Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis

Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phase-binding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several microm. The basic amino-terminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by co-immunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound - and does not bind - to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques.

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Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.111.6.3049 ◽

1990 ◽

Vol 111 (6) ◽

pp. 3049-3064 ◽

Cited By ~ 85

Author(s):

P A Coulombe ◽

Y M Chan ◽

K Albers ◽

E Fuchs

Keyword(s):

Intermediate Filament ◽

Intermediate Filament Proteins ◽

Amino Terminal ◽

Filament Assembly ◽

Keratin Filaments ◽

Keratin Filament ◽

Filament Networks ◽

10 Nm Filaments

To investigate the sequences important for assembly of keratins into 10-nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.

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Structural Elements the Amino-Terminal Head Domain of Vimentin Essential for Intermediate Filament Formation in Vivo and in Vitro

Experimental Cell Research ◽

10.1006/excr.1994.1182 ◽

1994 ◽

Vol 213 (1) ◽

pp. 128-142 ◽

Cited By ~ 29

Author(s):

Michael Beuttenmüller ◽

Ming Chen ◽

Alfred Janetzko ◽

Siegfried Kühn ◽

Peter Traub

Keyword(s):

Intermediate Filament ◽

Structural Elements ◽

Filament Formation ◽

Amino Terminal ◽

Head Domain

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Enteropathogenic EscherichiacoliVirulence Factor Bundle-Forming Pilus Has a Binding Specificity for Phosphatidylethanolamine

Infection and Immunity ◽

10.1128/iai.69.11.6573-6579.2001 ◽

2001 ◽

Vol 69 (11) ◽

pp. 6573-6579 ◽

Cited By ~ 19

Author(s):

C. Khursigara ◽

M. Abul-Milh ◽

B. Lau ◽

J. A. Girón ◽

C. A. Lingwood ◽

...

Keyword(s):

Epithelial Cells ◽

Solid Phase ◽

Specific Interaction ◽

Laboratory Strain ◽

Host Cells ◽

Wild Type ◽

Binding Assays ◽

Thin Layer Chromatogram ◽

Cultured Epithelial Cells ◽

Overlay Assay

ABSTRACT The bundle-forming pilus (BFP) of enteropathogenicEscherichia coli (EPEC), an established virulence factor encoded on the EPEC adherence factor (EAF) plasmid, has been implicated in the formation of bacterial autoaggregates and in the localized adherence of EPEC to cultured epithelial cells. While understanding of the pathogenic mechanism of this organism is rapidly improving, a receptor ligand for BFP has not yet been identified. We now report, using both solid-phase and liposome binding assays, that BFP expression correlates with phosphatidylethanolamine (PE) binding. In a thin-layer chromatogram overlay assay, specific recognition of PE was documented for BFP-expressing strains, including E2348/69, a wild-type EPEC clinical isolate, as well as a laboratory strain, HB101, transformed with a bfp-carrying plasmid. Strains which did not express BFP did not bind PE, including a bfpAdisruptional mutant of E2348/69, EAF plasmid-cured E2348/69, and HB101. E2348/69 also aggregated PE-containing liposomes but not phosphatidylcholine- or phosphatidylserine-containing liposomes, while BFP-negative strains did not produce aggregates with any tested liposomes. Purified BFP preparations bound commercial PE standards as well as a PE-containing band within lipid extracts from human epithelial cells and from E2348/69. Our results therefore indicate a specific interaction between BFP and PE and suggest that PE may serve as a BFP receptor for bacterial autoaggregation and may promote localized adherence to host cells, both of which contribute to bacterial pathogenesis.

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Differential effect of arginine modification with 1,2-cyclohexanedione on the capacity of vimentin and desmin to assemble into intermediate filaments and to bind to nucleic acids

Journal of Cell Science ◽

10.1242/jcs.65.1.1 ◽

1984 ◽

Vol 65 (1) ◽

pp. 1-20

Author(s):

P. Traub ◽

C.E. Vorgias

Keyword(s):

Nucleic Acids ◽

Intermediate Filaments ◽

Intermediate Filament ◽

Specific Interaction ◽

Inhibitory Effect ◽

Affinity Column ◽

Intermediate Filament Proteins ◽

Amino Terminal ◽

Short Period ◽

Arginine Residues

When the intermediate filament proteins vimentin and desmin were reacted for a short period of time with the arginine-specific reagent 1,2-cyclohexanedione, the modification had a severe, inhibitory effect on the assembly of intermediate filaments and on the susceptibility of the basic, amino-terminal polypeptide of both proteins to degradation by the intermediate filament-specific, Ca2+-activated proteinase. However, it had only a slightly inhibitory effect on the binding of vimentin and desmin to ribosomal RNA from Ehrlich ascites tumour cells. Since the Ca2+-activated proteinase is very likely to be a trypsin-like enzyme, with a preference for arginyl and lysyl peptide bonds, the results indicate that the arginine residues of the amino-terminal polypeptide of vimentin and desmin are highly essential for filament assembly but largely dispensable for the binding of both proteins to nucleic acids. This was supported by the observation that two breakdown products of vimentin lacking a 5 X 10(3) Mr and an 8 X 10(3) Mr polypeptide from the amino terminus, respectively, did not assemble into intermediate filaments but were still capable of binding to rRNA. Both polypeptides also bound to single-stranded DNA-cellulose under non-denaturing conditions, but passed the affinity column in the presence of 6 M-urea. Thus, the binding of vimentin to nucleic acids appears to be based on two components: a non-specific electrostatic interaction mediated by the positively charged arginine residues of the amino-terminal polypeptide that is insensitive to denaturation by urea, and a specific interaction that is sensitive to denaturation by urea.

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Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells

Molecular and Cellular Biology ◽

10.1128/mcb.2.9.1104-1114.1982 ◽

1982 ◽

Vol 2 (9) ◽

pp. 1104-1114

Author(s):

D L Gard ◽

E Lazarides

Keyword(s):

Cyclic Amp ◽

Intermediate Filament ◽

Specific Activity ◽

Fold Increase ◽

Major Protein ◽

Myogenic Cells ◽

Intermediate Filament Proteins ◽

Dependent Protein

The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation.

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Characterization of rat mammary cell types in primary culture: lectins and antisera to basement membrane and intermediate filament proteins as indicators of cellular heterogeneity

Journal of Cell Science ◽

10.1242/jcs.79.1.287 ◽

1985 ◽

Vol 79 (1) ◽

pp. 287-304

Author(s):

M.J. Warburton ◽

S.A. Ferns ◽

C.M. Hughes ◽

P.S. Rudland

Keyword(s):

Membrane Proteins ◽

Epithelial Cells ◽

Basement Membrane ◽

Intermediate Filament ◽

Cell Types ◽

Myoepithelial Cells ◽

Epithelioid Cells ◽

Intermediate Filament Proteins ◽

Basement Membrane Proteins

Three morphologically distinct major cell types were observed in primary cultures obtained from the mammary parenchyma of glands from virgin rats. These cell types consisted of small cuboidal epithelial cells, larger epithelioid cells and elongated cells. We have investigated the distribution of the basement membrane proteins laminin and type IV collagen, and the intermediate filament proteins vimentin and prekeratin, in these three cell types using immunofluorescence techniques. Antisera to the basement membrane proteins stain the large epithelioid cells and the elongated cells, but do not stain the small cuboidal cells. Polyclonal antiserum to keratin stains all the small cuboidal and large epithelioid cells, but only a small subpopulation of the elongated cells. However, a monoclonal antibody to keratin, LP34, stains only the large cuboidal and a proportion of the elongated cells. Vimentin antiserum fails to stain the small cuboidal cells but stains all the large epithelioid and elongated cells. In addition, peanut lectin, which binds only to ductal lining epithelial cells in the virgin rat mammary gland in vivo after their treatment with neuraminidase, binds to the small cuboidal cells after neuraminidase treatment but not to the other cell types. However, Griffonia simplicifolia agglutinin I, which specifically stains myoepithelial cells in vivo, binds to the large epithelioid and elongated cells but not to the small cuboidal cells. These results suggest that the small cuboidal cells are related to mammary ductal epithelial cells whereas the large epithelial and elongated cells have some characteristics of myoepithelial cells.

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A new model for nuclear lamina organization

Biochemical Society Transactions ◽

10.1042/bst0361339 ◽

2008 ◽

Vol 36 (6) ◽

pp. 1339-1343 ◽

Cited By ~ 48

Author(s):

Martin W. Goldberg ◽

Jindriska Fiserova ◽

Irm Huttenlauch ◽

Reimer Stick

Keyword(s):

Mechanical Strength ◽

Nuclear Membrane ◽

Intermediate Filament ◽

Xenopus Oocytes ◽

Nuclear Lamina ◽

Intermediate Filament Proteins ◽

New Model ◽

10 Nm Filaments

Lamins are intermediate filament proteins that form a network lining the inner nuclear membrane. They provide mechanical strength to the nuclear envelope, but also appear to have many other functions as reflected in the array of diseases caused by lamin mutations. Unlike other intermediate filament proteins, they do not self-assemble into 10 nm filaments in vitro and their in vivo organization is uncertain. We have recently re-examined the organization of a simple B-type lamina in Xenopus oocytes [Goldberg, Huttenlauch, Hutchison and Stick (2008) J. Cell Sci. 121, 215–225] and shown that it consists of tightly packed 8–10 nm filaments with regular cross-connections, tightly opposed to the membrane. When lamin A is expressed in oocytes, it forms organized bundles on top of the B lamina. This has led to a new model for lamina organization which is discussed in the present paper.

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In Vivo and In Vitro Analysis of Age-Associated Changes and Somatic Cellular Senescence in Renal Epithelial Cells

PLoS ONE ◽

10.1371/journal.pone.0088071 ◽

2014 ◽

Vol 9 (2) ◽

pp. e88071 ◽

Cited By ~ 26

Author(s):

Birgit Berkenkamp ◽

Nathan Susnik ◽

Arpita Baisantry ◽

Inna Kuznetsova ◽

Christoph Jacobi ◽

...

Keyword(s):

Epithelial Cells ◽

Cellular Senescence ◽

Renal Epithelial Cells ◽

Vitro Analysis ◽

In Vitro Analysis

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In vitro translation of the intermediate filament proteins desmin and vimentin

Molecular and Cellular Biology ◽

10.1128/mcb.1.4.303-309.1981 ◽

1981 ◽

Vol 1 (4) ◽

pp. 303-309

Author(s):

C M O'Connor ◽

D J Asai ◽

C N Flytzanis ◽

E Lazarides

Keyword(s):

Smooth Muscle ◽

Intermediate Filament ◽

Messenger Rna ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

In Vitro Translation ◽

Messenger Rnas ◽

Intermediate Filament Proteins

Polyadenylated ribonucleic acid (RNA) was isolated from chicken skeletal and smooth muscle and translated in a cell-free rabbit reticulocyte system. Both types of muscle tissue contain messenger RNAs that code for the intermediate filament proteins desmin and vimentin, and the relative concentrations of the two translation products reflect the prevalence of the two proteins in vivo. Desmin synthesis represents a greater proportion of the total protein synthesis from smooth muscle RNA than from skeletal muscle RNA, whereas the converse is true of vimentin synthesis. Fractionation of the RNA on formamide-containing sucrose gradients before translation indicates that the desmin messenger RNA is larger than the vimentin messenger RNA and contains an extensive noncoding segment. The desmin and vimentin messages code predominantly for the non-phosphorylated forms of desmin and vimentin. However, the ratio of phosphorylated to unphosphorylated forms of the proteins could be increased by adding cyclic adenosine monophosphate-dependent kinase activity to the translation mixtures. These results suggest that desmin and vimentin are each synthesized from a single messenger RNA species and that posttranslational phosphorylation generates the additional isoelectric variants of each which are observed in vivo.

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