Intermediate filament networks: in vitro and in vivo assembly models

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

Journal of Cell Science ◽

10.1242/jcs.113.13.2471 ◽

2000 ◽

Vol 113 (13) ◽

pp. 2471-2483 ◽

Cited By ~ 3

Author(s):

I. Hofmann ◽

C. Mertens ◽

M. Brettel ◽

V. Nimmrich ◽

M. Schnolzer ◽

...

Keyword(s):

Epithelial Cells ◽

Intermediate Filament ◽

Solid Phase ◽

Specific Interaction ◽

Binding Assays ◽

Intermediate Filament Proteins ◽

Amino Terminal ◽

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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Vimentin in a cold-water fish, the rainbow trout: highly conserved primary structure but unique assembly properties

Journal of Cell Science ◽

10.1242/jcs.109.3.569 ◽

1996 ◽

Vol 109 (3) ◽

pp. 569-578 ◽

Cited By ~ 2

Author(s):

H. Herrmann ◽

M.D. Munick ◽

M. Brettel ◽

B. Fouquet ◽

J. Markl

Keyword(s):

Rainbow Trout ◽

Intermediate Filament ◽

Cold Water ◽

White Blood Cells ◽

Cellular Functions ◽

Filamentous Form ◽

Rainbow Trout Oncorhynchus Mykiss ◽

Bona Fide

We have isolated from a rainbow trout (Oncorhynchus mykiss) spleen cDNA library a clone coding for vimentin. The deduced amino acid sequence reveals a high degree of identity with vimentin from carp (81%), frog (71%), chick and human (73% each). Large stretches in the central alpha-helical rod are identical within all four classes of vertebrates, but in 17 residues spread over the entire rod, the two fish differ distinctly from the tetrapod species. In addition, in the more diverged non-helical head domain, a nonapeptide motif previously shown to be important for regular filament formation is conserved. Recombinant trout vimentin assembles into bona fide filaments in vitro, with a temperature optimum between 18 and 24 degrees C. Above 27 degrees C, however, filament assembly is abruptly abolished and short filaments with thickened ends as well as structures without typical intermediate filament appearance are formed. This distinguishes its assembly properties significantly from amphibian, avian and mammalian vimentin. Also in vivo, after cDNA transfection into vimentin-free mammalian epithelial cells, trout vimentin does not form typical intermediate filament arrays at 37 degrees C. At 28 degrees C, and even more pronounced at 22 degrees C, the vimentin-positive material in the transfected cells is reorganized in the perinuclear region with a partial fibrillar appearance, but typical intermediate filament arrays are not formed. Together with immunoblotting and immunolocalization data from trout tissues, where vimentin is predominantly found in glial and white blood cells, we conclude that vimentin is indeed important in its filamentous form in fish and other vertebrates, possibly fulfilling cellular functions not directly evident in gene targeting experiments carried out in mice.

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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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Neuronal intermediate filament expression in rat dorsal root ganglia sensory neurons: An in vivo and in vitro study

Neuroscience ◽

10.1016/j.neuroscience.2008.02.080 ◽

2008 ◽

Vol 153 (4) ◽

pp. 1153-1163 ◽

Cited By ~ 42

Author(s):

M. Fornaro ◽

J.M. Lee ◽

S. Raimondo ◽

S. Nicolino ◽

S. Geuna ◽

...

Keyword(s):

Sensory Neurons ◽

Dorsal Root Ganglia ◽

Intermediate Filament ◽

Dorsal Root ◽

In Vitro Study ◽

Vitro Study

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A 295-kDA intermediate filament-associated protein in radial glia and developing muscle cells in vivo and in vitro

Developmental Dynamics ◽

10.1002/1097-0177(2000)9999:9999<::aid-dvdy1078>3.0.co;2-0 ◽

2000 ◽

Vol 219 (4) ◽

pp. 514-525 ◽

Cited By ~ 27

Author(s):

Grazyna Chanas-Sacr� ◽

Marc Thiry ◽

Sandrine Pirard ◽

Bernard Rogister ◽

Gustave Moonen ◽

...

Keyword(s):

Intermediate Filament ◽

Radial Glia ◽

Muscle Cells ◽

Developing Muscle

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alpha-Internexin, a 66-kD intermediate filament-binding protein from mammalian central nervous tissues.

The Journal of Cell Biology ◽

10.1083/jcb.101.4.1316 ◽

1985 ◽

Vol 101 (4) ◽

pp. 1316-1322 ◽

Cited By ~ 74

Author(s):

J S Pachter ◽

R K Liem

Keyword(s):

Optic Nerve ◽

Intermediate Filaments ◽

Intermediate Filament ◽

Peptide Mapping ◽

Exchange Chromatography ◽

Filament Assembly ◽

Antigen Antibody

In this paper we describe a 66-kD protein that co-purifies with intermediate filaments from rat optic nerve and spinal cord but can be separated further by ion-exchange chromatography. This protein is distinct from the 68-kD neurofilament subunit protein as judged by isoelectric focusing, immunoblotting, peptide mapping, and tests of polymerization competence. This protein is avidly recognized by the monoclonal anti-intermediate filament antigen antibody, previously demonstrated to recognize a common antigenic determinant in all five known classes of intermediate filaments. Also, when isolated this protein binds to various intermediate filament subunit proteins, which suggests an in vivo interaction with the intermediate filament cytoskeleton, and it appears to be axonally transported in the rat optic nerve. Because of this ability to bind to intermediate filaments in situ and in vitro we have named this protein alpha-internexin. A possible functional role for the protein in organizing filament assembly and distribution is discussed.

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Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin

Biochemical Journal ◽

10.1042/bj20120209 ◽

2012 ◽

Vol 444 (1) ◽

pp. 89-96 ◽

Cited By ~ 20

Author(s):

Keefe T. Chan ◽

David W. Roadcap ◽

Nicholas Holoweckyj ◽

James E. Bear

Keyword(s):

Binding Site ◽

Leading Edge ◽

Actin Binding ◽

Filamentous Actin ◽

Unique Region ◽

Equivalent Residue ◽

Filament Networks ◽

Actin Binding Site

Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg30) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo. However, whether this finding translates to other coronins has not been well defined. Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C. By mutagenesis and biochemical competition, we have identified a second actin-binding site in the unique region of Coronin 1C. Interestingly, leading-edge localization of Coronin 1C in fibroblasts requires the conserved site in the β-propeller, but not the site in the unique region. Furthermore, in contrast with Coronin 1A and Coronin 1B, Coronin 1C displays highly co-operative binding to actin filaments. In the present study, we highlight a novel mode of coronin regulation, which has implications for how coronins orchestrate cytoskeletal dynamics.

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