Concentration of an integral membrane protein, CD43 (leukosialin, sialophorin), in the cleavage furrow through the interaction of its cytoplasmic domain with actin-based cytoskeletons.

S Yonemura; A Nagafuchi; N Sato; S Tsukita

doi:10.1083/jcb.120.2.437

Concentration of an integral membrane protein, CD43 (leukosialin, sialophorin), in the cleavage furrow through the interaction of its cytoplasmic domain with actin-based cytoskeletons.

The Journal of Cell Biology ◽

10.1083/jcb.120.2.437 ◽

1993 ◽

Vol 120 (2) ◽

pp. 437-449 ◽

Cited By ~ 97

Author(s):

S Yonemura ◽

A Nagafuchi ◽

N Sato ◽

S Tsukita

Keyword(s):

Cell Cycle ◽

Membrane Protein ◽

Actin Filaments ◽

Integral Membrane Protein ◽

Cytoplasmic Domain ◽

Electron Microscopic Level ◽

Cleavage Furrow ◽

Electron Microscopic ◽

Attachment Sites ◽

E Cadherin

In leukocytes such as thymocytes and basophilic leukemia cells, a glycosilated integral membrane protein called CD43 (leukosialin or sialophorin), which is defective in patients with Wiskott-Aldrich syndrome, was highly concentrated in the cleavage furrow during cytokinesis. Not only at the mitotic phase but also at interphase, CD43 was precisely colocalized with ezrin-radixin-moesin family members. (ERM), which were previously reported to play an important role in the plasma membrane-actin filament association in general. At the electron microscopic level, throughout the cell cycle, both CD43 and ERM were tightly associated with microvilli, providing membrane attachment sites for actin filaments. We constructed a cDNA encoding a chimeric molecule consisting of the extracellular domain of mouse E-cadherin and the transmembrane/cytoplasmic domain of rat CD43, and introduced it into mouse L fibroblasts lacking both endogenous CD43 and E-cadherin. In dividing transfectants, the chimeric molecules were concentrated in the cleavage furrow together with ERM, and both proteins were precisely colocalized throughout the cell cycle. Furthermore, using this transfection system, we narrowed down the domain responsible for the CD43-concentration in the cleavage furrow. Based on these findings, we conclude that CD43 is concentrated in the cleavage furrow through the direct or indirect interaction of its cytoplasmic domain with ERM and actin filaments.

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Occludin: a novel integral membrane protein localizing at tight junctions.

The Journal of Cell Biology ◽

10.1083/jcb.123.6.1777 ◽

1993 ◽

Vol 123 (6) ◽

pp. 1777-1788 ◽

Cited By ~ 1628

Author(s):

M Furuse ◽

T Hirase ◽

M Itoh ◽

A Nagafuchi ◽

S Yonemura ◽

...

Keyword(s):

Membrane Protein ◽

Tight Junctions ◽

Plasma Membranes ◽

Protein A ◽

Adherens Junction ◽

Integral Membrane Protein ◽

Electron Microscopic Level ◽

Electron Microscopic ◽

Membrane Contact ◽

Membrane Spanning

Recently, we found that ZO-1, a tight junction-associated protein, was concentrated in the so called isolated adherens junction fraction from the liver (Itoh, M., A. Nagafuchi, S. Yonemura, T. Kitani-Yasuda, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 121:491-502). Using this fraction derived from chick liver as an antigen, we obtained three monoclonal antibodies specific for a approximately 65-kD protein in rats. This antigen was not extractable from plasma membranes without detergent, suggesting that it is an integral membrane protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this approximately 65-kD membrane protein was exclusively localized at tight junctions of both epithelial and endothelial cells: at the electron microscopic level, the labels were detected directly over the points of membrane contact in tight junctions. To further clarify the nature and structure of this membrane protein, we cloned and sequenced its cDNA. We found that the cDNA encoded a 504-amino acid polypeptide with 55.9 kDa. A search of the data base identified no proteins with significant homology to this membrane protein. A most striking feature of its primary structure was revealed by a hydrophilicity plot: four putative membrane-spanning segments were included in the NH2-terminal half. This hydrophilicity plot was very similar to that of connexin, an integral membrane protein in gap junctions. These findings revealed that an integral membrane protein localizing at tight junctions is now identified, which we designated as "occludin."

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Detergent/Nanodisc Screening for High-Resolution NMR Studies of an Integral Membrane Protein Containing a Cytoplasmic Domain

PLoS ONE ◽

10.1371/journal.pone.0054378 ◽

2013 ◽

Vol 8 (1) ◽

pp. e54378 ◽

Cited By ~ 31

Author(s):

Christos Tzitzilonis ◽

Cédric Eichmann ◽

Innokentiy Maslennikov ◽

Senyon Choe ◽

Roland Riek

Keyword(s):

High Resolution ◽

Membrane Protein ◽

Integral Membrane Protein ◽

Cytoplasmic Domain ◽

Nmr Studies ◽

High Resolution Nmr

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Lipocortin 1 (annexin 1) in patches associated with the membrane of a lung adenocarcinoma cell line and in the cell cytoplasm

Journal of Cell Science ◽

10.1242/jcs.111.10.1405 ◽

1998 ◽

Vol 111 (10) ◽

pp. 1405-1418 ◽

Cited By ~ 1

Author(s):

V. Traverso ◽

J.F. Morris ◽

R.J. Flower ◽

J. Buckingham

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Lung Adenocarcinoma ◽

Western Blotting ◽

Signal Sequence ◽

Membrane Surface ◽

Integral Membrane Protein ◽

Subcellular Fractionation ◽

Cell Fractionation ◽

Electron Microscopic

Lipocortin 1 (annexin I) is a calcium- and phospholipid-binding annexin protein which can be externalised from cells despite the lack of a signal sequence. To determine its cellular distribution lipocortin 1 in A549 human lung adenocarcinoma cells was localised by light- and electron-microscopic immunocytochemistry and by cell fractionation and western blotting. Lipocortin 1 immunoreactivity is concentrated in prominent patches associated with the plasma membrane. The intensity of these patches varied with the confluence and duration of the culture and was not detectably diminished by an EDTA wash before fixation. Tubulin and cytokeratin 8 were colocalized with lipocortin 1 in the patches. Within the cells lipocortin 1 was distributed throughout the cytoplasm. Electron microscopy revealed prominent immunoreactivity along the plasma membrane with occasional large clusters of gold particles in contact with the membrane surface of the cells; within the cytoplasm the membrane of some vesicle/vacuole structures and some small electron-dense bodies was immunoreactive, but no immunogold particles were associated with the multilamellar bodies. Subcellular fractionation, extraction and western blotting showed that lipocortin 1 in the membrane pellet was present as two distinct fractions; one, intimately associated with the lipid bilayer, which behaved like an integral membrane protein and one loosely attached which behaved like a peripheral membrane protein. The results show that a substantial amounts of lipocortin 1 is concentrated in focal structures associated with and immediately beneath the plasma membrane. These might form part of the mechanism by which lipocortin 1 is released from the cells.

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An integral membrane protein antigen associated with the membrane attachment sites of actin microfilaments is identified as an integrin beta-chain

Molecular and Cellular Biology ◽

10.1128/mcb.8.2.564-570.1988 ◽

1988 ◽

Vol 8 (2) ◽

pp. 564-570

Author(s):

P A Maher ◽

S J Singer

Keyword(s):

Molecular Weight ◽

Membrane Protein ◽

Protein Complex ◽

Apparent Molecular Weight ◽

Integral Membrane Protein ◽

Beta Chain ◽

Attachment Sites ◽

Wide Range ◽

Membrane Attachment

A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101:785-801, 1985) which identified an integral membrane glycoprotein of chicken cells that was associated with a wide variety of sites of actin microfilament attachments to membranes. In this report, we present a further characterization of this integral protein. An immunochemical comparison was made of MAb 30B6 binding properties with those of two other MAbs, JG9 and JG22, which identify a component of a membrane protein complex that interacts with extracellular matrix proteins including fibronectin. We showed that the 110-kilodalton protein recognized by MAb 30B6 in extracts of chicken gizzard smooth muscle is identical, or closely related, to the protein that reacts with MAbs JG9 and JG22. These 110-kilodalton proteins are also structurally closely similar, if not identical, to one another as demonstrated by 125I-tryptic peptide maps. However, competition experiments showed that MAb 30B6 recognizes a different epitope from those recognized by MAbs JG9 and JG22. In addition, the 30B6 antigen is part of a complex that can be isolated on fibronectin columns. These results together establish that the 30B6 antigen is the same as, or closely similar to, the beta-chain of the protein complex named integrin, which is the complex on chicken fibroblast membranes that binds fibronectin. Although the 30B6 antigen is present in a wide range of tissues, its apparent molecular weight on gels varies in different tissues. These differences in apparent molecular weight are due, in large part, to differences in glycosylation.

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Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique.

The Journal of Cell Biology ◽

10.1083/jcb.105.4.1771 ◽

1987 ◽

Vol 105 (4) ◽

pp. 1771-1780 ◽

Cited By ~ 35

Author(s):

T Nakata ◽

N Hirokawa

Keyword(s):

Actin Filaments ◽

Plasma Membranes ◽

Human Platelets ◽

Electron Microscopic Level ◽

Immunocytochemical Study ◽

Cytoskeletal Reorganization ◽

Electron Microscopic ◽

Activated Platelets ◽

Mixed Polarity ◽

Cross Bridges

We studied the cytoskeletal reorganization of saponized human platelets after stimulation by using the quick-freeze deep-etch technique, and examined the localization of myosin in thrombin-treated platelets by immunocytochemistry at the electron microscopic level. In unstimulated saponized platelets we observed cross-bridges between: adjoining microtubules, adjoining actin filaments, microtubules and actin filaments, and actin filaments and plasma membranes. After activation with 1 U/ml thrombin for 3 min, massive arrays of actin filaments with mixed polarity were found in the cytoplasm. Two types of cross-bridges between actin filaments were observed: short cross-bridges (11 +/- 2 nm), just like those observed in the resting platelets, and longer ones (22 +/- 3 nm). Actin filaments were linked with the plasma membrane via fine short filaments and sometimes ended on the membrane. Actin filaments and microtubules frequently ran close to the membrane organelles. We also found that actin filaments were associated by end-on attachments with some organelles. Decoration with subfragment 1 of myosin revealed that all the actin filaments associated end-on with the membrane pointed away in their polarity. Immunocytochemical study revealed that myosin was present in the saponin-extracted cytoskeleton after activation and that myosin was localized on the filamentous network. The results suggest that myosin forms a gel with actin filaments in activated platelets. Close associations between actin filaments and organelles in activated platelets suggests that contraction of this actomyosin gel could bring about the observed centralization of organelles.

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Biotinylphallotoxins: preparation and use as actin probes.

Journal of Histochemistry & Cytochemistry ◽

10.1177/37.7.2499619 ◽

1989 ◽

Vol 37 (7) ◽

pp. 1035-1045 ◽

Cited By ~ 7

Author(s):

H Faulstich ◽

S Zobeley ◽

U Bentrup ◽

B M Jockusch

Keyword(s):

Actin Filaments ◽

Ultrastructural Level ◽

Microscopic Level ◽

Electron Microscopic Level ◽

Electron Microscopic ◽

Permeabilized Cells ◽

High Affinity ◽

Double Label ◽

The One

We describe the synthesis of four phalloidin derivatives conjugated with biotin. An aminomethyldithiolane derivative of ketophalloidin was used as a reactive starter compound, and biotin residues were coupled to this molecule either directly, separated by spacer chains comprised of one or two glycyl residues, or of a 12-atom long chain constructed from succinic acid and hexamethylendiamine. Although all products still displayed a high affinity for F-actin, as seen in competition experiments with [3H]-demethylphalloidin, only the one with the longest spacer (BHPP) showed specific and high-affinity decoration of actin filaments in permeabilized cells, in conjunction with FITC-coupled avidin and fluorescence microscopy. Combined with gold-streptavidin, BHPP decorated the actin filament system at the light and electron microscopic level faithfully and with satisfactory density. Actin filaments polymerized in vitro from purified protein were not as densely labeled as had been expected. However, in all these experiments the new phalloidin probe, when combined with avidin or streptavidin, yielded clear and highly specific labeling of F-actin. Therefore, this system is useful to identify and localize actin unambiguously in microfilaments, independent of actin antibodies, and should facilitate double-label experiments on cytoskeletal components at the ultrastructural level.

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The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly.

The Journal of Cell Biology ◽

10.1083/jcb.120.4.909 ◽

1993 ◽

Vol 120 (4) ◽

pp. 909-922 ◽

Cited By ~ 22

Author(s):

C P Chia ◽

A Shariff ◽

S A Savage ◽

E J Luna

Keyword(s):

Membrane Protein ◽

Actin Filaments ◽

Actin Polymerization ◽

Plasma Membranes ◽

Specific Activity ◽

Lipid Vesicles ◽

Integral Membrane Protein ◽

Actin Binding ◽

Actin Assembly ◽

Nucleation Activity

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F-actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.

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Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex.

The Journal of Cell Biology ◽

10.1083/jcb.131.1.165 ◽

1995 ◽

Vol 131 (1) ◽

pp. 165-178 ◽

Cited By ~ 255

Author(s):

C M Field ◽

B M Alberts

Keyword(s):

Cell Cycle ◽

Actin Filaments ◽

Myosin Ii ◽

Drosophila Embryo ◽

Cell Cortex ◽

Cleavage Furrow ◽

Protein Fragments ◽

Culture Cells ◽

Acid Protein ◽

Sds Page

We report the cDNA sequence and localization of a protein first identified by actin filament chromatography of Drosophila embryo extracts as ABP8 (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). The cDNA encodes a 1201-amino acid protein which we name anillin. Anillin migrates at 190 kD on SDS-PAGE. Anillin is expressed throughout Drosophila development and in tissue culture cells. By immunofluorescence, anillin localizes to the nucleus of interphase cells, except in the syncytial embryo where it is always cytoplasmic. During metaphase, it is present in the cytoplasm and cortex, and during anaphase-telophase it becomes highly enriched in the cleavage furrow along with myosin II. In the syncytial embryo, anillin, along with myosin-II, is enriched in cortical areas undergoing cell cycle regulated invagination including metaphase furrows and the cellularization front. In contractile rings, metaphase furrows, and nascent ring canals, anillin remains bound to the invaginated cortex suggesting a stabilizing role. Anillin is not expressed in cells that have left the cell cycle. Anillin isolated from embryo extracts binds directly to actin filaments. The domain responsible for this binding has been mapped to a region of 244 amino acids by expression of protein fragments in bacteria. This domain, which is monomeric in solution, also bundles actin filaments. We speculate that anillin plays a role in organizing and/or stabilizing the cleavage furrow and other cell cycle regulated, contractile domains of the actin cytoskeleton.

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