The focal adhesions of chick retinal pigmented epithelial cells

1985 ◽  
Vol 63 (6) ◽  
pp. 553-563 ◽  
Author(s):  
Michał Opas
Keyword(s):  
Plasma Membrane ◽  
Incident Light ◽  
Focal Adhesions ◽  
Rpe Cells ◽  
Surface Reflection ◽  
Immersion Medium ◽  
Microfilament Bundles ◽  
Associated Proteins ◽  
Definition Of

Retinal pigmented epithelial (RPE) cells obtained from the eyes of chick embryos form colonies in vitro in which cells at the periphery of the colony express an undifferentiated, well-spread morphology and develop extremely large areas of cell–substratum adhesion. These adhesions can be classified as focal on the basis of the following: (a) their black surface reflection interference image, the contrast of which is not affected by changes in either the wavelength of the incident light or the refractive index of the immersion medium; (b) their association with the termini of actin-containing microfilament bundles; and (c) their ability to be labelled with antiserum against vinculin, a protein specific for adhesions of the focal type. The focal adhesions of RPE cells comprise laterally associated individual focal contacts, the mechanism by which this association is achieved and maintained is yet unknown. Because of the unusually large size and excellent microscopical definition of their focal adhesions, I used RPE cells to investigate the role of other actin-associated proteins in adhesion complexes. One of these, nonerythroid spectrin (fodrin), a protein suggested to play a role in anchoring actin filaments to the plasma membrane, was neither concentrated in nor excluded from the focal adhesions of RPE cells. Thus, at least in this cell type, spectrin seems unlikely to serve as a link between the major actin-containing microfilament bundles and the plasma membrane in the regions of cell-to-substratum contacts.

scholarly journals Domain architecture of the smooth-muscle plasma membrane: regulation by annexins

2005 ◽  
Vol 387 (2) ◽  
pp. 309-314 ◽  
Author(s):  
Annette DRAEGER ◽  
Susan WRAY ◽  
Eduard B. BABIYCHUK
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Membrane Lipids ◽  
Domain Architecture ◽  
Focal Adhesions ◽  
Membrane Lipid ◽  
Dependent Manner ◽  
Detailed Structural Analysis ◽  
Specific Subset ◽  
Associated Proteins

Individual signalling events are processed in distinct, spatially segregated domains of the plasma membrane. In a smooth muscle, the sarcolemma is divided into domains of focal adhesions alternating with caveolae-rich zones, both harbouring a specific subset of membrane-associated proteins. Recently, we have demonstrated that the sarcolemmal lipids are similarly segregated into domains of cholesterol-rich lipid rafts and glycerophospholipid-rich non-raft regions. In the present study, we provide a detailed structural analysis of the relationship between these proteinaceous and lipid domains. We demonstrate that the segregation of plasmalemmal protein constituents is intimately linked to that of the membrane lipids. Our results imply that lipid segregation is critical for the preservation of membrane protein architecture and essential for directional translocation of proteins to the sarcolemma. We show that the membrane lipid segregation is supported by the annexin protein family in a Ca2+-dependent manner. Eukaryotic cells harbour numerous, tissue-specific subsets of annexins. By examining the significance of this variety in a smooth muscle, we demonstrate that four different annexins target membrane sites of distinct lipid composition and that each annexin requires a different [Ca2+] for its translocation to the sarcolemma. Our results suggest that the interactions of annexins with distinct plasma membrane regions promote membrane segregation and, in combination with their individual Ca2+ sensitivity, might allow a spatially confined, graded response to a multitude of extra- or intracellular stimuli.

The Cytoskeletal Binding Domain of Band 3 Is Required for Multiprotein Complex Formation and Retention during Erythropoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4003-4003
Author(s):  
Timothy J Satchwell ◽  
Bethan R Hawley ◽  
Amanda J Bell ◽  
Maria Leticia Ribeiro ◽  
Ashley M Toye
Keyword(s):  
Plasma Membrane ◽  
Erythrocyte Membrane ◽  
Band 3 ◽  
Multiprotein Complex ◽  
Wild Type ◽  
Membrane Expression ◽  
Protein 4.2 ◽  
Type Band ◽  
Associated Proteins

Abstract The bicarbonate/chloride exchanger protein band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex required for vertical association between the plasma membrane and the underlying spectrin cytoskeleton. A wealth of knowledge, derived from a host of varied studies including in vitro binding assays, work on mature erythrocytes and in other cellular systems have identified a number of binding partners including ankyrin, adducin and protein 4.2 amongst others. However, studies of the role that band 3 and the establishment of its connectivity with the cytoskeleton play both in assembly of multiprotein complexes during erythropoiesis and in particular in protein retention during enucleation have been understandably limited by the technical challenges associated with study of this protein within its unique native cellular context. The complete absence of band 3 in human erythrocytes has only been reported once, in a Portuguese patient with severe hereditary spherocytosis and distal renal tubular acidosis resulting from homozygosity for a V488M band 3 mutation (band 3 Coimbra). In this study, we used in vitro culture of erythroblasts derived from this patient as well as shRNA mediated depletion of band 3 to investigate the development of a band 3 deficient erythrocyte membrane and to specifically assess the formation, stability and retention of band 3 dependent protein complexes in the absence of this core protein during erythropoiesis and erythroblast enucleation. We demonstrate that the mutant band 3 Coimbra protein is expressed at very low but detectable levels during erythropoiesis but does not reach the cell surface and is not rescued by interaction with wild type protein. Failure to traffic to the plasma membrane and rapid degradation during erythropoiesis accounts for the absence of band 3 in Coimbra erythrocytes. The absence of plasma membrane expression of band 3 results in secondary deficiencies of a host of band 3 associated membrane proteins that we quantitatively show result predominantly from reduced plasma membrane expression during erythropoiesis compounded by impaired retention in the nascent reticulocyte membrane during erythroblast enucleation. In order to explore the importance of the capacity of band 3 to associate with the cytoskeleton for surface expression of this protein and its associated multiprotein complex binding proteins, immature band 3 Coimbra patient erythroblasts were lentivirally transduced with N terminally GFP-tagged wild type band 3 or band 3 mutants with absent or impaired ability to associate with the cytoskeleton. We demonstrate for the first time the ability to restore expression of band 3 to normal levels in this uniquely compromised patient and to rescue key secondary protein deficiencies arising from the absence of band 3 in reticulocytes. Exogenous expression levels of band 3, monitored by GFP intensity, correlate directly with degree of rescue of a variety of band 3 associated proteins. When expressed in band 3 deficient Coimbra erythroblasts, the band 3 membrane domain, which is unable to associate with the cytoskeleton, exhibits an increased partitioning to the plasma membrane surrounding the extruded nuclei compared to wild type band 3 and fails to rescue reticulocyte membrane retention of band 3 associated proteins. Expression of the kidney isoform of band 3, which is unable to bind ankyrin but retains the binding site for the cytoskeletal accessory protein, protein 4.2 results in partial rescue of the protein 4.2 dependent CD47 only. This demonstrates the importance of band 3 association with the cytoskeleton for efficient retention of band 3 associated proteins during erythroblast enucleation. Interestingly, whilst both exhibit reduced reticulocyte membrane retention relative to wild type, a significant proportion of both band 3 membrane domain and kidney band 3 is retained in the reticulocyte membrane following erythroblast enucleation indicating that cytoskeletal attachment of band 3 is not the sole determinant of partitioning during this complex process. This study advances our understanding of the mechanisms by which the properties of band 3 influence the sculpting and composition of the erythrocyte membrane and highlights the role of this protein as a core for assembly and stabilisation of key membrane proteins in both the early and late stages of terminal erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

Proteins involved in the attachment of actin to the plasma membrane

1982 ◽  
Vol 299 (1095) ◽  
pp. 291-299 ◽  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Cell Types ◽  
Brain Protein ◽  
Cultured Fibroblasts ◽  
Microfilament Bundles ◽  
Different Cell Types ◽  
Adhesion Plaque ◽  
The Brain

Proteins that may be involved in two types of actin-membrane association are discussed. The first set includes α-actinin, vinculin, fimbrin and a new cytoskeletal protein that are all concentrated in adhesion plaques, those regions of cultured fibroblasts where bundles of actin microfilaments terminate and where the plasma membrane comes close to the underlying substrate. The properties of non-muscle α-actinin suggest that it functions to cross-link actin filaments and thereby stabilize microfilament bundles rather than functioning in their attachment to the membrane. Fimbrin also appears to be involved in bundling of filaments rather than in attachment. In contrast, vinculin binds to the ends of actin filaments in vitro and is probably the best candidate for a role in the attachment of actin to membranes at the adhesion plaque. The discovery of a new protein, 215k, of unknown function, in the adhesion plaque suggests that many more proteins remain to be identified in this region. Attachment of actin filaments to other regions of the plasma membrane is also considered and a protein is described that seems to be a spectrin homologue in brain and other tissues. The brain protein resembles erythrocyte spectrin in its physical properties, in binding actin, in being associated with cell membranes and in crossreacting immunologically. We suggest that the brain protein and erythrocyte spectrin both belong to a family of related proteins (the spectrins) which function in the attachment of actin to membranes in many different cell types.

246 EVIDENCE OF FOCAL ADHESION ASSEMBLY IN BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 221
Author(s):  
B. R. Sessions ◽  
H. Rutigliano ◽  
C. J. Davies ◽  
K. L. White
Keyword(s):  
Plasma Membrane ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Signalling Pathways ◽  
Bovine Oocyte ◽  
Signalling Molecules ◽  
Sperm Binding ◽  
Bovine Oocytes ◽  
Oocyte Membrane

Integrins are located on the plasma membrane of bovine oocytes and have been implicated in bovine fertilization and activation (Campbell et al. 2000; Sessions et al. 2006). In response to signals from the extracellular matrix, activated integrins cluster at focal adhesions and interact with the cytoskeleton via signalling molecules, including focal adhesion kinase (FAK), α-actinin, phosphotyrosine, and talin. Immunohistochemistry was used to illustrate the formation of focal adhesions at the site of sperm binding in bovine oocytes during fertilization. To confirm antibody specificity, proteins were recovered from bovine oocyte lysate, separated by electrophoreses, and exposed to Western blotting. Specificity was confirmed based on antibody binding to the correctly sized bovine protein in the blot. Bovine oocytes were recovered, matured in vitro, and exposed to spermatozoa as described previously. Antibodies were then used to directly identify co-localization of FAK, α-actinin, phophotyrosine, and talin with each other in addition to localization with β-integrin isotypes within the bovine oocyte plasma membrane. Appropriate controls were performed in which all steps were duplicated with the elimination of the primary antibodies, to confirm that immunohistochemistry was specific for the proteins of interest. The results of immunohistochemistry indicate a co-localization of FAK protein clusters that involves specific oocyte membrane integrin proteins during fertilization. These findings illustrate the formation of focal adhesions via colocalization of signalling molecules with integrin β subunits in bovine oocytes and further support the hypothesis that induction of signalling pathways is initiated with sperm–oocyte binding in the bovine.

scholarly journals Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR

2016 ◽  
Vol 113 (9) ◽  
pp. E1152-E1161 ◽  
Author(s):  
Loes M. Stevers ◽  
Chan V. Lam ◽  
Seppe F. R. Leysen ◽  
Femke A. Meijer ◽  
Daphne S. van Scheppingen ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Binding Site ◽  
Binding Motif ◽  
Protein Protein Interaction ◽  
Biochemical Assays ◽  
Binding Groove ◽  
Definition Of ◽  
R Domain

Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein–protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3–CFTR interface might offer an approach for cystic fibrosis therapeutics.

scholarly journals The actin cytoskeleton and plasma membrane connection: PtdIns(4,5)P(2) influences cytoskeletal protein activity at the plasma membrane

2000 ◽  
Vol 113 (21) ◽  
pp. 3685-3695 ◽  
Author(s):  
A.S. Sechi ◽  
J. Wehland
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Adhesion Molecules ◽  
Focal Adhesion ◽  
Small Gtpases ◽  
Cytoskeletal Protein ◽  
Protein Activity ◽  
Associated Proteins ◽  
Tight Connection

The co-ordination of rearrangements of the actin cytoskeleton depends on its tight connection to the plasma membrane. Phosphatidylinositol 4,5-bisphosphate is thought to transmit signals originating at the plasma membrane to the underlying actin cytoskeleton. This lipid binds to, and influences the activity of, several actin-associated proteins in vitro that regulate the architecture of the actin cytoskeleton. Signalling intermediates in this process include focal adhesion molecules such as vinculin and members of two families of proteins, ERM and WASP. These proteins interact with phosphatidylinositol 4,5-bisphosphate and appear to be regulated by interplay between small GTPases and phosphatidylinositol 4,5-bisphosphate metabolism, and thus link the plasma membrane with cytoskeletal remodelling.

Accumulation of profilin II at the surface of Listeria is concomitant with the onset of motility and correlates with bacterial speed

2000 ◽  
Vol 113 (8) ◽  
pp. 1415-1426 ◽  
Author(s):  
M. Geese ◽  
K. Schluter ◽  
M. Rothkegel ◽  
B.M. Jockusch ◽  
J. Wehland ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Cellular Motility ◽  
Binding Properties ◽  
Bacterial Surface ◽  
Associated Proteins ◽  
Infected Cells ◽  
Green Fluorescent

The spatial and temporal activity of the actin cytoskeleton is precisely regulated during cell motility by several microfilament-associated proteins of which profilin plays an essential role. We have analysed the distribution of green fluorescent protein (GFP)-tagged profilins in cultured and in Listeria-infected cells. Among the different GFP-profilin fusion proteins studied, only the construct in which the GFP moiety was fused to the carboxy terminus of profilin II (profilin II-GFP) was recruited by intracellular Listeria. The in vitro ligand-binding properties of this construct, e.g. the binding to monomeric actin, poly-L-proline and phosphatidylinositol 4,5-bisphosphate (PIP2), were unaffected by GFP. Profilin II-GFP co-localised with vinculin and Mena to the focal adhesions in REF-52 fibroblasts and was distributed as a thin line at the front of protruding lamellipodia in B16-F1 mouse melanoma cells. In Listeria-infected cells, profilin II-GFP was recruited, in an asymmetric fashion, to the surface of Listeria at the onset of motility whereas it was not detectable on non-motile bacteria. In contrast to the vasodilator-stimulated phosphoprotein (VASP), profilin II-GFP localised at the bacterial surface only on motile Listeria. Moreover, the fluorescence intensity of profilin II-GFP directly correlated with the speed of the bacteria. Thus, the use of GFP-tagged profilin II provides new insights into the role of profilins in cellular motility.

scholarly journals Intermediate filament-associated cytolinker plectin 1c destabilizes microtubules in keratinocytes

2013 ◽  
Vol 24 (6) ◽  
pp. 768-784 ◽  
Author(s):  
Rocio G. Valencia ◽  
Gernot Walko ◽  
Lubomir Janda ◽  
Jirka Novacek ◽  
Eva Mihailovska ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Cell Shape ◽  
Ex Vivo ◽  
Specific Binding ◽  
Focal Adhesions ◽  
Cellular Functions ◽  
Inhibitory Function ◽  
Associated Proteins ◽  
Experimental Approaches

The transition of microtubules (MTs) from an assembled to a disassembled state plays an essential role in several cellular functions. While MT dynamics are often linked to those of actin filaments, little is known about whether intermediate filaments (IFs) have an influence on MT dynamics. We show here that plectin 1c (P1c), one of the multiple isoforms of the IF-associated cytolinker protein plectin, acts as an MT destabilizer. We found that MTs in P1c-deficient (P1c−/−) keratinocytes are more resistant toward nocodazole-induced disassembly and display increased acetylation. In addition, live imaging of MTs in P1c−/−, as well as in plectin-null, cells revealed decreased MT dynamics. Increased MT stability due to P1c deficiency led to changes in cell shape, increased velocity but loss of directionality of migration, smaller-sized focal adhesions, higher glucose uptake, and mitotic spindle aberrations combined with reduced growth rates of cells. On the basis of ex vivo and in vitro experimental approaches, we suggest a mechanism for MT destabilization in which isoform-specific binding of P1c to MTs antagonizes the MT-stabilizing and assembly-promoting function of MT-associated proteins through an inhibitory function exerted by plectin's SH3 domain. Our results open new perspectives on cytolinker-coordinated IF-MT interaction and its physiological significance.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

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