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

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

Journal of Cell Science ◽

10.1242/jcs.112.13.2233 ◽

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.

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Effects of truncated neurofilament proteins on the endogenous intermediate filaments in transfected fibroblasts

Journal of Cell Science ◽

10.1242/jcs.99.2.335 ◽

1991 ◽

Vol 99 (2) ◽

pp. 335-350 ◽

Cited By ~ 7

Author(s):

S.S. Chin ◽

P. Macioce ◽

R.K. Liem

Keyword(s):

Intermediate Filament ◽

Dominant Negative ◽

Deletion Mutants ◽

Tail Domain ◽

Cultured Fibroblasts ◽

Mutant Proteins ◽

Amino Terminal ◽

Novel Approach ◽

Carboxyl Terminal

The expression and assembly characteristics of carboxyl- and amino-terminal deletion mutants of rat neurofilament low Mr (NF-L) and neurofilament middle Mr (NF-M) proteins were examined by transient transfection of cultured fibroblasts. Deletion of the carboxyl-terminal tail domain of either protein indicated that this region was not absolutely essential for co-assembly into the endogenous vimentin cytoskeleton. However, deletion into the alpha-helical rod domain resulted in an inability of the mutant proteins to co-assemble with vimentin into filamentous structures. Instead, the mutant proteins appeared to be assembled into unusual tubular-vesicular structures. Additionally, these latter deletions appeared to act as dominant negative mutants which induced the collapse of the endogenous vimentin cytoskeleton as well as the constitutively expressed NF-H and NF-M cytoskeletons in stably transfected cell lines. Thus, an intact alpha-helical rod domain was essential for normal IF co-assembly whereas carboxyl-terminal deletions into this region resulted in dramatic alterations of the existing type III and IV intermediate filament cytoskeletons in vivo. Deletions from the amino-terminal end into the alpha-helical rod region gave different results. With these deletions, the transfected protein was not co-assembled into filaments and the endogenous vimentin IF network was not disrupted, indicating that these deletion mutants are recessive. The dominant negative mutants may provide a novel approach to studying intermediate filament function within living cells.

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Paranemin and the organization of desmin filament networks

Journal of Cell Science ◽

10.1242/jcs.114.6.1079 ◽

2001 ◽

Vol 114 (6) ◽

pp. 1079-1089 ◽

Cited By ~ 1

Author(s):

S.C. Schweitzer ◽

M.W. Klymkowsky ◽

R.M. Bellin ◽

R.M. Robson ◽

Y. Capetanaki ◽

...

Keyword(s):

Cell Lines ◽

Intermediate Filament ◽

Transgene Expression ◽

De Novo ◽

Muscle Cells ◽

The Other ◽

Intermediate Filament Proteins ◽

Mouse Fibroblasts ◽

Filament Networks ◽

Filament Network

De novo expression of vimentin, GFAP or peripherin leads to the assembly of an extended intermediate filament network in intermediate filament-free SW13/cl.2 cells. Desmin, in contrast, does not form extended filament networks in either SW13/cl.2 or intermediate filament-free mouse fibroblasts. Rather, desmin formed short thickened filamentous structures and prominent spot-like cytoplasmic aggregates that were composed of densely packed 9–11 nm diameter filaments. Analysis of stably transfected cell lines indicates that the inability of desmin to form extended networks is not due to a difference in the level of transgene expression. Nestin, paranemin and synemin are large intermediate filament proteins that coassemble with desmin in muscle cells. Although each of these large intermediate filament proteins colocalized with desmin when coexpressed in SW-13 cells, expression of paranemin, but not synemin or nestin, led to the formation of an extended desmin network. A similar rescue of desmin network organization was observed when desmin was coexpressed with vimentin, which coassembles with desmin, or with keratins, which formed a distinct filament network. These studies demonstrate that desmin filaments differ in their organizational properties from the other vimentin-like intermediate filament proteins and appear to depend upon coassembly with paranemin, at least when they are expressed in non-muscle cells, in order to form an extended filament network.

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A cDNA from Drosophila melanogaster encodes a lamin C-like intermediate filament protein

Journal of Cell Science ◽

10.1242/jcs.104.4.1263 ◽

1993 ◽

Vol 104 (4) ◽

pp. 1263-1272 ◽

Cited By ~ 4

Author(s):

C.A. Bossie ◽

M.M. Sanders

Keyword(s):

Drosophila Melanogaster ◽

Intermediate Filament ◽

Blot Analysis ◽

Polytene Chromosomes ◽

Intermediate Filament Protein ◽

Chromosome 2 ◽

Cross Hybridization ◽

Intermediate Filament Proteins ◽

Nuclear Lamins ◽

Filament Protein

A novel intermediate filament cDNA, pG-IF, has been isolated from a Drosophila melanogaster embryonic expression library screened with a polyclonal antiserum produced against a 46 kDa cytoskeletal protein isolated from Kc cells. This 46 kDa protein is known to be immunologically related to vertebrate intermediate filament proteins. The screen resulted in the isolation of four different cDNA groups. Of these, one has been identified as the previously characterized Drosophila nuclear lamin cDNA, Dm0, and a second, pG-IF, demonstrates homology to Dm0 by cross hybridization on Southern blots. DNA sequence analysis reveals that pG-IF encodes a newly identified intermediate filament protein in Drosophila. Its nucleotide sequence is highly homologous to nuclear lamins with lower homology to cytoplasmic intermediate filament proteins. pG-IF predicts a protein of 621 amino acids with a predicted molecular mass of 69,855 daltons. In vitro transcription and translation of pG-IF yielded a protein with a SDS-PAGE estimated molecular weight of approximately 70 kDa. It contains sequence principles characteristic of class V intermediate filament proteins. Its near neutral pI (6.83) and the lack of a terminal CaaX motif suggests that it may represent a lamin C subtype in Drosophila. In situ hybridization to polytene chromosomes detects one band of hybridization on the right arm of chromosome 2 at or near 51A. This in conjunction with Southern blot analysis of various genomic digests suggests one or more closely placed genes while Northern blot analysis detects two messages in Kc cells.

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

Journal of Cell Science ◽

10.1242/jcs.107.6.1593 ◽

1994 ◽

Vol 107 (6) ◽

pp. 1593-1607 ◽

Cited By ~ 7

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.

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

Biochemistry and Cell Biology ◽

10.1139/o99-003 ◽

1999 ◽

Vol 77 (1) ◽

pp. 41-45 ◽

Cited By ~ 54

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.

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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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