scholarly journals Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts.

1982 ◽  
Vol 92 (2) ◽  
pp. 398-408 ◽  
Author(s):  
I I Singer
Keyword(s):  
Time Course ◽  
Immunofluorescence Microscopy ◽  
Stress Fibers ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Adhesive Surface ◽  
Contrast Microscopy ◽  
Double Label ◽  
Fetal Bovine ◽  
Perinuclear Area

We have recently observed a transmembrane association between extracellular fibronectin (FN) fibers and elongated focal patches or fibers of vinculin (VN) in G1-arrested stationary Nil 8 hamster fibroblasts, with double-label immunofluorescence microscopy (Singer and Paradiso, 1981, Cell. 24:481-492). We hypothesized that these FN-VN complexes might correspond to focal contacts, the membrane sites that are probably mainly responsible for attaching cells to their substrata, because vinculin is often localized in focal contacts. However, because fibronectin-vinculin associations may not be restricted to the substrate adhesive surface of the cell, it became necessary to determine whether some or all of the various kinds of FN-VN complexes which we described are in proximity to the substrate. Using interference reflection optics and double-label immunofluorescence microscopy for fibronectin and vinculin, many elongated (up to 38 micrometer) FN-VN associations were found to be strikingly coincident with focal contacts in the perinuclear area of extremely flattened arrested Nil 8 fibroblasts in 0.3% fetal bovine serum (FBS). In addition, the long FN-VN adhesion complexes were precisely aligned with the major phase-dense stress fibers observed at the ventral surfaces of these stationary cells with phase contrast microscopy. Fibronectin was neither associated with vinculin-containing focal contacts of Nil 8 cells cultured in medium with 5% FBS nor with vinculin-negative focal contacts located at the extreme edges of stationary cells arrested in 0.3 FBS. Our time-course experiments suggest that early FN-VN lacking-focal contacts, which form at the cellular margins, develop into mature substrate adhesion complexes containing both fibronectin and vinculin, localized in the major stress fibers at the centers of sessile fibroblasts.

Macromolecular specializations that mediate lateral interactions between microfilaments and the membrane at focal contacts

1992 ◽  
Vol 50 (1) ◽  
pp. 724-725
Author(s):  
Steven J. Samuelsson ◽  
Paul W. Luther ◽  
David W. Pumplin ◽  
Robert J. Bloch
Keyword(s):  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Focal Contact ◽  
Lateral Interactions ◽  
Matrix Assembly ◽  
Cytoplasmic Surface ◽  
Focal Contacts ◽  
Specific Proteins

Focal contacts are membrane specializations of cultured cells where stress fibers terminate and where the cell is most closely applied to the substrate. The organization of this cytoskeletal-membrane-extracellular matrix assembly has been well characterized. Immunofluorescence microscopy has shown that two focal contact-specific proteins, vinculin and talin, colocalize with microfilaments for several microns before the stress fiber terminates. This result raises the question of whether microfilament-membrane interactions are limited to the ends of microfilaments, or if lateral interactions also occur. We addressed this question by examining the cytoplasmic surface of isolated focal contacts in detail.

scholarly journals Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extracellular matrix accumulation.

1988 ◽  
Vol 106 (6) ◽  
pp. 2171-2182 ◽  
Author(s):  
I I Singer ◽  
S Scott ◽  
D W Kawka ◽  
D M Kazazis ◽  
J Gailit ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Cell Attachment ◽  
Cellular Migration ◽  
Surface Distribution ◽  
Fibronectin Receptor ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Double Label ◽  
Coated Surfaces

We used antibodies against the alpha subunits of the human fibronectin receptor (FNR) and vitronectin receptor (VNR) to localize simultaneously FNR and VNR at major substrate adhesion sites of fibroblasts and melanoma cells with double-label immunofluorescence microscopy. In early (2-6-h) serum-containing cultures, both FNR and VNR coaccumulated in focal contacts detected by interference reflection microscopy. Under higher resolution immunoscanning electron microscopy, FNR and VNR were also observed to be distributed randomly on the dorsal cell surface. As fibronectin-containing extracellular matrix fibers accumulated beneath the cells at 24 h, FNR became concentrated at contacts with these fibers and was no longer detected at focal contacts. VNR was not observed at matrix contacts but remained strikingly localized in focal contacts of the 24-h cells. Since focal contacts represent the sites of strongest cell-to-substrate adhesion, these results suggest that FNR and VNR together play critical roles in the maintenance of stable contacts between the cell and its substrate. In addition, the accumulation of FNR at extracellular matrix contacts implies that this receptor might also function in the process of cellular migration along fibronectin-containing matrix cables. To define the factors governing accumulation of FNR and VNR at focal contacts, fibroblasts in serum-free media were plated on substrates coated with purified ligands. Fibronectin-coated surfaces fostered accumulation of FNR but not VNR at focal contacts. On vitronectin-coated surfaces, or substrata derivatized with a tridecapeptide containing the cell attachment sequence Arg-Gly-Asp, both FNR and VNR became concentrated at focal contacts. These observations suggest that the availability of ligand is critical to the accumulation of FNR and VNR at focal contacts, and that FNR might also recognize substrate-bound vitronectin.

Fibronexus-Like Adhesion Sites are Present at the Invasive Zones of Human Epidermoid Carcinomas

1982 ◽  
Vol 40 ◽  
pp. 106-109
Author(s):  
Irwin I. Singer
Keyword(s):  
Cell Cycle ◽  
Serum Concentration ◽  
Substrate Binding ◽  
High Serum ◽  
Adhesion Sites ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Adhesion Complex ◽  
Low Serum

Our previous results indicate that two types of fibronectin-cytoskeletal associations may be formed at the fibroblast surface: dorsal matrixbinding fibronexuses generated in high serum (5% FBS) cultures, and ventral substrate-adhering units formed in low serum (0.3% FBS) cultures. The substrate-adhering fibronexus consists of at least vinculin (VN) and actin in its cytoplasmic leg, and fibronectin (FN) as one of its major extracellular components. This substrate-adhesion complex is localized in focal contacts, the sites of closest substratum approach visualized with interference reflection microscopy, which appear to be the major points of cell-tosubstrate adhesion. In fibroblasts, the latter substrate-binding complex is characteristic of cultures that are arrested at the G1 phase of the cell cycle due to the low serum concentration in their medium. These arrested fibroblasts are very well spread, flattened, and immobile.

Ultrastructure of multiple microtubule initiation sites in mouse neuroblastoma cells

1981 ◽  
Vol 47 (1) ◽  
pp. 1-24
Author(s):  
G.A. Sharp ◽  
M. Osborn ◽  
K. Weber
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Immunofluorescence Microscopy ◽  
Neuroblastoma Cells ◽  
Biological Significance ◽  
Optimal Conditions ◽  
Serial Sectioning ◽  
Mouse Neuroblastoma ◽  
Microtubule Organizing Centres ◽  
Perinuclear Area

Morphologically undifferentiated and differentiated mouse neuroblastoma N115 and N18 cells were examined after serial sectioning by electron microscopy. A sizeable percentage of the cells revealed multiple centrioles, usually clustered together in the perinuclear area with 2 preferential locations, i.e. above and below the largest nuclear diameter. These results indicate that the multiple microtubule-organizing centres previously visualized by immunofluorescence microscopy with tubulin antibody in neuroblastoma cells recovering from Colcemid poisoning are most likely in majority related to multiple centrioles. This interpretation is further strengthened by experiments in which cells are first recorded in the fluorescence microscope and then after serial sectioning in the electron microscope. The results show that under optimal conditions immunofluorescence microscopy is able to visualize single centrioles. The possible biological significance of the combined electron and immunofluorescence microscopical results is discussed.

scholarly journals A CYTOCHEMICAL DESCRIPTION OF THE MULTIPLICATION OF MENGOVIRUS IN L-929 CELLS

1962 ◽  
Vol 12 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Richard M. Franklin
Keyword(s):  
Phase Contrast ◽  
Basic Protein ◽  
Virus Production ◽  
Cytological Change ◽  
Virus Particles ◽  
Contrast Microscopy ◽  
Infected Cells ◽  
Cytological Changes ◽  
Perinuclear Area

A correlation of cytochemical changes with virus production has been studied in L cells infected with Mengovirus. After a latent period of about 2 hours, virus was produced rapidly, reaching maximum titers of up to 12,000 particles per cell in 6 to 8 hours. The earliest cytological change was in the nucleus and consisted of a slight condensation of chromatin. There is no evidence, however, for the multiplication of either the viral RNA or protein in the nucleus. RNA, of high molecular weight, accumulated in the perinuclear area of the cytoplasm and was later found in inclusions. The perinuclear RNA was digestible with RNase and may be located in or on ribosomes. The inclusion RNA was resistant to RNase but could be removed by pepsin or potassium permanganate; it is probably in completed virus particles. Viral antigen was first observed in a perinuclear location and later in the above-mentioned inclusions. Although the viral protein contains appreciable amounts of arginine and lysine, it is not a basic protein of the histone type. Phase-contrast microscopy of living cells clearly demonstrated the role of the inclusions in release of virus from infected cells. A comparison is made between these cytological changes in Mengo-infected cells and those which have been found by other workers in polio-infected cells. There are many very similar changes.

Selective distributions of proteoglycans and their ligands in pericellular matrix of cultured fibroblasts. Implications for their roles in cell-substratum adhesion

1993 ◽  
Vol 106 (1) ◽  
pp. 55-65 ◽  
Author(s):  
M. Yamagata ◽  
S. Saga ◽  
M. Kato ◽  
M. Bernfield ◽  
K. Kimata
Keyword(s):  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
The Other ◽  
Stress Fibers ◽  
Chondroitin Sulfate Proteoglycan ◽  
Pericellular Matrix ◽  
Sulfate Proteoglycan ◽  
Cultured Fibroblasts ◽  
Focal Contacts ◽  
The Difference

We showed previously that a large chondroitin sulfate proteoglycan, PG-M (also known as versican), inhibits cell-substratum adhesion, while basement membrane heparan sulfate proteoglycan (recently named perlecan) does not (Yamagata et al. (1989) J. Biol. Chem. 264, 8012–8018). To extend our understanding of the adhesive function of these proteoglycans, we examined the pericellular localization of the proteoglycans and their ligands and also that of some matrix receptors and cytoskeletal molecules in various fibroblast culture systems. PG-M was abundant in the subcellular space of fibroblasts, but was excluded selectively from focal contacts where vinculin, integrins and fibronectin were localized. Hyaluronan, CD44 and tenascin were distributed similarly as PG-M. In contrast, perlecan was associated with fibronectin and was included in focal contacts. Syndecan-1, a membrane heparan sulfate/chondroitin sulfate proteoglycan, was associated with fibronectin at the cell surface, partly at focal contacts and in association with stress fibers. Thus, complexes of PG-M with hyaluronan, tenascin and CD44, are not involved in focal contacts. On the other hand, perlecan and syndecan-1 together with fibronectin may participate in focal contacts. The difference in localization between these proteoglycans may be related to their glycosaminoglycan content and to their distinctive roles in cell-substratum adhesion.

Protein phosphatases independently regulate vesicle movement and microtubule subpopulations in hepatocytes

1996 ◽  
Vol 271 (3) ◽  
pp. C929-C943 ◽  
Author(s):  
S. F. Hamm-Alvarez ◽  
X. Wei ◽  
N. Berndt ◽  
M. Runnegar
Keyword(s):  
Immunofluorescence Microscopy ◽  
Maximum Dose ◽  
Protein Phosphatases ◽  
Differential Interference Contrast Microscopy ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Microscopy Analysis ◽  
High Doses ◽  
Vesicle Movement ◽  
Cellular Components

To investigate the regulation of microtubule (MT)-based vesicle transport and the interphase MT array in hepatocytes, we have used okadaic acid (OKA) and microcystin (MCYST), two toxins that inhibit serine-threonine protein phosphatases (PP) 1 and 2A, to alter cellular phosphorylation. Video-enhanced differential interference contrast microscopy analysis revealed that both toxins inhibited the frequency, velocity, and run length of MT-dependent vesicle movements dose dependently between 50 and 500 nM. At our maximum dose of 500 nM, both toxins significantly decreased PP2A activity (OKA to 45 +/- 12% and MCYST to 57 +/- 2%), whereas PP1 was inhibited only by MCYST. Because no additional effects on vesicle movements were caused by MCYST over the changes caused by OKA, these data implicate PP2A in the regulation of MT-dependent vesicle movement. To understand whether the changes in parameters of vesicle movements were due to changes in the MT array, the effects of these toxins on MT distribution were examined by immunofluorescence microscopy. Although lower doses of OKA produced no effects, treatment with 500 nM OKA altered MT organization and also caused fragmentation and loss of acetylated (stable) MTs. In contrast, MCYST concentrations up to 500 nM elicited no changes in MT organization in general or in the acetylated (stable) array. From these findings we conclude that inhibition of MT-dependent vesicle movement by the PP inhibitors, MCYST and OKA, in hepatocytes cannot result from changes or disruption in the MT array. Because OKA (an inhibitor of PP2A only in our system) at high doses caused loss of stable MTs, whereas MCYST (an inhibitor of both PP1 and PP2A) did not, we conclude that the control of the preservation of the stable MT array in hepatocytes is complex. Stable MTs require active PP2A for maintenance, but the disruption of the array through inhibition of PP2A can be prevented if PP1 is also inhibited, suggesting that the relative degree of phosphorylation of multiple cellular components is the determinant of MT stability.

Integrin-Mediated Fibroblast Adhesion Strength: Role of the β1 Subunit

1993 ◽  
Vol 331 ◽  
Author(s):  
Kelly A. Ward ◽  
Jun-Lin Guan ◽  
Daniel A. Hammer
Keyword(s):  
Extracellular Matrix ◽  
Adhesion Strength ◽  
Cytoplasmic Domain ◽  
Integrin Receptors ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Cell Substrate ◽  
Extracellular Matrix Molecules ◽  
Integrin Ligand ◽  
Cell Substrate Adhesion

AbstractCell-substratum adhesion is important in wound healing [4], embryogenic development [11], tissue architecture [6], and metastasis [7]. Integrins constitute a major class of heterodimeric cell-surface glycoproteins involved in receptor-mediated adhesion to the extracellular matrix (ECM). Focal contacts are regions of the cell-substratum adhesion in which clusters of integrin receptors connect the cytoskeleton to extracellular matrix molecules such as fibronectin. Focal contacts strengthen cell-substrate adhesion, and are sites of biochemical activity. Since cell adhesion strength in part depends on the cell's ability to cluster receptors and cytoskeleton into focal contacts, the integrity of the focal contact, and hence a cell's adhesive strength, will depend both on integrin-cytoskeletal binding as well as integrin-ligand binding.Using a centrifugation assay, we have quantified cell-substratum adhesion strength of mouse 3T3 cells transfected with the avian β1 integrin receptor (wild type), including various deletion mutants of its cytoplasmic domain, to surfaces containing varying concentrations of CSAT, a monoclonal antibody against the extracellular domain of the avian β1 subunit. For all the transfectants, adhesion strength decreases with decreasing CSAT concentration and increasing centrifugal strength. Different truncations of the cytoplasmic domain lead to different levels of adhesion. There is no simple correlation between the length of the cytoplasmic domain and the strength of adhesion.

scholarly journals Centrosomal protein centrin is not detectable during early pre-implantation development but reappears during late blastocyst stage in porcine embryos

Reproduction ◽  
2006 ◽  
Vol 132 (3) ◽  
pp. 423-434 ◽  
Author(s):  
G Manandhar ◽  
D Feng ◽  
Y-J Yi ◽  
L Lai ◽  
J Letko ◽  
...  
Keyword(s):  
Western Blotting ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
De Novo ◽  
Germinal Vesicle ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Embryonic Cells ◽  
Perinuclear Area

Centrin is an evolutionarily conserved 20 kDa, Ca+2-binding, calmodulin-related protein associated with centrioles and basal bodies of phylogenetically diverse eukaryotic cells. Earlier studies have shown that residual centrosomes of non-rodent mammalian spermatozoa retain centrin and, in theory, could contribute this protein for the reconstruction of the zygotic centrosome after fertilization. The present work shows that CEN2 and CEN3 mRNA were detected in germinal vesicle-stage (GV) oocytes, MII oocytes, and pre-implantation embryos from the two-cell through the blastocyst stage, but not in spermatozoa. Boar ejaculated spermatozoa possess centrin as revealed by immunofluorescence microscopy and western blotting. Immature, GV oocytes possess speckles of centrin particles in the perinuclear area, visualized by immunofluorescence microscopy and exhibit a 19 kDa band revealed by western blotting. Mature MII stage oocytes lacked centrin that could be detected by immunofluorescence or western blotting. The sperm centrin was lost in zygotes afterin vitrofertilization. It was not detectable in embryos by immunofluorescence microscopy until the late blastocyst stage. Embryonic centrin first appeared as fine speckles in the perinuclear area of some interphase blastocyst cells and as putative centrosomes of the spindle poles of dividing cells. The cells of the hatched blastocysts developed centrin spots comparable with those of the cultured cells. Some blastomeres displayed undefined curved plate-like centrin-labeled structures. Anti-centrin antibody labeled interphase centrosomes of cultured pig embryonic fibroblast cells as distinct spots in the juxtanuclear area. Enucleated pig oocytes reconstructed by electrofusion with pig fibroblasts displayed centrin of the donor cell during the early stages of nuclear decondensation but became undetectable in the late pronuclear or cleavage stages. These observations suggest that porcine zygotes and pre-blastocyst embryonic cells lack centrin and do not retain exogenously incorporated centrin. The early embryonic centrosomes function without centrin. Centrin in the blastocyst stage embryos is likely a result ofde novosynthesis at the onset of differentiation of the pluripotent blastomeres.

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