[35S]Methionine Metabolic Labeling to Study Axonal Transport of Neuronal Intermediate Filament Proteins In Vivo

2004 ◽  
pp. 555-571 ◽  
Author(s):  
Stéphanie Millecamps ◽  
Jean-Pierre Julien
Keyword(s):  
Axonal Transport ◽  
Intermediate Filament ◽  
Metabolic Labeling ◽  
Intermediate Filament Proteins ◽  
Neuronal Intermediate Filament Proteins

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

2000 ◽  
Vol 113 (13) ◽  
pp. 2471-2483 ◽  
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.

scholarly journals Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments

1993 ◽  
Vol 122 (6) ◽  
pp. 1323-1335 ◽  
Author(s):  
GY Ching ◽  
RK Liem
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Neuronal Cell ◽  
Intermediate Filament Protein ◽  
Amino Acid Residues ◽  
Intermediate Filament Proteins ◽  
Transfected Cells ◽  
Type Iv

We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha-internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.

scholarly journals Quantitative phosphoproteomic analysis of neuronal intermediate filament proteins (NF‐M/H) in Alzheimer's disease by iTRAQ

2010 ◽  
Vol 24 (11) ◽  
pp. 4396-4407 ◽  
Author(s):  
Parvathi Rudrabhatla ◽  
Philip Grant ◽  
Howard Jaffe ◽  
Michael J. Strong ◽  
Harish C. Pant
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Intermediate Filament ◽  
Intermediate Filament Proteins ◽  
Neuronal Intermediate Filament Proteins

scholarly journals Characterisation of neurofilament protein NF70 mRNA from the gastropod Helix aspersa reveals that neuronal and non-neuronal intermediate filament proteins of cerebral ganglia arise from separate lamin-related genes

1995 ◽  
Vol 108 (11) ◽  
pp. 3581-3590 ◽  
Author(s):  
J. Adjaye ◽  
U. Plessmann ◽  
K. Weber ◽  
H. Dodemont
Keyword(s):  
Sequence Analysis ◽  
Intermediate Filament ◽  
Cerebral Ganglion ◽  
Helix Aspersa ◽  
Neurofilament Protein ◽  
Intermediate Filament Proteins ◽  
Nuclear Lamins ◽  
Untranslated Sequence ◽  
Cerebral Ganglia ◽  
Neuronal Intermediate Filament Proteins

The neuronal cytoplasmic intermediate filament (IF) protein HeNF70 of the gastropod Helix aspersa is identified by sequence analysis of the corresponding 4,600 bp cDNA isolated from a cerebral ganglion cDNA library. HeNF70 shares 60% sequence identity with the neuronal LoNF70 protein of the cephalopod Loligo pealei and only 36% identity with the Helix non-neuronal IF-A protein. All three molluscan IF proteins display the lamin-type extended coil 1b subdomain harbouring six additional heptads and all have long C-terminal sequences with substantial homology to the tail domains of nuclear lamins. The lamin-like tail domains of the two neurofilament proteins share a unique motif comprising 13 residues, which is absent from Helix IF-A and all other known non-neuronal IF proteins. HeNF70 is encoded by a 9.5 kb RNA transcript. The very long 7.2 kb 3′-untranslated sequence contains a unique 26 nucleotide repeat extending over 0.5 kb in its 5′-region. The HeNF70 mRNA is expressed at low abundancy in cerebral ganglia but not in any of the 13 non-neuronal tissues tested. In contrast, all tissues express at fairly high levels the same 4.6 and 4.2 kb mRNAs encoding the Helix non-neuronal IF-A/B proteins. Blot hybridisation studies on genomic DNA and ganglion mRNA with subprobes from the cloned HeNF70 cDNA, as well as sequence analysis of an RT-PCR generated partial cDNA encoding a putative HeNF60 protein, indicate at least two different neuronal IF genes in Helix.

Analysis of the roles of the head domains of type IV rat neuronal intermediate filament proteins in filament assembly using domain-swapped chimeric proteins

1999 ◽  
Vol 112 (13) ◽  
pp. 2233-2240
Author(s):  
G.Y. Ching ◽  
R.K. Liem
Keyword(s):  
Intermediate Filament ◽  
Self Assembly ◽  
Chimeric Proteins ◽  
Filament Formation ◽  
Intermediate Filament Proteins ◽  
L Protein ◽  
Type Iv ◽  
Filament Assembly ◽  
Head Domain ◽  
Neuronal Intermediate Filament Proteins

Type IV neuronal intermediate filament proteins consist of alpha-internexin, which can self-assemble into filaments and the neurofilament triplet proteins, which are obligate heteropolymers, at least in rodents. These IF proteins therefore provide good systems for elucidating the mechanism of intermediate filament assembly. To analyze the roles of the head domains of these proteins in contributing to their differential assembly properties, we generated chimeric proteins by swapping the head domains between rat alpha-internexin and either rat NF-L or NF-M and examined their assembly properties in transfected cells that lack their own cytoplasmic intermediate filament network. Lalphaalpha and Malphaalpha, the chimeric proteins generated by replacing the head domain of alpha-internexin with those of NF-L and NF-M, respectively, were unable to self-assemble into filaments. In contrast, alphaLL, a chimeric NF-L protein generated by replacing the head domain of NF-L with that of alpha-internexin, was able to self-assemble into filaments, whereas MLL, a chimeric NF-L protein containing the NF-M head domain, was unable to do so. These results demonstrate that the alpha-internexin head domain is essential for alpha-internexin's ability to self-assemble. While coassembly of Lalphaalpha with NF-M and coassembly of Malphaalpha with NF-L resulted in formation of filaments, coassembly of Lalphaalpha with NF-L and coassembly of Malphaalpha with NF-M yielded punctate patterns. These coassembly results show that heteropolymeric filament formation requires that one partner has the NF-L head domain and the other partner has the NF-M head domain. Thus, the head domains of rat NF-L and NF-M play important roles in determining the obligate heteropolymeric nature of filament formation. The data obtained from these self-assembly and coassembly studies provide some new insights into the mechanism of intermediate filament assembly.

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

1990 ◽  
Vol 111 (6) ◽  
pp. 3049-3064 ◽  
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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