. To gain insight into the structure and function of desmosomes, their morphogenesis has been studied in a primary mouse epidermal (PME) cell culture system. When these cells are grown in approximately 0.1 mM Ca2+, they contain no desmosomes. They are induced to form desmosomes when the Ca2+ level in the culture medium is raised to approximately 1.2 mM Ca2+. PME cells in medium containing low levels of Ca2+, and then processed for indirect immunofluorescence using antibodies directed against desmoplakins (desmosomal plaque proteins), display a pattern of discrete fluorescent spots concentrated mainly in the perinuclear region. Double label immunofluorescence using keratin and desmoplakin antibodies reveals that the desmoplakin-containing spots and the cytoplasmic network of tonofibrils (bundles of intermediate filaments [IFB]) are in the same juxtanuclear region. Within 1 h after the switch to higher levels of Ca2+, the spots move toward the cell surface, primarily to areas of cell-cell contact and not to free cell surfaces. This reorganization occurs at the same time that tonofibrils also move toward cell surfaces in contact with neighboring cells. Once the desmoplakin spots have reached the cell surface, they appear to aggregate to form desmosomes. These immunofluorescence observations have been confirmed by immunogold ultrastructural localization. Preliminary biochemical and immunological studies indicate that desmoplakin appears in whole cell protein extracts and in Triton high salt insoluble residues (i.e., cytoskeletal preparations consisting primarily of IFB) prepared from PME cells maintained in medium containing both low and normal Ca2+ levels. These findings show that certain desmosome components are preformed in the cytoplasm of PME cells. These components undergo a dramatic reorganization, which parallels the changes in IFB redistribution, upon induction of desmosome formation. The reorganization depends upon both the extracellular Ca2+ level and the establishment of cell-to-cell contacts. Furthermore, the data suggests that desmosomes do not act as organizing centers for the elaboration of IFB. Indeed, we postulate that the movement of IFB and preformed desmosomal components to the cell surface is an important initiating event in desmosome morphogenesis.

Effect of glycoPEGylation on Factor VIIa Binding and Internalization

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1029-1029
Author(s):  
Prosenjit Sen ◽  
Samit Ghosh ◽  
Pernille K Holm ◽  
Mirella Ezban ◽  
Usha Pendurthi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Cho Cells ◽  
Radioligand Binding ◽  
Cell Protein ◽  
Activate Factor ◽  
Factor X ◽  
Severe Hemophilia ◽  
Cell Surfaces ◽  
Binding Studies

Abstract Frequent spontaneous joint bleedings in severe hemophilia leads to chronic arthopathy, which severely reduces the quality of life of severe hemophiliacs. Prophylactic administration of FVIII/FIX concentrates was introduced in the early 1960’s to convert severe hemophilia into a moderate form that cause less frequent joint bleedings. Recent data in hemophilia patients with inhibitors have provided evidence for that daily dose of rFVIIa can act in secondary prophylaxis in patients with frequent bleeds. The plasma half-life of rFVIIa is approx 2–3 hours which thus may limit its efficacy and convenience in prevention of bleeds. Therefore, development of rFVIIa molecules that could remain in the circulation for a prolonged period may potentially improve prophylactic options of rFVIIa. PEGylation is an established and clinically proven strategy for prolonging the circulatory life-time of bio-therapeutic proteins. Recently, GlycoPEGylation technology was used to generate PEGylated rFVIIa derivatives (Stennicke et al., 2008 Thromb. Haemost, in press) and they appeared to activate factor X at a similar rate as of rFVIIa (Stennicke et al., 2008 Thromb. Haemost, in press; and Ghosh et al., 2008, J. Thromb. Haemost, in press). In the present study, we further characterized the glycoPEGylated rFVIIa, rFVIIa-10K PEG and rFVIIa-40K PEG, particularly in reference to their interaction with tissue factor (TF) and endothelial cell protein C receptor (EPCR) on cell surfaces and their catabolism. rFVIIa and glycoPEGylated rFVIIa were labeled with 125I and the radio-iodinated proteins were used to monitor rFVIIa binding and uptake in endothelial cells, CHO cells stably transfected with EPCR, and fibroblasts. PEG modification of rFVIIa resulted in a marked decrease in the rate of rFVIIa uptake in endothelial cells. The reduction in the uptake of rFVIIa following attachment of PEG was primarily due to the reduced association of rFVIIa to the cell surface. The level of glycoPEGylated rFVIIa binding to endothelial cells was about 20 to 30% of that was obtained with the rFVIIa. No significant differences were found between rFVIIa-10K PEG and rFVIIa-40K PEG in their association with endothelial cells. rFVIIa-40K PEG uptake was slightly lower compared to rFVIIa-10K PEG. The reduction in the uptake of glycoPEGylated rFVIIa by endothelial cells appeared to be the result of a decreased rate of glycoPEGylated rFVIIa binding to EPCR. Consistent with this glycoPEGylated rFVIIa binding, relative to rFVIIa, was markedly lower to CHO cells expressing EPCR whereas no significant differences were found in the basal binding of rFVIIa and glycoPEGylated rFVIIa to wild-type CHO cells. The radioligand binding studies also revealed marked differences between rFVIIa and glycoPEGylated rFVIIa binding to TF on fibroblasts. Although the total amounts of radioligands associated with cell surface TF at saturating concentrations were similar, the affinity of rFVIIa-10K PEG and rFVIIa-40K PEG to TF, relative to rFVIIa affinity to TF, was lowered by about 6 to 20-fold, respectively. Surprisingly, if the binding affinities were evaluated in factor X activation studies, the differences between rFVIIa and glycoPEGylated rFVIIa were minimal. The differences between rFVIIa and glycoPEGylated rFVIIa binding to TF in these two different assay systems suggest that rFVIIa and glycoPEGylated rFVIIa may interact with active TF with equal affinity whereas they interact differently with cryptic TF. Alternatively, glycoPEGylated rFVIIa binds initially to cell surface TF with a similar affinity as of rFVIIa, but may dissociate readily from TF upon the washing that is required to measure the binding of radioligands to cells. Additional studies yielded data that favor the later possibility. Overall the present data suggest that the reduced affinity of rFVIIa associated with glycoPEGylation on binding to EPCR and TF is likely attributable to either steric hindrance or changes in electrostatic binding properties rather than modification of binding sites itself. Further, the reduced uptake of glycoPEGylated rFVIIa by cells stems primarily from the decreased association of the modified rFVIIa to cell surfaces rather than specific impairment in their internalization.

Form and function of the glycocalyx on free cell surfaces

1974 ◽  
Vol 268 (891) ◽  
pp. 55-66 ◽  
Keyword(s):  
Free Cell ◽  
Surface Coat ◽  
Cell Surfaces ◽  
Cell Coat ◽  
Fresh Tissue ◽  
Functional Roles ◽  
Form And Function ◽  
Freeze Etching ◽  
Coat Layer ◽  
And Function

Selected examples of the glycocalyx or cell coat on rickettsiae, bacteria, amoebae, sea-urchin eggs and the cat intestinal microvilli are illustrated and their functional roles are discussed. The differences in the form of various surface coats are noted; while many surface components are truely extraneous expendable coatings, others are so firmly attached that they seem to be a permanent part of the cell. The fuzzy surface coat on the cat intestinal microvilli have been considered in some detail and some new observations on the form of the glycocalyx are presented. The enteric surface coat is not readily visualized in fractured surface replicas of glycerinated tissue but fixed cells frozen in distilled water when replicated after freeze-etching reveal a flamboyant array of a filamentous meshwork attached to the microvilli. This fuzzy coat layer is at least twice as thick in the freeze-etched preparations when compared to thin sectioned material. Fresh tissue frozen without fixation or glycerin treatment did not have a thick fuzzy coat. In its place a thin amorphous blanket-like layer was found.

scholarly journals Ultrastructural localization of lectin receptors on the surface of the rat retinal pigment epithelium. Decreased sensitivity of the avidin-biotin method due to cell surface charge.

1984 ◽  
Vol 32 (8) ◽  
pp. 862-868 ◽  
Author(s):  
R M Pino
Keyword(s):  
Bone Marrow ◽  
Retinal Pigment Epithelium ◽  
Cell Surface ◽  
Retinal Pigment Epithelial ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Ultrastructural Localization ◽  
Cell Surfaces ◽  
Lectin Receptors ◽  
Lectin Receptor

Monosaccharides on the apical processes of the retinal pigment epithelium were examined using lectin-affinity cytochemical methods. Lectin receptor sugars were localized with lectin-horseradish peroxidase (HRP) and lectin-ferritin conjugates as well as with biotinylated lectins, avidin, and biotinylated HRP. In contrast, only wheat germ agglutinin (WGA) receptors were identified with biotinylated WGA followed by avidin-ferritin or free avidin and biotinylated ferritin. Labeling with avidin-ferritin subsequent to biotinylated lectin treatment was dependent upon the source and lot of the reagent. These findings are similar to those reported for the endothelium of bone marrow sinusoids (Pino RM: Am J Anat, 169:259, 1984). Since both the retinal pigment epithelial and bone marrow sinusoidal surfaces are highly anionic (negative), we investigated the possibility that the charge of the lectin reagents and cell surfaces might affect the localization of monosaccharides on cell surfaces. Analytical isoelectric focusing revealed that biotinylated ferritin and some avidin-ferritins are highly anionic, while the other lectin reagents have more cationic (positive) components. Based on this information, a less charged biotinylated ferritin marker was made that made it possible to localize biotinylated lectins bound to the cell surface.

scholarly journals Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras.

1991 ◽  
Vol 112 (5) ◽  
pp. 873-889 ◽  
Author(s):  
C A Schoenenberger ◽  
A Zuk ◽  
D Kendall ◽  
K S Matlin
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Discrete Distribution ◽  
Free Cell ◽  
Transformed Cells ◽  
Cell Contact ◽  
Ras Oncogene ◽  
Oncogene Expression ◽  
Transformed Cell Line ◽  
Flux Measurements

The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.

scholarly journals The Small GTPase Rab13 Regulates Assembly of Functional Tight Junctions in Epithelial Cells

2002 ◽  
Vol 13 (6) ◽  
pp. 1819-1831 ◽  
Author(s):  
Anne-Marie Marzesco ◽  
Irene Dunia ◽  
Rudy Pandjaitan ◽  
Michel Recouvreur ◽  
Daniel Dauzonne ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Tight Junctions ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Freeze Fracture ◽  
Small Gtpase ◽  
Cell Contact ◽  
Junctional Complexes ◽  
And Function

Junctional complexes such as tight junctions (TJ) and adherens junctions are required for maintaining cell surface asymmetry and polarized transport in epithelial cells. We have shown that Rab13 is recruited to junctional complexes from a cytosolic pool after cell–cell contact formation. In this study, we investigate the role of Rab13 in modulating TJ structure and functions in epithelial MDCK cells. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delayed the formation of electrically tight epithelial monolayers as monitored by transepithelial electrical resistance (TER) and induced the leakage of small nonionic tracers from the apical domain. It also disrupted the TJ fence diffusion barrier. Freeze-fracture EM analysis revealed that tight junctional structures did not form a continuous belt but rather a discontinuous series of stranded clusters. Immunofluorescence studies showed that the expression of Rab13Q67L delayed the localization of the TJ transmembrane protein, claudin1, at the cell surface. In contrast, the inactive Rab13T22N mutant did not disrupt TJ functions, TJ strand architecture nor claudin1 localization. Our data revealed that Rab13 plays an important role in regulating both the structure and function of tight junctions.

scholarly journals The relationship between intermediate filaments and microfilaments before and during the formation of desmosomes and adherens-type junctions in mouse epidermal keratinocytes.

1987 ◽  
Vol 104 (5) ◽  
pp. 1389-1402 ◽  
Author(s):  
K J Green ◽  
B Geiger ◽  
J C Jones ◽  
J C Talian ◽  
R D Goldman
Keyword(s):  
Adherens Junction ◽  
Epidermal Keratinocytes ◽  
Cell Contact ◽  
Microfilament Bundles ◽  
Cell Substrate ◽  
Primary Mouse ◽  
Filament Bundles ◽  
Double Label ◽  
Mouse Keratinocytes ◽  
Cell Cell

Actin, keratin, vinculin and desmoplakin organization were studied in primary mouse keratinocytes before and during Ca2+-induced cell contact formation. Double-label fluorescence shows that in cells cultured in low Ca2+ medium, keratin-containing intermediate filament bundles (IFB) and desmoplakin-containing spots are both concentrated towards the cell center in a region bounded by a series of concentric microfilament bundles (MFB). Within 5-30 min after raising Ca2+ levels, a discontinuous actin/vinculin-rich, submembranous zone of fluorescence appears at cell-cell interfaces. This zone is usually associated with short, perpendicular MFB, which become wider and longer with time. Later, IFB and the desmoplakin spots are seen aligned along the perpendicular MFB as they become redistributed to cell-cell interfaces where desmosomes form. Ultrastructural analysis confirms that before the Ca2+ switch, IFB and desmosomal components are found predominantly within the perimeter defined by the outermost of the concentric MFB. Individual IF often splay out, becoming interwoven into these MFB in the region of cell-substrate contact. In the first 30 min after the Ca2+ switch, areas of submembranous dense material (identified as adherens junctions), which are associated with the perpendicular MFB, can be seen at newly formed cell-cell contact sites. By 1-2 h, IFB-desmosomal component complexes are aligned with the perpendicular MFB as the complexes become redistributed to cell-cell interfaces. Cytochalasin D treatment causes the redistribution of actin into numerous patches; keratin-containing IFB undergo a concomitant redistribution, forming foci that coincide with the actin-containing aggregates. These results are consistent with an IF-MF association before and during desmosome formation in the primary mouse epidermal keratinocyte culture system, and with the temporal and spatial coordination of desmosome and adherens junction formation.

Mechanisms That Regulate the Function of the Selectins and Their Ligands

1999 ◽  
Vol 79 (1) ◽  
pp. 181-213 ◽  
Author(s):  
DIETMAR VESTWEBER ◽  
JAMES E. BLANKS
Keyword(s):  
Cell Surface ◽  
Molecular Mechanisms ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Cell Contact ◽  
Endothelial Cell Surface ◽  
And Function ◽  
Blocking Antibodies ◽  
Selectin Binding

Vestweber, Dietmar, and James E. Blanks. Mechanisms That Regulate the Function of the Selectins and Their Ligands. Physiol. Rev. 79: 181–213, 1999. — Selectins are a family of three cell adhesion molecules (L-, E-, and P-selectin) specialized in capturing leukocytes from the bloodstream to the blood vessel wall. This initial cell contact is followed by the selectin-mediated rolling of leukocytes on the endothelial cell surface. This represents the first step in a cascade of molecular interactions that lead to leukocyte extravasation, enabling the processes of lymphocyte recirculation and leukocyte migration into inflamed tissue. The central importance of the selectins in these processes has been well documented in vivo by the use of adhesion-blocking antibodies as well as by studies on selectin gene-deficient mice. This review focuses on the molecular mechanisms that regulate expression and function(s) of the selectins and their ligands. Cell-surface expression of the selectins is regulated by a variety of different mechanisms. The selectins bind to carbohydrate structures on glycoproteins, glycolipids, and proteoglycans. Glycoproteins are the most likely candidates for physiologically relevant ligands. Only a few glycoproteins are appropriately glycosylated to allow strong binding to the selectins. Recently, more knowledge about the structure and the regulated expression of some of the carbohydrates on these ligands necessary for selectin binding has been accumulated. For at least one of these ligands, the physiological function is now well established. A novel and exciting aspect is the signaling function of the selectins and their ligands. Especially in the last two years, convincing data have been published supporting the idea that selectins and glycoprotein ligands of the selectins participate in the activation of leukocyte integrins.

Spatial control of actin-based motility through plasmalemmal PtdIns(4,5)P2-rich raft assemblies.

2005 ◽  
Vol 72 ◽  
pp. 119-127 ◽  
Author(s):  
Tamara Golub ◽  
Caroni Pico
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Lipid Rafts ◽  
Patch Clustering ◽  
Pkc Protein Kinase C ◽  
Membrane Bound ◽  
And Function ◽  
Cytoskeleton Dynamics ◽  
Syndrome Protein

The interactions of cells with their environment involve regulated actin-based motility at defined positions along the cell surface. Sphingolipid- and cholesterol-dependent microdomains (rafts) order proteins at biological membranes, and have been implicated in most signalling processes at the cell surface. Many membrane-bound components that regulate actin cytoskeleton dynamics and cell-surface motility associate with PtdIns(4,5)P2-rich lipid rafts. Although raft integrity is not required for substrate-directed cell spreading, or to initiate signalling for motility, it is a prerequisite for sustained and organized motility. Plasmalemmal rafts redistribute rapidly in response to signals, triggering motility. This process involves the removal of rafts from sites that are not interacting with the substrate, apparently through endocytosis, and a local accumulation at sites of integrin-mediated substrate interactions. PtdIns(4,5)P2-rich lipid rafts can assemble into patches in a process depending on PtdIns(4,5)P2, Cdc42 (cell-division control 42), N-WASP (neural Wiskott-Aldrich syndrome protein) and actin cytoskeleton dynamics. The raft patches are sites of signal-induced actin assembly, and their accumulation locally promotes sustained motility. The patches capture microtubules, which promote patch clustering through PKA (protein kinase A), to steer motility. Raft accumulation at the cell surface, and its coupling to motility are influenced greatly by the expression of intrinsic raft-associated components that associate with the cytosolic leaflet of lipid rafts. Among them, GAP43 (growth-associated protein 43)-like proteins interact with PtdIns(4,5)P2 in a Ca2+/calmodulin and PKC (protein kinase C)-regulated manner, and function as intrinsic determinants of motility and anatomical plasticity. Plasmalemmal PtdIns(4,5)P2-rich raft assemblies thus provide powerful organizational principles for tight spatial and temporal control of signalling in motility.

scholarly journals Bend, Push, Stretch: Remarkable Structure and Mechanics of Single Intermediate Filaments and Meshworks

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1960
Author(s):  
K. Tanuj Sapra ◽  
Ohad Medalia
Keyword(s):  
Intermediate Filaments ◽  
Eukaryotic Cell ◽  
Coiled Coils ◽  
Cellular Functions ◽  
Mechanical Pressure ◽  
Mechanical Feature ◽  
Cellular Processes ◽  
Nuclear Lamins ◽  
Filament Networks ◽  
And Function

The cytoskeleton of the eukaryotic cell provides a structural and functional scaffold enabling biochemical and cellular functions. While actin and microtubules form the main framework of the cell, intermediate filament networks provide unique mechanical properties that increase the resilience of both the cytoplasm and the nucleus, thereby maintaining cellular function while under mechanical pressure. Intermediate filaments (IFs) are imperative to a plethora of regulatory and signaling functions in mechanotransduction. Mutations in all types of IF proteins are known to affect the architectural integrity and function of cellular processes, leading to debilitating diseases. The basic building block of all IFs are elongated α-helical coiled-coils that assemble hierarchically into complex meshworks. A remarkable mechanical feature of IFs is the capability of coiled-coils to metamorphize into β-sheets under stress, making them one of the strongest and most resilient mechanical entities in nature. Here, we discuss structural and mechanical aspects of IFs with a focus on nuclear lamins and vimentin.

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