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

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.     

Characterization of dominant and recessive assembly-defective mutations in mouse neurofilament NF-M

We have generated a set of amino- and carboxy-terminal deletions of the neurofilament NF-M gene and determined the molecular consequences of forced expression of these mutant constructs in mouse fibroblasts. To follow the expression of mutant NF-M subunits in transfected cells, a 12 amino acid epitope (from the human c-myc protein) was expressed at the carboxy terminus of each mutant. We show that NF-M molecules missing up to 90 or 70% of the nonhelical carboxy-terminal tail or amino-terminal head domains, respectively, incorporate readily into an intermediate filament network comprised either of vimentin or NF-L, whereas deletions into either the amino- or carboxy-terminal alpha- helical rod region generate assembly-incompetent polypeptides. Carboxy- terminal deletions into the rod domain invariably yield dominant mutants which rapidly disrupt the array of filaments comprised of NF-L or vimentin. Accumulation of these mutant NF-M subunits disrupts vimentin filament arrays even when present at approximately 1% the level of the wild-type subunits. In contrast, the amino-terminal deletions into the rod produce pseudo-recessive mutants that perturb the wild-type NF-L or vimentin arrays only modestly. The inability of such amino-terminal mutants to disrupt wild-type subunits defines a region near the amino-terminal alpha-helical rod domain (residues 75- 126) that is required for the earliest steps in filament assembly.     

Expression of rat neurofilament proteins NF-L and NF-M in transfected non-neuronal cells

Two cDNA clones fully encoding the rat neurofilament proteins NF-L and NF-M were subcloned into eukaryotic expression vectors behind the strong constitutive viral promoters from SV40 and Rous sarcoma viruses. Transient transfection of L tk- and Cos cell lines with these expression constructs resulted in cells expressing the neurofilament proteins in an intermediate filament-type pattern. Additionally, a putative juxtanuclear organizing center or region was observed in the transfected cells, most noticeable shortly after the transfection procedure. Stable transfections were performed on mouse L tk- and Swiss 3T6 cells using NF-L and NF-M constructs bearing an SV40 early promoter driven neomycin selectable marker. Although G418-resistant clones were recovered with both the NF-L and the NF-M constructs, only clones expressing immunofluorescently stainable amounts of NF-M were detected and established. Immunoelectron microscopic analysis revealed NF-M and vimentin proteins to be colocalized on the same intermediate filaments.     

Assembly Properties of Amino- and Carboxyl-Terminally Truncated Neurofilament NF-H Proteins with NF-L and NF-M in the Presence and Absence of Vimentin

Abstract: To understand the assembly characteristics of the high-molecular-weight neurofilament protein (NF-H), carboxyl- and amino-terminally deleted NF-H proteins were examined by transiently cotransfecting mutant NF-H constructs with the other neurofilament triplet proteins, low- and middle-molecular-weight neurofilament protein (NF-L and NF-M, respectively), in the presence or absence of cytoplasmic vimentin. The results confirm that NF-H can coassemble with vimentin and NF-L but not with NF-M into filamentous networks. Deletions from the amino-terminus show that the N-terminal head is necessary for the coassembly of NF-H with vimentin, NF-L, or NF-M/vimentin. However, headless NF-H or NF-H from which the head and a part of the rod is removed can still incorporate into an NF-L/vimentin network. Deletion of the carboxyl-terminal tail of NF-H shows that this region is not essential for coassembly with vimentin but is important for coassembly with NF-L into an extensive filamentous network. Carboxyl-terminal deletion into the α-helical rod results in a dominant-negative mutant, which disrupts all the intermediate filament networks. These results indicate that NF-L is the preferred partner of NF-H over vimentin and NF-M, the head region of NF-H is important for the formation of NF-L/NF-H filaments, and the tail region of NF-H is important to form an extensive network of NF-L/NF-H filaments.     

Two distinct functions of the carboxyl-terminal tail domain of NF-M upon neurofilament assembly: cross-bridge formation and longitudinal elongation of filaments

Neurofilaments are the major cytoskeletal elements in the axon that take highly ordered structures composed of parallel arrays of 10-nm filaments linked to each other with frequent cross-bridges, and they are believed to maintain a highly polarized neuronal cell shape. Here we report the function of rat NF-M in this characteristic neurofilament assembly. Transfection experiments were done in an insect Sf9 cell line lacking endogenous intermediate filaments. NF-L and NF-M coassemble to form bundles of 10-nm filaments packed in a parallel manner with frequent cross-bridges resembling the neurofilament domains in the axon when expressed together in Sf9 cells. Considering the fact that the expression of either NF-L or NF-M alone in these cells results in neither formation of any ordered network of 10-nm filaments nor cross- bridge structures, NF-M plays a crucial role in this parallel filament assembly. In the case of NF-H the carboxyl-tail domain has been shown to constitute the cross-bridge structures. The similarity in molecular architecture between NF-M and NF-H suggests that the carboxyl-terminal tail domain of NF-M also constitutes cross-bridges. To examine this and to further investigate the function of the carboxyl-terminal tail domain of NF-M, we made various deletion mutants that lacked part of their tail domains, and we expressed these with NF-L. From this deletion mutant analysis, we conclude that the carboxyl-terminal tail domain of NF-M has two distinct functions. First, it is the structural component of cross-bridges, and these cross-bridges serve to control the spacing between core filaments. Second, the portion of the carboxyl- terminal tail domain of NF-M that is directly involved in cross-bridge formation affects the core filament assembly by helping them to elongate longitudinally so that they become straight.     

Plasticin, a type III neuronal intermediate filament protein, assembles as an obligate heteropolymer: implications for axonal flexibility

The assembly characteristics of the neuronal intermediate filament protein plasticin were studied in SW13 cells in the presence and absence of a cytoplasmic filament network. Full-length plasticin cannot polymerize into homopolymers in filament-less SW13c1.2Vim(-) cells but efficiently coassembles with vimentin in SW13c1.1Vim(-) cells. By cotransfecting plasticin and vimentin in SW13c1.1Vim(-) cells, we show that plasticin assembly requires vimentin in noncatalytic amounts. Differing effects on assembly were seen with point mutations of plasticin monomers that were analogous to the keratin mutations that cause epidermolysis bullosa simplex (EBS). In particular, plasticin monomers with point mutations analogous to those in EBS do not uniformly inhibit neurofilament (NF) network formation. A point mutation in the helix termination sequence resulted in complete filament aggregation when coexpressed with vimentin but showed limited coassembly with low- and medium-molecular-weight NF proteins (NF-L and NF-M, respectively). In transfected SW13c1.1Vim(+) cells, a point mutation in the first heptad of the alpha-helical coil region formed equal amounts of filaments, aggregates, and a mixture of filaments and aggregates. Furthermore, coexpression of this point mutation with NF-L and NF-M was associated with a shift toward increased numbers of aggregates. These results suggest that there are important structural differences in assembly properties between homologous fish and mammalian intermediate filament proteins. These structural differences may contribute to the distinctive growth characteristics of the teleost visual pathway.     

Neurofilaments are obligate heteropolymers in vivo

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.     

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

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.     

Disruption of Type IV Intermediate Filament Network in Mice Lacking the Neurofilament Medium and Heavy Subunits

To clarify the role of the neurofilament (NF) medium (NF-M) and heavy (NF-H) subunits, we generated mice with targeted disruption of both NF-M and NF-H genes. The absence of the NF-M subunit resulted in a two- to threefold reduction in the caliber of large myelinated axons, whereas the lack of NF-H subunits had little effect on the radial growth of motor axons. In NF-M-/- mice, the velocity of axonal transport of NF light (NF-L) and NF-H proteins was increased by about two-fold, whereas the steady-state levels of assembled NF-L were reduced. Although the NF-M or NF-H subunits are each dispensable for the formation of intermediate filaments, the absence of both subunits in double NF-M; NF-H knockout mice led to a scarcity of intermediate filament structures in axons and to a marked approximately twofold increase in the number of microtubules. Protein analysis indicated that the levels of NF-L and alpha-internexin proteins were reduced dramatically throughout the nervous system. Immunohistochemistry of spinal cord from the NF-M-/-;NF-H-/- mice revealed enhanced NF-L staining in the perikaryon of motor neurons but a weak NF-L staining in axons. In addition, axonal transport studies carried out by the injection of [35S]methionine into spinal cord revealed after 30 days very low levels of newly synthesized NF-L proteins in the sciatic nerve of NF-M-/-;NF-H-/- mice. The combined results demonstrate a requirement of the high-molecular-weight subunits for the assembly of type IV intermediate filament proteins and for the efficient translocation of NF-L proteins into the axonal compartment.     

Antibody labeling of bovine neurofilaments: implications on the structure of neurofilament sidearms.

We carried out immunolabeling studies of purified bovine spinal cord neurofilaments (NFs) and filaments reconstituted from several combinations of the NF triplet polypeptides, NF-H, NF-M, and NF-L. Six antibodies with known epitopes in either the rod domains or the tailpiece extensions of the NF triplet were used in these studies, and the immune complexes were visualized directly by the glycerol-spray, rotary shadowing technique, which permitted unambiguous identification of the NF sidearms. Antibodies directed against the tailpiece extensions of NF-H and NF-M labeled the sidearms of native NFs and reconstituted filaments containing those two polypeptides, but not the backbone of the filaments. Combining these two antibodies in the same labeling experiment resulted in more intense labeling than either of the antibodies alone, indicating that both NF-H and NF-M are capable of forming sidearms. The anti-NF-L tailpiece antibody recognized only a limited number of sites along native NFs, but labeled reconstituted NF-L homopolymers uniformly and heavily. This suggests that the NF-L tailpiece extension is relatively inaccessible in native filaments, but is accessible in reconstituted homopolymers. One possible explanation is that, in native NFs, the NF-H- and NF-M-containing sidearms curtailed antibody access to NF-L. A second possibility that is not mutually exclusive with the first is that, when both NF-L and another triplet polypeptide are present, they preferentially form heterodimers such that the NF-L tailpiece epitope becomes hidden. Taken collectively, and in combination with published structural information, our data are consistent with a subunit packing scheme in which an NF-L-containing dimer serves as the fundamental building block of most mammalian NFs, such that their sidearms consist of pairs of NF-H/NF-L, NF-M/NF-L, or NF-L/NF-L tailpiece extensions.     

Characterization of NF-L and betaIISigma1-spectrin interaction in live cells.

Neurofilaments (NFs) are neuron-specific intermediate filaments (IFs) composed of three different subunits, NF-L, NF-M, and NF-H. NFs move down the axon with the slow component of axonal transport, together with microtubules, microfilaments, and alphaII/betaII-spectrin (nonerythroid spectrin or fodrin). It has been shown that alphaII/betaII-spectrin is closely associated with NFs in vivo and that betaII-spectrin subunit binds to NF-L filaments in vitro. In the present study we seek to elucidate the relationship between NF-L and betaII-spectrin in vivo. We transiently transfected full-length NF-L and carboxyl-terminal deleted NF-L mutants in SW13 Cl.2 Vim- cells, which lack an endogenous IF network and express alphaII/betaIISigma1-spectrin. Double-immunofluorescence and electron microscopy studies showed that a large portion of betaIISigma1-spectrin colocalizes with the structures formed by NF-L proteins. We found a similar association between NF-L proteins and actin. However, coimmunoprecipitation experiments in transfected cells and the yeast two-hybrid system results failed to demonstrate a direct interaction of NF-L with betaIISigma1-spectrin in vivo. The presence of another protein that acts as a bridge between the membrane skeleton and neurofilaments or modulating their association may therefore be required.     

Structure and evolutionary origin of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein

We describe the complete sequence of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein. The coding sequence is interrupted by two intervening sequences which align perfectly with the first two intervening sequences in the gene encoding NF-L (the low-molecular-mass neurofilament protein); there is no intron in the gene encoding NF-M corresponding to the third intron in NF-L. Therefore, both the number of introns and their arrangement in the genes coding NF-L and NF-M contrast sharply with the number and arrangement of introns in the genes of known sequence, encoding other members of the intermediate filament multigene family (desmin, vimentin, glial fibrillary acidic protein and the acidic and basic keratins); with the exception of a single truncated keratin gene that lacks an encoded tailpiece, these genes all contain eight introns, of which at least six are placed at homologous locations. Assuming the existence of a primordial intermediate filament gene containing most (if not all) the introns found in contemporary non-neurofilament intermediate filament genes, it seems likely that an RNA-mediated transposition event was involved in the generation of an ancestral gene encoding the NF polypeptides. A combination of insertional transposition and gene-duplication events could then explain the anomalous number and placement of introns within these genes. Consistent with this notion, we show that the genes encoding NF-M and NF-L are linked.     

Assembly of amino-terminally deleted desmin in vimentin-free cells

To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin- free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.     

Coil-1 of rod domain of NF-L is essential for its assemblyin vivo

Neurofilaments take highly ordered structures composed of parallel mays of 10 nm filaments linked to each other with frequent cross-bridge. It is composed of three components named NF-L, NF-M and NF-H. NF-L is able to form filamentous network alone in Sf9 cells, while M could not. To identify which domain is essential for the assembly of NF-L, two chimera proteins named ML and MML were constructed: ML was composed of the head domain of NF-M and other domains of NF-L; MML was composed of the head and Coil-1 domains of NF-M and Coil-2 and tail domains of NF-L. ML was not only able to form filaments in Sf9 cells, but also co-assemble with NF-M into parallel filamentous bundles. MML could not assemble into filaments. Thus the Coil-1 domain of NF-L was essential for its assembly.     

Coil-1 of rod domain of NF-L is essential for its assembly in vivo

Neurofilaments take highly ordered structures composed of parallel arrays of 10 nm filaments linked to each other with frequent cross-bridge. It is composed of three components named NF-L, NF-M and NF-H. NF-L is able to form filamentous network alone in Sf9 cells, while M could not. To identify which domain is essential for the assembly of NF-L, two chimera proteins named ML and MML were constructed: ML was composed of the head domain of NF-M and other domains of NF-L; MML was composed of the head and Coil-1 domains of NF-M and Coil-2 and tail domains of NFL. ML was not only able to form filaments in Sf9 cells, but also co-assemble with NF-M into parallel filamentous bundies. MML could not assemble into filaments. Thus the Coil-1 domain of NF-L was essential for its assembly.     

Phosphorylation of the head domain of neurofilament protein (NF-M): a factor regulating topographic phosphorylation of NF-M tail domain KSP sites in neurons

In neurons the phosphorylation of neurofilament (NF) proteins NF-M and NF-H is topographically regulated. Although kinases and NF subunits are synthesized in cell bodies, extensive phosphorylation of the KSP repeats in tail domains of NF-M and NF-H occurs primarily in axons. The nature of this regulation, however, is not understood. As obligate heteropolymers, NF assembly requires interactions between the core NF-L with NF-M or NF-H subunits, a process inhibited by NF head domain phosphorylation. Phosphorylation of head domains at protein kinase A (PKA)-specific sites seems to occur transiently in cell bodies after NF subunit synthesis. We have proposed that transient phosphorylation of head domains prevents NF assembly in the soma and inhibits tail domain phosphorylation; i.e. assembly and KSP phosphorylation in axons depends on prior dephosphorylation of head domain sites. Deregulation of this process leads to pathological accumulations of phosphorylated NFs in the soma as seen in some neurodegenerative disorders. To test this hypothesis, we studied the effect of PKA phosphorylation of the NF-M head domain on phosphorylation of tail domain KSP sites. In rat cortical neurons we showed that head domain phosphorylation of endogenous NF-M by forskolin-activated PKA inhibits NF-M tail domain phosphorylation. To demonstrate the site specificity of PKA phosphorylation and its effect on tail domain phosphorylation, we transfected NIH3T3 cells with NF-M mutated at PKA-specific head domain serine residues. Epidermal growth factor stimulation of cells with mutant NF-M in the presence of forskolin exhibited no inhibition of NF-tail domain phosphorylation compared with the wild type NF-M-transfected cells. This is consistent with our hypothesis that transient phosphorylation of NF-M head domains inhibits tail domain phosphorylation and suggests this as one of several mechanisms underlying topographic regulation.     

Coil-1 of rod domain of NF-L is essential for its assembly in vivo

Neurofilaments take highly ordered structures composed of parallel mays of 10 nm filaments linked to each other with frequent cross-bridge. It is composed of three components named NF-L, NF-M and NF-H. NF-L is able to form filamentous network alone in Sf9 cells, while M could not. To identify which domain is essential for the assembly of NF-L, two chimera proteins named ML and MML were constructed: ML was composed of the head domain of NF-M and other domains of NF-L; MML was composed of the head and Coil-1 domains of NF-M and Coil-2 and tail domains of NF-L. ML was not only able to form filaments in Sf9 cells, but also co-assemble with NF-M into parallel filamentous bundles. MML could not assemble into filaments. Thus the Coil-1 domain of NF-L was essential for its assembly. Project supported by the National Grant for Outstanding Young Scientists.     

Timing of neuronal intermediate filament proteins expression in the mouse vomeronasal organ during pre- and postnatal development. An immunohistochemical study

Several types of intermediate filament proteins are expressed in developing and mature neurons; they cooperate with other cytoskeletal components to sustain neuronal function from early neurogenesis onward. In this work the timing of expression of nestin, peripherin, internexin, and the neuronal intermediate filament triplet [polypeptide subunits of low (NF-L), medium (NF-M), and high (NF-H) molecular weight] was investigated in the developing fetal and postnatal mouse vomeronasal organ (VNO) by means of immunohistochemistry. The results show that the sequence of expression of intermediate filament proteins is internexin, nestin, and NF-M in the developing vomeronasal sensory epithelium; internexin, peripherin, and NF-M in the developing vomeronasal nerve; and nestin, internexin and peripherin, NF-L, and NF-M in the nerve supply to accessory structures of the VNO. At sexual maturity (2 months) NF-M is only expressed in vomeronasal neurons and NF-M, NF-L and peripherin are expressed in extrinsic nerves supplying VNO structures. The differential distribution of intermediate filament proteins in the vomeronasal sensory epithelium and nerve is discussed in terms of the cell types present therein. It is concluded that several intermediate filament proteins are sequentially expressed during intrauterine development of the VNO neural structures in a different pattern according to the different components of the VNO.