Interactions between peripherin and neurofilaments in cultured cells: disruption of peripherin assembly by the NF-M and NF-H subunits

1999 ◽  
Vol 77 (1) ◽  
pp. 41-45 ◽  
Author(s):  
Jean-Martin Beaulieu ◽  
Janice Robertson ◽  
Jean-Pierre Julien
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Intermediate Filament Protein ◽  
Physiological Conditions ◽  
Filament Protein ◽  
Filament Network ◽  
Filament Type

Neurofilaments are the principal intermediate filament type expressed by neurons. They are formed by the co-assembly of three subunits: NF-L, NF-M, and NF-H. Peripherin is another intermediate filament protein expressed mostly in neurons of the peripheral nervous system. In contrast to neurofilaments, peripherin can self-assemble to establish an intermediate filament network in cultured cells. The co-expression of neurofilaments and peripherin is found mainly during development and regeneration. We used SW13 cells devoid of endogenous cytoplasmic intermediate filaments to assess the exact assembly characteristics of peripherin with each neurofilament subunit. Our results demonstrate that peripherin can assemble with NF-L. In contrast, the co-expression of peripherin with the large neurofilament subunits interferes with peripherin assembly. These results confirm the existence of interactions between peripherin and neurofilaments in physiological conditions. Moreover, they suggest that perturbations in the stoichiometry of neurofilaments can have an impact on peripherin assembly in vivo.Key words: peripherin, neurofilament, SW13 cells, intermediate filament.

The presence or absence of a vimentin-type intermediate filament network affects the shape of the nucleus in human SW-13 cells

1994 ◽  
Vol 107 (6) ◽  
pp. 1593-1607 ◽  
Author(s):  
A.J. Sarria ◽  
J.G. Lieber ◽  
S.K. Nordeen ◽  
R.M. Evans
Keyword(s):  
Electron Microscopy ◽  
Intermediate Filament ◽  
Nuclear Morphology ◽  
Intermediate Filament Protein ◽  
Mosaic Pattern ◽  
Expression Plasmid ◽  
Vimentin Expression ◽  
Filament System ◽  
Filament Protein ◽  
Filament Network

Human SW-13 cells express the intermediate filament protein vimentin in a mosaic pattern (Hedberg, K. K. and Chen, L. B. (1986). Exp. Cell Res. 163, 509–517). We have isolated SW-13 clones that do (vim+) or do not (vim-) synthesize vimentin as analyzed using anti-intermediate filament immunofluorescence, electron microscopy and two-dimensional gel analysis of detergent-extracted preparations. Vimentin is the only cytoplasmic intermediate filament protein present in the vim+ cells, and the vim- cells do not contain any detectable cytoplasmic intermediate filament system. The presence or absence of intermediate filaments did not observably affect the distribution of mitochondria, endoplasmic reticulum, microtubules or actin stress fibers when these structures were visualized by fluorescence microscopy. However, electron microscopy and anti-lamin A/C immunofluorescence studies showed that nuclear morphology in vim- cells was frequently characterized by large folds or invaginations, while vim+ cells had a more regular or smooth nuclear shape. When vim- cells were transfected with a mouse vimentin expression plasmid, the synthesis of a mouse vimentin filament network restored the smooth nuclear morphology characteristic of vim+ cells. Conversely, when vim+ cells were transfected with a carboxy-terminally truncated mutant vimentin, expression of the mutant protein disrupted the organization of the endogenous vimentin filaments and resulted in nuclei with a prominently invaginated morphology. These results indicated that in SW-13 cells the vimentin filament system affects the shape of the nucleus.

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 Association of microtubule-associated protein 2 (MAP 2) with microtubules and intermediate filaments in cultured brain cells.

1983 ◽  
Vol 96 (6) ◽  
pp. 1523-1531 ◽  
Author(s):  
G S Bloom ◽  
R B Vallee
Keyword(s):  
Intermediate Filaments ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Immune Serum ◽  
Brain Cells ◽  
Intermediate Filament Protein ◽  
Antibody Staining ◽  
Filament Protein

The classification of MAP 2 as a microtubule-associated protein is based on its affinity for microtubules in vitro and its filamentous distribution in cultured cells. We sought to determine whether MAP 2 is also able to bind in situ to organelles other than microtubules. For this purpose, primary cultures of rat brain cells were stained for immunofluorescence microscopy with a rabbit anti-MAP 2 antibody prepared in our laboratory, as well as with antibodies to vimentin, an intermediate filament protein, and to tubulin, the major subunit of microtubules. MAP 2 was present on cytoplasmic fibers in neurons and in a subpopulation of the flat cells present in the cultures. Our observations were concentrated on the flat cells because of their suitability for high-resolution immunofluorescence microscopy. Double antibody staining revealed co-localization of MAP 2 with both tubulin and vimentin in the flat cells. Pretreatment of the cultures with vinblastine resulted in the redistribution of MAP 2 into perinuclear cables that contained vimentin. Tubulin paracrystals were not stained by anti-MAP 2. In cells extracted with digitonin, the normal fibrillar distribution of MAP 2 was resistant to several treatments (PIPES buffer plus 10 mM Ca++, phosphate buffer at pH 7 or 9) that induced depolymerization of microtubules, but not intermediate filaments. Staining of the primary brain cells was not observed with preimmune serum nor with immune serum adsorbed prior to use with pure MAP 2. We detected MAP 2 on intermediate filaments not only with anti-MAP 2 serum, but also with affinity purified anti-MAP 2 and with a monoclonal anti-MAP 2 prepared in another laboratory. We conclude from these experiments that material recognized by anti-MAP 2 antibodies associates with both microtubules and intermediate filaments. We propose that one function of MAP 2 is to cross-link the two types of cellular filaments.

scholarly journals Intermediate filaments EXC-2 and IFA-4 Maintain Luminal Structure of the Tubular Excretory Canals in Caenorhabditis elegans

2018 ◽  
Author(s):  
Hikmat I. Al-Hashimi ◽  
David H. Hall ◽  
Brian D. Ackley ◽  
Erik A. Lundquist ◽  
Matthew Buechner
Keyword(s):  
Caenorhabditis Elegans ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Light And Electron Microscopy ◽  
Intermediate Filament Protein ◽  
Apical Surface ◽  
Intermediate Filament Proteins ◽  
Canal Diameter ◽  
Excretory Canals ◽  
Filament Protein

ABSTRACTThe excretory canals of Caenorhabditis elegans are a model for understanding the maintenance of apical morphology in narrow single-celled tubes. Light and electron microscopy shows that mutants in exc-2 start to form canals normally, but these swell to develop large fluid-filled cysts that lack a complete terminal web at the apical surface, and accumulate filamentous material in the canal lumen. Here, whole-genome sequencing and gene rescue show that exc-2 encodes intermediate filament protein IFC-2. EXC-2/IFC-2 protein, fluorescently tagged via CRISPR/Cas9, is located at the apical surface of the canals independently of other intermediate filament proteins. EXC-2 is also located in several other tissues, though the tagged isoforms are not seen in the larger intestinal tube. Tagged EXC-2 binds via pulldown to intermediate filament protein IFA-4, which is also shown to line the canal apical surface. Overexpression of either protein results in narrow but shortened canals. These results are consistent with a model whereby three intermediate filaments in the canals, EXC-2, IFA-4, and IFB-1, restrain swelling of narrow tubules in concert with actin filaments that guide the extension and direction of tubule outgrowth, while allowing the tube to bend as the animal moves.Article SummaryThe C. elegans excretory canals form a useful model for understanding formation of narrow tubes. exc-2 mutants start to form normal canals that then swell into fluid-filled cysts. We show that exc-2 encodes a large intermediate filament (IF) protein previously not thought to be located in the canals. EXC-2 is located at the apical (luminal) membrane, binds to another IF protein, and appears to be one of three IF proteins that form a flexible meshwork to maintain the thin canal diameter. This work provides a genetically useful model for understanding the interactions of IF proteins with other cytoskeletal elements to regulate tube size and growth.

scholarly journals Neurofilaments are obligate heteropolymers in vivo

1993 ◽  
Vol 122 (6) ◽  
pp. 1337-1350 ◽  
Author(s):  
MK Lee ◽  
Z Xu ◽  
PC Wong ◽  
DW Cleveland
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Dna Transfection ◽  
Tail Domain ◽  
In Vitro Assembly ◽  
Mature Neurons ◽  
Filament Network

Neurofilaments (NFs), composed of three distinct subunits NF-L, NF-M, and NF-H, are neuron-specific intermediate filaments present in most mature neurons. Using DNA transfection and mice expressing NF transgenes, we find that despite the ability of NF-L alone to assemble into short filaments in vitro NF-L cannot form filament arrays in vivo after expression either in cultured cells or in transgenic oligodendrocytes that otherwise do not contain a cytoplasmic intermediate filament (IF) array. Instead, NF-L aggregates into punctate or sheet like structures. Similar nonfilamentous structures are also formed when NF-M or NF-H is expressed alone. The competence of NF-L to assemble into filaments is fully restored by coexpression of NF-M or NF-H to a level approximately 10% of that of NF-L. Deletion of the head or tail domain of NF-M or substitution of the NF-H tail onto an NF-L subunit reveals that restoration of in vivo NF-L assembly competence requires an interaction provided by the NF-M or NF-H head domains. We conclude that, contrary to the expectation drawn from earlier in vitro assembly studies, NF-L is not sufficient to assemble an extended filament network in an in vivo context and that neurofilaments are obligate heteropolymers requiring NF-L and NF-M or NF-H.

Vimentin intermediate filaments in fish melanophores

1987 ◽  
Vol 88 (5) ◽  
pp. 649-655
Author(s):  
F.K. Gyoeva ◽  
E.V. Leonova ◽  
V.I. Rodionov ◽  
V.I. Gelfand
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Polyclonal Antibodies ◽  
Cell Types ◽  
Immunofluorescence Staining ◽  
Intermediate Filament Protein ◽  
Transmission Electron ◽  
Filament Protein ◽  
Pterophyllum Scalare ◽  
Cell Centre

The distribution and chemical composition of intermediate filaments in cultured melanophores of two teleost species - Gymnocorymbus ternetzi and Pterophyllum scalare - were studied by immunofluorescence staining and immunoblotting techniques. The immunofluorescence staining of the melanophores with monoclonal and polyclonal antibodies to the intermediate filament protein vimentin revealed a system of fibrils radiating from the cell centre. These fibrils were resistant to 0.6 M-KCl and nocodazole treatments as has been found in other cell types. Transmission electron microscopy confirmed the presence of intermediate filaments in melanophores. Immunoblotting experiments showed the presence of the intermediate filament protein vimentin in melanophore lysates. Therefore, teleost melanophores possess a developed radial system of vimentin intermediate filaments.

The appearance and distribution of intermediate filament proteins during differentiation of the central nervous system, skin and notochord of Xenopus laevis

Development ◽  
1986 ◽  
Vol 97 (1) ◽  
pp. 201-223
Author(s):  
S. F. Godsave ◽  
B. H. Anderton ◽  
C. C. Wylie
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Intermediate Filament ◽  
Ependymal Cells ◽  
Intermediate Filament Protein ◽  
Neurofilament Protein ◽  
Differentiation Markers ◽  
Intermediate Filament Proteins ◽  
The Central Nervous System ◽  
Filament Protein

Antibodies against various intermediate filament proteins have been used to follow cell differentiation in the early Xenopus embryo. Three new monoclonal antibodies against Xenopus cytokeratins raised against Triton-insoluble material from tadpoles (RD35/2a, RD35/3a and D3/3a), two antibodies against mammalian cytokeratins (LE65 and LP3K), monoclonal anti-(rat 200K neurofilament protein), rabbit anti-(rat glial filament acidic protein), and rabbit antibodies to hamster and calf vimentin were used. We show that cytokeratins are present in the early central nervous system (CNS) and persist in the ependymal cells of the adult CNS. We also show that the notochord contains cytokeratin. The ontogeny of intermediate filament protein appearance in the CNS, skin and notochord between neural fold stage and swimming tadpole stage are described. These results are discussed in particular with regard to the use of the antibodies as differentiation markers.

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