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

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Roles of head and tail domains in alpha-internexin's self-assembly and coassembly with the neurofilament triplet proteins

Journal of Cell Science ◽

10.1242/jcs.111.3.321 ◽

1998 ◽

Vol 111 (3) ◽

pp. 321-333 ◽

Cited By ~ 1

Author(s):

G.Y. Ching ◽

R.K. Liem

Keyword(s):

Intermediate Filament ◽

Self Assembly ◽

Deletion Mutant ◽

Deletion Mutants ◽

Intermediate Filament Protein ◽

Intermediate Filament Proteins ◽

Amino Terminal ◽

Head Domain ◽

Normal Filament ◽

Filament Network

The roles of the head and tail domains of alpha-internexin, a type IV neuronal intermediate filament protein, in its self-assembly and coassemblies with neurofilament triplet proteins, were examined by transient transfections with deletion mutants in a non-neuronal cell line lacking an endogenous cytoplasmic intermediate filament network. The results from the self-assembly studies showed that the head domain was essential for alpha-internexin's ability to self-assemble into a filament network and the tail domain was important for establishing a proper filament network. The data from the coassembly studies demonstrated that alpha-internexin interacted differentially with the neurofilament triplet protein subunits. Wild-type NF-L or NF-M, but not NF-H, was able to complement and form a normal filament network with the tailless alpha-internexin mutant, the alpha-internexin head-deletion mutant, or the alpha-internexin mutant missing the entire tail and some amino-terminal portion of the head domain. In contrast, neither the tailless NF-L mutant nor the NF-L head-deletion mutant was able to form a normal filament network with any of these alpha-internexin deletion mutants. However, coassembly of the tailless NF-M mutant with the alpha-internexin head-deletion mutant and coassembly of the NF-M head-deletion mutant with the tailless alpha-internexin mutant resulted in the formation of a normal filament network. Thus, the coassembly between alpha-internexin and NF-M exhibits some unique characteristics previously not observed with other intermediate filament proteins: only one intact tail and one intact head are required for the formation of a normal filament network, and they can be present within the same partner or separately in two partners.

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The endless story of the glial fibrillary acidic protein

Journal of Cell Science ◽

10.1242/jcs.107.8.2299 ◽

1994 ◽

Vol 107 (8) ◽

pp. 2299-2311 ◽

Cited By ~ 7

Author(s):

W.J. Chen ◽

R.K. Liem

Keyword(s):

Glial Fibrillary Acidic Protein ◽

Intermediate Filament ◽

Self Assembly ◽

Acidic Protein ◽

Terminal Deletion ◽

Deletion Mutants ◽

Tail Domain ◽

Intermediate Filament Proteins ◽

Type Iii ◽

Head Domain

All intermediate filament proteins consist of an alpha-helical rod domain flanked by non-helical N-terminal head and C-terminal tail domains. The roles of the non-helical domains of various intermediate filament proteins in the assembly and co-assembly of higher-order filamentous structures have been studied by many groups but with quite contradictory results. Type III intermediate filaments are unique in that they can form homopolymers both in vitro and in vivo. The expression and assembly characteristics of carboxy- and amino-terminal deletion mutants of glial fibrillary acidic protein (GFAP), an astrocyte-specific type III intermediate filament protein, were examined by transient transfections of either vimentin-positive or vimentin-negative variants of human adrenocarcinoma-derived SW13 cell lines. Whereas complete deletion of the C-terminal tail domain of GFAP results in the formation of polymorphic aggregates, both intranuclear and cytoplasmic in self-assembly experiments, efficient co-assembly of these tail-less GFAP mutants with vimentin can be achieved as long as the KLLEGEE sequence at the C-terminal end of the rod domain is preserved. Up to one-fifth of the C-terminal end of the tail domain can be deleted without affecting the capability of GFAP to self-assemble. The highly conserved RDG-containing motif in the tail domain may be important for self-assembly but is not sufficient. The entire head domain seems to be required for self-assembly. All N-terminal deletion mutants of GFAP share the same phenotype of diffuse cytoplasmic staining when expressed in vimentin-negative SW13 cells. Although co-assembly with vimentin can still be achieved with completely head-less GFAP, preservation of some of the head domain greatly enhanced the efficiency. Our results form the basis for further, more detailed mapping of the essential regions in filament assembly of GFAP and other type III IFs.

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Differential Regulation of Two Classes of Neuronal Intermediate Filament Proteins During Optic Nerve Regeneration

Journal of Neurochemistry ◽

10.1046/j.1471-4159.1994.63062307.x ◽

2002 ◽

Vol 63 (6) ◽

pp. 2307-2313 ◽

Cited By ~ 7

Author(s):

Christina M. Hall

Keyword(s):

Optic Nerve ◽

Nerve Regeneration ◽

Intermediate Filament ◽

Differential Regulation ◽

Intermediate Filament Proteins ◽

Optic Nerve Regeneration ◽

Neuronal Intermediate Filament Proteins

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Peripherin assembles into homopolymers in SW13 cells

Journal of Cell Science ◽

10.1242/jcs.108.10.3279 ◽

1995 ◽

Vol 108 (10) ◽

pp. 3279-3284 ◽

Cited By ~ 3

Author(s):

C. Cui ◽

P.J. Stambrook ◽

L.M. Parysek

Keyword(s):

Intermediate Filament ◽

Network Formation ◽

Full Length ◽

Intermediate Filament Proteins ◽

Filament Assembly ◽

Mature Neurons ◽

Carboxyl Terminal ◽

Relationship Of ◽

Filament Network ◽

Carboxyl Tail

The properties of full-length and mutant peripherins were studied in intermediate filament-less SW13 cells to define regions of peripherin that are essential for initiation of filament assembly. A full-length rat peripherin gene transfected into SW13 cells resulted in filament formation, consistent with the close structural relationship of peripherin to other type III intermediate filament proteins that readily form homopolymers. Translation of full-length rat peripherin is initiated predominantly at the second of two inframe AUGs. Deletions within the amino terminus of wild-type peripherin abolished its ability to form filaments in SW13 cells. In contrast, deletion of the entire carboxyl-terminal tail of peripherin did not affect its ability to form filamentous arrays in transfected SW13 cells. These results indicate that, of the intermediate filament proteins that are expressed in mature neurons, only peripherin and alpha-internexin are capable of making homopolymer intermediate filaments. In addition, mutations of the carboxyl tail of peripherin generally do not interfere with filament network formation.

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

The Journal of Cell Biology ◽

10.1083/jcb.122.6.1323 ◽

1993 ◽

Vol 122 (6) ◽

pp. 1323-1335 ◽

Cited By ~ 179

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.

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