To bead or not to bead? Lens-specific intermediate filaments revisited

1997 ◽  
Vol 110 (21) ◽  
pp. 2629-2634 ◽  
Author(s):  
S.D. Georgatos ◽  
F. Gounari ◽  
G. Goulielmos ◽  
U. Aebi
Keyword(s):  
Plasma Membrane ◽  
Intermediate Filaments ◽  
Cultured Cells ◽  
Fiber Cells ◽  
New Information ◽  
Specific Proteins ◽  
Beaded Filaments ◽  
Nodular Appearance

For nearly three decades cytoplasmic intermediate filaments (IFs) have been described as 10 nm thick, unbranched ropes radiating from the cell nucleus and extending to the plasma membrane. This stereotype is now being challenged by the discovery and molecular characterization of the beaded filaments (BFs), a novel class of IFs composed of the lens-specific proteins filensin and phakinin. In contrast to ‘mainstream’ IFs, BFs have a distinctly nodular appearance and form a meshwork underneath the plasma membrane of the lens fiber cells. In vitro assembly studies, expression of filensin and phakinin in cultured cells, and analysis of the corresponding genes reveal that these proteins have evolved from two different subfamilies of IF proteins, thus yielding a unique structure. The new information provides a basis for understanding how the various forms of tissue-specific IF proteins might have developed adopting to the constraints of a specialized environment.

scholarly journals 2P-050 Morphological analysis of the intermediate filaments formed by lens-specific proteins filensin and phakinin in vitro(The 46th Annual Meeting of the Biophysical Society of Japan)

2008 ◽  
Vol 48 (supplement) ◽  
pp. S82-S83
Author(s):  
Ryoichi Nakamuta ◽  
Hiroyuki Ainobu ◽  
Masaya Wada ◽  
Taketsune Matsuzaki ◽  
Yushi Oishi ◽  
...  
Keyword(s):  
Annual Meeting ◽  
Intermediate Filaments ◽  
Morphological Analysis ◽  
Biophysical Society ◽  
Specific Proteins

scholarly journals Intermediate filaments in non-neuronal cells of invertebrates: isolation and biochemical characterization of intermediate filaments from the esophageal epithelium of the mollusc Helix pomatia.

1985 ◽  
Vol 101 (2) ◽  
pp. 427-440 ◽  
Author(s):  
E Bartnik ◽  
M Osborn ◽  
K Weber
Keyword(s):  
Positive Reaction ◽  
Intermediate Filaments ◽  
Biochemical Characterization ◽  
Helix Pomatia ◽  
Molar Ratio ◽  
Negative Stain ◽  
Epithelial Morphology ◽  
Lower Chordate

To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.

scholarly journals Effects of cytoplasmic acidification on clathrin lattice morphology.

1989 ◽  
Vol 108 (2) ◽  
pp. 401-411 ◽  
Author(s):  
J Heuser
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Membrane Receptors ◽  
The Other ◽  
Culture Dish ◽  
Initial Curvature ◽  
Internal Ph ◽  
Cytoplasmic Acidification

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.

Brush border myosin-I microinjected into cultured cells is targeted to actin-containing surface structures

1994 ◽  
Vol 107 (6) ◽  
pp. 1623-1631 ◽  
Author(s):  
M. Footer ◽  
A. Bretscher
Keyword(s):  
Plasma Membrane ◽  
Brush Border ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Surface Structures ◽  
Specific Expression ◽  
Cultured Fibroblasts ◽  
Myosin I ◽  
Section Electron

The isolated intestinal microvillus cytoskeleton (core) consists of four major proteins: actin, villin, fimbrin and brush border myosin-I. These proteins can assemble in vitro into structures resembling native microvillus cores. Of these components, villin and brush border myosin-I show tissue-specific expression, so they may be involved in the morphogenesis of intestinal microvilli. When introduced into cultured cells that normally lack the protein, villin induces a reorganization of the actin filaments to generate large surface microvilli. Here we examine the consequences of microinjecting brush border myosin-I either alone or together with villin into cultured fibroblasts. Injection of brush border myosin-I has no discernible effect on the overall morphology of the cells, but does become localized to either normal or villin-induced microvilli and other surface structures containing an actin cytoskeleton. Since some endogenous myosin-Is have been found associated with cytoplasmic vesicles, these results show that brush border myosin-I has a domain that specifically targets it to the plasma membrane in both intestinal and cultured cell systems. Ultrastructural examination of microvilli on control cultured cells revealed that they contain a far more highly ordered bundle of microfilaments than had been previously appreciated. The actin filaments in microvilli of villin-injected cells appeared to be more tightly cross-linked when examined by thin-section electron microscopy. In intestinal microvilli, the core bundle is separated from the plasma membrane by about 30 nm due to the presence of brush border myosin-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular map of the desmosomal plaque

1999 ◽  
Vol 112 (23) ◽  
pp. 4325-4336 ◽  
Author(s):  
A.J. North ◽  
W.G. Bardsley ◽  
J. Hyam ◽  
E.A. Bornslaeger ◽  
H.C. Cordingley ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intermediate Filaments ◽  
Protein Interactions ◽  
Immunogold Labelling ◽  
Molecular Approaches ◽  
Molecular Map ◽  
Carboxy Terminal ◽  
Desmoglein 3 ◽  
Terminal Domains

Recent biochemical and molecular approaches have begun to establish the protein interactions that lead to desmosome assembly. To determine whether these associations occur in native desmosomes we have performed ultrastructural localisation of specific domains of the major desmosomal components and have used the results to construct a molecular map of the desmosomal plaque. Antibodies directed against the amino- and carboxy-terminal domains of desmoplakin, plakoglobin and plakophilin 1, and against the carboxy-terminal domains of desmoglein 3, desmocollin 2a and desmocollin 2b, were used for immunogold labelling of ultrathin cryosections of bovine nasal epidermis. For each antibody, the mean distance of the gold particles, and thus the detected epitope, from the cytoplasmic surface of the plasma membrane was determined quantitatively. Results showed that: (i) plakophilin, although previously shown to bind intermediate filaments in vitro, is localised extremely close to the plasma membrane, rather than in the region where intermediate filaments are seen to insert into the desmosomal plaque; (ii) while the ‘a’ form of desmocollin overlaps with plakoglobin and desmoplakin, the shorter ‘b’ form may be spatially separated from them; (iii) desmoglein 3 extends across the entire outer plaque, beyond both desmocollins; (iv) the amino terminus of desmoplakin lies within the outer dense plaque and the carboxy terminus some 40 nm distant in the zone of intermediate filament attachment. This is consistent with a parallel arrangement of desmoplakin in dimers or higher order aggregates and with the predicted length of desmoplakin II, indicating that desmoplakin I may be folded or coiled. Thus several predictions from previous work were borne out by this study, but in other cases our observations yielded unexpected results. These results have significant implications relating to molecular interactions in desmosomes and emphasise the importance of applying multiple and complementary approaches to biological investigations.

scholarly journals Identification and characterization of plasma membrane aquaporins isolated from fiber cells of Calotropis procera

2013 ◽  
Vol 14 (7) ◽  
pp. 586-595 ◽  
Author(s):  
Usman Aslam ◽  
Asia Khatoon ◽  
Hafiza Masooma Naseer Cheema ◽  
Aftab Bashir
Keyword(s):  
Plasma Membrane ◽  
Calotropis Procera ◽  
Fiber Cells ◽  
Identification And Characterization

Prospective In Vivo Identification of Osteogenic Stem/Progenitor Cells from Bone Marrow-Derived Lin−/Sca-1+/CD45− Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1409-1409
Author(s):  
Zhuo Wang ◽  
Junghun Jung ◽  
Magdalena Kucia ◽  
Junhui Song ◽  
Yusuke Shiozawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Formation ◽  
Cultured Cells ◽  
Fluorescent Microscopy ◽  
Micro Ct ◽  
Mineralized Tissue

Abstract We previously developed an in vivo prospective assay for identification of non-cultured cells with MSC potential. Using this assay we identified a population of cells that were slowly cycling and of low density that were capable of multilineage differentiation both in vitro and in vivo (Z. Wang et al, Stem Cells. 2006 24(6):1573). Further characterization of these cells suggested that they resemble a homogenous population of rare Lin−/Sca-1+/CD45− cells that have the morphology and express several markers of undifferentiated embryonic-like stem cells. In vitro the Lin−/Sca-1+/CD45− cells may differentiate into cells from all three germ-layers (M. Kucia et al, Leukemia. 2007 21(2):297). To determine the in vivo fate of this population, we transplanted 500 or 5,000 Lin−/Sca-1+/CD45− cells from a GFP mouse into SCID mice in each group (n=3) immediately after cell sorting to evaluate tissue generation in vivo. At 4 weeks the regenerative potential of these populations was evaluated by micro-CT and histology, and cells were tracked by gross examination of the harvested tissues by fluorescent microscopy. The results showed that a large number of GFP+ cells are located in the implants, indicating that the transplanted cells maintain the ability to contribute to the generation of new tissue. Bone-like tissue was observed in the Lin−/Sca-1+/CD45− group with as low as 500-cells/implant, while 5,000 Lin−/Sca-1+/CD45− cells generated significantly larger mineralized tissue volume, which was confirmed by micro-CT. Lin−/Sca-1+/CD45+ cell only implantation did not form any mineralized tissue, however, while mixed with 2x106 whole bone morrow cells, positive mineralized tissue occurred. Whole bone marrow mixture also improve bone formation in Lin−/Sca-1+/CD45− cell implants compared the actual bone volumes measured by micro-CT. This study demonstrates that non-cultured BM-derived Lin−/Sca-1+/CD45− cells exhibit the capacity to form bone in vivo with as low as 500 cells/implant. Whole bone marrow mixtures can enhance the bone formation, presumably through the interaction of other populations cells. Based on these findings, it is proposed that non-cultured BM-derived Lin−/Sca-1+/CD45− cells are enriched osteogenic cells that can be applied to bone regeneration in vivo.

scholarly journals Fodrin: axonally transported polypeptides associated with the internal periphery of many cells.

1981 ◽  
Vol 90 (3) ◽  
pp. 631-642 ◽  
Author(s):  
J Levine ◽  
M Willard
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cortical Actin ◽  
Cultured Fibroblasts ◽  
Tissue Sections ◽  
Parallel Arrays ◽  
Guinea Pig Brain

Fodrin (formerly designated 26 and 27) comprises two polypeptides (250,000 and 240,000 mol wt) that are axonally transported at a maximum time-averaged velocity of 40 mm/d--slower than the most rapidly moving axonally transported proteins, but faster than at least three additional groups of proteins. In this communication, we report the intracellular distribution of fodrin. Fodrin was purified from guinea pig brain, and a specific antifodrin antibody was produced in rabbit and used to localize fodrin in tissue sections and cultured cells by means of indirect immunofluorescence. Fodrin antigens were highly concentrated in the cortical cytoplasm of neurons and also nonneuronal tissues (e.g., skeletal muscle, uterus, intestinal epithelium). Their disposition resembles a lining of the cell: hence, the designation fodrin (from Greek fodros, lining). In cultured fibroblasts, immunofluorescently labeled fodrin antigens were arranged in parallel arrays of bands in the plane of the plasma membrane, possibly reflecting an exclusion of labeled fodrin from some areas occupied by stress fibers. The distribution of fodrin antigens in mouse 3T3 cells transformed with simian virus 40 was more diffuse, indicating that the disposition of fodrin is responsive to altered physiological states of the cell. When mixtures of fodrin and F-actin were centrifuged, fodrin cosedimented with the actin, indicating that these proteins interact in vitro. We conclude that fodrin is a specific component of the cortical cytoplasm of many cells and consider the possibilities: (a) that fodrin may be indirectly attached to the plasma membrane via cortical actin filaments; (b) that fodrin may be mobile within the cortical cytoplasm and that, in axons, a cortical lining may be in constant motion relative to the internal cytoplasm; and (c) that fodrin could serve to link other proteins and organelles to a submembrane force-generating system.

scholarly journals Isolation and preliminary characterization of the major membrane boundaries of the endocytic pathway in lymphocytes.

1990 ◽  
Vol 111 (5) ◽  
pp. 1811-1823 ◽  
Author(s):  
B D Beaumelle ◽  
A Gibson ◽  
C R Hopkins
Keyword(s):  
Plasma Membrane ◽  
Protein Composition ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Selective Labeling ◽  
Specific Proteins ◽  
T Lymphoma ◽  
Cellular Compartments ◽  
Density Shift

Plasma membrane, coated pits, endosomes, and lysosomes were isolated from a mouse T lymphoma cell line using a density shift protocol in which these compartments were selectively loaded with gold conjugates. The plasma membrane was prepared after selective labeling for 1 h at 2 degrees C with gold-ricin and gave a yield of 40% according to enzymatic and antigenic markers. Endosomes were obtained by loading the cells for 2 h at 22 degrees C with gold complexed to an antimouse transferrin receptor mAb. Coated pits were isolated using a similar procedure, but after an incubation at 10 degrees C, which allowed deep invagination of the pits but prevented internalization. The yield (calculated using the recovery of [125I]transferrin) was 32% for endosomes and 10% for coated pits. Finally lysosomes were prepared by loading the cells for 18 h at 37 degrees C with gold low density lipoproteins (LDLs) followed by a 3-h chase at 37 degrees C with LDL alone. The final lysosome yield (based on the recovery of lysosomal enzymes) was 16%. Studies of the protein composition of these cellular compartments on two-dimensional gels showed that while some major proteins are present throughout the pathway, specific proteins can be identified in each of the isolated fractions. The greatest change in the pattern of protein constituents seen along the pathway was between endosomal and lysosomal preparations.

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