Peripherin assembles into homopolymers in SW13 cells

1995 ◽  
Vol 108 (10) ◽  
pp. 3279-3284 ◽  
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.

Paranemin and the organization of desmin filament networks

2001 ◽  
Vol 114 (6) ◽  
pp. 1079-1089 ◽  
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.

Keratin intermediate filament dynamics in cell heterokaryons reveals diverse behaviour of different keratins

1997 ◽  
Vol 110 (9) ◽  
pp. 1099-1111 ◽  
Author(s):  
J.M. Paramio ◽  
M.L. Casanova ◽  
A. Alonso ◽  
J.L. Jorcano
Keyword(s):  
Protein Synthesis ◽  
Intermediate Filament ◽  
Dynamic Nature ◽  
Filament Dynamics ◽  
Filament Assembly ◽  
Different Types ◽  
Form Part ◽  
Filament Network ◽  
Keratin Intermediate Filaments ◽  
Complete Process

To study the dynamics of keratin intermediate filaments, we fused two different types of epithelial cells (PtK2 and BMGE+H) and studied how the keratins from the parental cells recombine and copolymerize to form the heterokaryon cytoskeleton. The behaviour of the keratins during this process was followed by immunofluorescence using specific antibodies. After fusion, the parental cytoskeletons undergo a depolymerization process most apparent in the region adjacent to the fusion area. The depolymerized subunits spread throughout the heterokaryon and copolymerize into a new hybrid cytoskeleton. The complete process is very rapid, occurring in 3–4 hours, thus demonstrating the highly dynamic nature of the keratin cytoskeleton. Although newly synthesised subunits contribute to the formation of the hybrid cytoskeleton, the process takes place with similar kinetics in the absence of protein synthesis, showing the dynamic nature of the keratins from pre-existing cytoskeletons. During this process, specific keratins behave differently. Keratins K8, K18, K5 and K10 are mobilised from the parental cytoskeletons and reassemble rapidly into the hybrid cytoskeleton (3–6 hours), whereas K14 requires a substantially longer period (9–24 hours). Thus, different keratins, even when they form part of the same heterodimeric/tetrameric complexes, as is the case for K5 and K14, exhibit different dynamics. This suggests that individual polypeptides or homopolymeric complexes rather than exclusively heterodimeric/ tetrameric subunits, as is currently thought, can also take part in keratin intermediate filament assembly and dynamics. Biochemical analysis performed in the absence of protein synthesis revealed greater amounts of K5 than of K14 in the soluble pool of BMGE+H cells. Crosslinking and immunoprecipitation experiments indicated an excess of monomeric K5, as well as of K5/K14 heterodimers and K5 homodimers in the soluble pool. These results are in agreement with the different dynamic behaviour of these keratins observed in immunofluorescence. On the contrary, the phosphorylation levels of K5 and K14 are similar in both the soluble pool and the polymerized fraction, suggesting that phosphorylation does not play an important role in the different dynamics displayed by these two proteins. In summary, our results demonstrate that, following fusion, the keratin intermediate filament network reshapes rather rapidly and that keratins are highly dynamic proteins, although this mobility depends on each particular polypeptide.

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.

Identification of two N-terminal non-alpha-helical domain motifs important in the assembly of glial fibrillary acidic protein

1994 ◽  
Vol 107 (7) ◽  
pp. 1935-1948 ◽  
Author(s):  
J.E. Ralton ◽  
X. Lu ◽  
A.M. Hutcheson ◽  
R.A. Quinlan
Keyword(s):  
Intermediate Filament ◽  
Consensus Sequence ◽  
Intermediate Filament Protein ◽  
Intermediate Filament Proteins ◽  
Type Iii ◽  
Conserved Sequence ◽  
Terminal Domain ◽  
Filament Assembly ◽  
Arginine Residues ◽  
Filament Protein

The non-alpha-helical N-terminal domain of intermediate filament proteins plays a key role in filament assembly. Previous studies have identified a nonapeptide motif, SSYRRIFGG, in the non-alpha-helical N-terminal domain of vimentin that is required for assembly. This motif is also found in desmin, peripherin and the type IV intermediate filament proteins. GFAP is the only type III intermediate filament protein in which this motif is not readily identified. This study has identified two motifs in the non-alpha-helical N-terminal domain of mouse GFAP that play important roles in GFAP assembly. One motif is located at the very N terminus and has the consensus sequence, MERRRITS-ARRSY. It has some characteristics in common with the vimentin nonapeptide motif, SSYRRIFGG, including its location in the non-alpha-helical N-terminal domain and a concentration of arginine residues. Unlike the vimentin motif in which even conserved sequence changes affect filament assembly, the GFAP consensus sequence, MERRRITS-ARRSY, can be replaced by a completely unrelated sequence; namely, the heptapeptide, MVRANKR, derived from the lambda cII protein. When fused to GFAP sequences with sequential deletions of the N-terminal domain, the lambda cII heptapeptide was used to help identify a second motif, termed the RP-box, which is located just upstream of the GFAP alpha-helical rod domain. This RP-box affected the efficiency of filament assembly as well as protein-protein interactions in the filament, as shown by sedimentation assays and electron microscopy. These results are supported by previous data, which showed that the dramatic reorganization of GFAP within cells was due to phosphorylation-dephosphorylation of a site located in this RP-box. The results in this study suggest the RP-box motif to be a key modulator in the mechanism of GFAP assembly, and support a role for this motif in both the nucleation and elongation phases of filament assembly. The RP-box motif in GFAP has the consensus sequence, RLSL-RM-PP. Sequences similar to the GFAP RP-box motif are also to be found in vimentin, desmin and peripherin. Like GFAP, these include phosphorylation and proteolysis sites and are adjacent to the start of the central alpha-helical rod domain, suggesting that this motif of general importance to type III intermediate filament protein assembly.

scholarly journals Dynamics of counterion-induced attraction between vimentin filaments followed in microfluidic drops

Lab on a Chip ◽  
2014 ◽  
Vol 14 (15) ◽  
pp. 2681-2687 ◽  
Author(s):  
Christian Dammann ◽  
Sarah Köster
Keyword(s):  
Spatial Resolution ◽  
Intermediate Filament ◽  
Network Formation ◽  
Temporal And Spatial ◽  
Filament Network ◽  
Vimentin Filaments

The dynamics of intermediate filament network formation are studied in microfluidic drops at high temporal and spatial resolution.

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

1998 ◽  
Vol 111 (3) ◽  
pp. 321-333 ◽  
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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