A Rapid HPLC Method to Separate the Triplet Proteins of Neurofilament

In this article a fast HPLC technique to separate the individual neurofilament proteins is described. Highly pure fractions of the three neurofilament proteins can be obtained. As much as 50 mg of each neurofilament polypeptide can be separated from a crude neurofilament protein preparation in one step in less than 2 h. The short separation time is of importance in minimizing degradation, especially of the 150-kilodalton neurofilament polypeptide.     

Polyclonal Antisera to the Individual Neurofilament Triplet Protdins: A Characterization Using ELISA and Immunoblotting

In this article, the preparation and characterization of polyclonal rabbit antisera against the individual polypeptides of bovine neurofilament (68, 150, and 200 kilodaltons) is described. Selected antisera against the 68- and 150-kilodalton neurofilament polypeptides were specific for the corresponding antigen in homogenates of bovine, rat, and human brain as judged by immunoblots. The antisera against the 200-kilodalton neurofilament polypeptide cross-reacted to some extent with the 150-kilodalton neurofilament polypeptide, especially with the human antigen. The most specific antisera were used to develop an enzyme-linked immunosorbent assay (ELISA), and the cross-reactivities between the antisera and the different bovine and rat neurofilament polypeptides were determined. Contrary to the results in the immunoblots, the antiserum against the 200-kilodalton neurofilament polypeptide was subunit-specific, as was the 150-kilodalton antiserum. The 68-kilodalton antiserum displayed a minute cross-reactivity against bovine 150- and 200-kilodalton neurofilaments, but it cross-reacted somewhat more with the rat 150- and 200-kilodalton antigens. Even so, the subunit specificity of the antisera is high enough to enable the development of a quantitative ELISA for determination of the individual bovine or rat neurofilament polypeptides in a mixture. This study is the necessary preparation for such an assay.     

Organization, Sequence, and Expression of a Gene Encoding Goldfish Neurofilament Medium Protein

Abstract: The goldfish visual pathway displays a remarkable capacity for continuous neurogenesis, plasticity, and regeneration. The intermediate filament protein composition of this system differs from that of higher vertebrates, which lack the capacity for continued nerve growth and development. In an effort to determine how intermediate filament proteins are regulated during nerve growth, we isolated and characterized cDNA and genomic clones representing the goldfish neurofilament medium (NF-M) protein. The tissue-specific expression of goldfish NF-M mRNA was analyzed by RNase protection assays and by in situ hybridization. The expression of goldfish NF-M is qualitatively the same as in other species. Although the intermediate filament protein composition of the goldfish visual pathway is unusual when compared with higher vertebrates, the goldfish NF-M protein is similar to higher vertebrate NF-M proteins. In addition, the organization of the goldfish NF-M gene is identical to the NF-M genes in all other vertebrate species. In contrast, the promoter region of the goldfish NF-M gene has several potential regulatory sequences that are not found in the promoter regions of higher vertebrate NF-M genes.     

A Comparison of In Vitro- and In Vivo Phosphorylated Neurofilament Polypeptides

Abstract: Neurofilament polypeptides phosphorylated in vitro by incubation of neurofilament-enriched preparations from rat CNS with [γ-³²P]ATP were compared with the corresponding polypeptides labeled in vivo by injection of ³²P_i into the lateral ventricles of rats. Autoradiography of sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed that the major phosphorylated species in both preparations were the three neurofilament subunits, which have molecular weights of 200K, 145K, and 68K. However, the relative levels of ³²P detected in the three in vitro -labeled subunits differed from the relative in vivo levels. The two larger neurofilament polypeptides displayed similar ³²P isoprotein distribution patterns on two-dimensional gels, whereas additional isoproteins were seen in the in vitro -labeled 68K species. Limited proteolysis in SDS-polyacrylamide gels revealed the presence of common phosphopeptides in the corresponding pairs of in vitro- and in vivo-labeled subunits, but the in vivo -labeled 145K and in vitro -labeled 200K polypeptides contained additional digestion products. Two-dimensional peptide mapping of the 68K polypeptide digested with a mixture of trypsin and chymotrypsin indicated that this component was phosphorylated at a single, identical site, both in vivo and in vitro. These results indicate that the protein kinase that copurifies with neurofilament preparations may be involved in their in vivo phosphorylation.     

Intracellular assembly and sorting of intermediate filament proteins: role of the 42 amino acid lamin insert

Nuclear and cytoplasmic intermediate filament (IF) proteins segregate into two independent cellular networks by mechanisms that are poorly understood. We examined the role of a 42 amino acid (aa) insert unique to vertebrate lamin rod domains in the coassembly of nuclear and cytoplasmic IF proteins by overexpressing chimeric IF proteins in human SW13+ and SW13- cells, which contain and lack endogenous cytoplasmic IF proteins, respectively. The chimeric IF proteins consisted of the rod domain of human nuclear lamin A/C protein fused to the amino and carboxyl-terminal domains of the mouse neurofilament light subunit (NF-L), which contained or lacked the 42 aa insert. Immunofluorescence microscopy was used to follow assembly and targeting of the proteins in cells. Chimeric proteins that lacked the 42 aa insert colocalized with vimentin, whereas those that contained the 42 aa insert did not. When overexpressed in SW13- cells, chimeric proteins containing the 42 aa formed very short or broken cytoplasmic filaments, whereas chimeric proteins that lacked the insert assembled efficiently into long, stable cytoplasmic filaments. To examine the roles of other structural motifs in intracellular targeting, we added two additional sequences to the chimera, a nuclear localization signal (NLS) and a CAAX motif, which are found in nuclear IF proteins. Addition of an NLS alone or an NLS in combination with the CAAX motif to the chimera with the 42 aa insert resulted in cagelike filament that assembled close to the nuclear envelope and nuclear lamina-like targeting, respectively. Our results suggest that the rod domains of eukaryotic nuclear and cytoplasmic IF proteins, which are related to each other, are still compatible upon deletion of the 42 aa insert of coassembly. In addition, NF-L end domains can substitute for the corresponding lamin domains in nuclear lamina targeting.     

Preparation of Neurofilament Protein from Guinea Pig Peripheral Nerve and Spinal Cord

Abstract: A simple and rapid method for preparation of enriched neurofilament protein from mammalian peripheral nerve or spinal cord is described. Tissue extracts from guinea pig nerve or spinal cord are fractionated by ammonium sulfate fractionation, chromatography on Sepharose 4B, and precipitation with ethanol. Molecular exclusion chromatography on Sepharose 4B, in which the neurofilament protein elutes quantitatively in the exclusion volume of the column, with little contamination by other proteins, is found to be a highly effective purification step. The protein is found to precipitate in ammonium sulfate fractions over a wide range of salt concentration, from 20 to 80% saturation. It is found to be quantitatively precipitated in 40% v/v ethanol-water. The preparative method described yields 0.25 mg of neurofilament protein per gram of nerve or spinal cord, with a purity of approximately 50%. The three principal neurofilament polypeptides, which have molecular weights by SDS-polyacrylamide gel electrophoresis of 200K, 145K, and 68K, are found to be present in the preparation in a molar ratio of 1:2:6. A variant form of neurofilament protein occurring in approximately 20% of Hartley strain guinea pigs is described, which has the polypeptide composition: 200K, 192K, 145K, 68K.     

Increased Solubility of High-Molecular-Mass Neurofilament Subunit by Suppression of Dephosphorylation: Its Relation to Axonal Transport

Abstract: To investigate the role of phosphorylation in the turnover and transport of neurofilament (NF) proteins in vivo, we studied their solubility properties and axonal transport in the rat sciatic nerve using phosphatase inhibitors to minimize dephosphorylation during preparation. About 20% of the 200-kDa subunit (NF-H) in the axon was soluble in the 1% Triton-containing buffer under the present conditions, whereas this amount was less and more variable in the absence of phosphatase inhibitors. The 68-kDa subunit (NF-L) was exclusively insoluble and not affected by the inhibitors. Such selective solubilization of NF-H by phosphorylation differed significantly from the in vitro phosphorylation with cyclic AMP-dependent protein kinase, which resulted in NF disassembly. The carboxy-terminal phosphorylation state of NF-H probed with the phosphorylation-sensitive antibodies was also not directly related to solubility. The solubility of NF-H did not differ along the nerve. In contrast, the solubility of l -[³⁵S]methionine-labeled, transported NF-H was lowest at the peak of radioactivity. Higher solubility at the leading edge, regardless of its location along the nerve, indicates that NF-H solubility is positively correlated with the rate of NF transport.     

A Unique Neurofilament from Torpedo Electric Lobe: Sequence, Expression, and Localization Analysis

A set of cDNA clones encoding a protein highly homologous to the mammalian middle-size class of neurofilaments (NF-M) was characterized. The amino acid similarity between the Torpedo and rat NF-M approaches 90% in the amino-terminal rod-like domain and is significantly lower in the carboxy-terminal tail. The Torpedo protein contains 13 tandem repeats of a unique six amino acid core, containing a Lys-Ser-Lys putative phosphorylation site. Surprisingly, the 3' untranslated region contains stretches of 80-90% nucleic acid homology with the mammalian, but not with the chicken sequences. This homology is greater than much of the coding region, suggesting that the 3' untranslated region of the message has an important functional role, perhaps governing RNA stability or localization. This Torpedo NF-M mRNA is expressed specifically in the electric lobe and was not detected in other tissues, including brain and spinal cord. A polyclonal antibody generated against a fusion protein synthesized in E. coli detects a 150-kDa protein in the electric lobe and organ, as well as a small amount of material in the brain. Cytochemical studies reveal immunoreactivity in electromotor neuron axons and terminals. Specific expression of neurofilament genes in subsets of central neurons may be important in determining the morphology and functional characteristics of specific neuronal subtypes.     

Early Effects of β,β'-Iminodipropionitrile on Tubulin Solubility and Neurofilament Phosphorylation in the Axon

Abstract: To elucidate the role of neurofilaments in microtubule stabilization in the axon, we studied the effects of β,β'-iminodipropionitrile (IDPN) on the solubility and transport of tubulin as well as neurofilament phosphorylation in the motor fibers of the rat sciatic nerve. IDPN is known to impair the axonal transport of neurofilaments, causing accumulation of neurofilaments in the proximal axon and segregation of neurofilaments to the peripheral axoplasm throughout the nerve. Administration of IDPN at various intervals after radioactive labeling of the spinal cord with l -[³⁵S]methionine revealed that transport inhibition occurred all along the nerve within 1–2 days. Transport of cold-insoluble tubulin, which accounts for 50% of axonal tubulin, was also affected. A significant increase in the proportion of cold-soluble tubulin was observed, reaching a maximum at 3 days after IDPN treatment and returning to the control level in the following weeks. Preceding this change in tubulin solubility, a transient decrease in the phosphorylation level of the 200-kDa neurofilament protein was detected in the ventral root using phosphorylation-dependent antibodies. These early changes agreed in timing with the onset of segregation and transport inhibition, suggesting that interaction between neurofilaments and microtubules possibly regulated by phosphorylation plays a significant role in microtubule stabilization.     

Neurofilament Protein Phosphorylation in Spinal Cord of Experimentally Diabetic Rats

This study was designed to determine if the known decrease in slow axonal transport of proteins in the sciatic nerve of experimentally diabetic rats is related to altered phosphorylation of neurofilament proteins (NFPs). Rats were rendered diabetic with 50 mg/kg of streptozotocin, i.p. At 3 and 6 weeks later, NFPs were prepared from spinal cord. The in vivo phosphorylation state of NFPs was examined by using phosphate-dependent (RT97) and -independent (RMd09) antibodies against high-molecular-mass NFPs on Western blots. Neurofilament-associated kinase activity was also measured in vitro by incubation of NFPs with [32P]ATP. Phosphorylation of all three NFPs (high, medium, and low molecular mass) occurred, as confirmed by gel electrophoresis and autoradiography. At 30 min of incubation, protein-bound radioactivity in NFPs from diabetic animals was reduced to 86.7 +/- 3.4 and 54.3 +/- 19.6% of that in nondiabetic animals at 3 and 6 weeks of diabetes, respectively (p less than 0.001 and p less than 0.05, respectively). NFPs were also incubated with acid phosphatase and rephosphorylated. Results showed that the increased in vivo phosphorylation contributed to the decreased in vitro phosphorylation. Extraction of protein kinases and addition back to the NFPs revealed, in addition, a reduced activity in the diabetic animals of the protein kinases measured in vitro.     

Simultaneous labelling of microtubules and fibrillar bundles in tobacco BY-2 cells by the anti-intermediate filament antibody,ME 101

Summary In previous studies on plant cells, antibodies directed against intermediate filaments (IFs) have shown that IF antigens are distributed in one of two quite distinct forms. The first co-distributes with each of the four microtubule arrays (cortical, preprophase band, spindle and phragmoplast), while the second form is associated with cytoplasmic paracrystalline fibrillar bundles (FBs) of 10 nm filaments. Conditions allowing one form to be labelled with antibody have generally proved unsuitable for labelling of the other; this has prevented the simultaneous visualization of the two forms of IF antigen in plants and the study of any possible physical relationships between them at the electron microscopic level. In this paper, we show that ME 101, which recognizes an epitope in the N-terminal portion of all classes of intermediate filaments, stains both forms of plant IF antigen simultaneously in tobacco suspension cells using immunofluorescence or immunogold labelling techniques. These cells contain in their cortex short (ca. l m) fibrillar bundles which stain with ME 101. These bundles appear to be independent of the microtubule-associated epitope which stains in a continuous linear manner with ME 101. When protoplasts are either cleaved open on grids or sequentially extracted with detergents prior to critical point drying, the short fibrillar bundles are specifically labelled by ME 101 tagged with colloidal gold. ME 101 also co-distributed with underlying linear filaments, which appeared to be microtubules. In addition to these structures, the cortex also contains a meshwork of variably-sized fine filaments but these are not labelled with ME 101 nor with an antibody raised against the plant cytoskeleton, which recognizes cytokeratin 8. These results confirm that the fibrillar bundles and the microtubule-associated form of plant IF antigens are present simultaneously rather than experimentally-interconvertible, and that they appear to be physically unconnected.Abbreviations DAPI 4,6-diamidino-2-phenylindole - FB fibrillar bundle - FITC fluorescein isothiocyanate - IF intermediate filaments - MTSB microtubule stabilizing buffer - TBS Tris-buffered-saline     

Selective solubilization of high-molecular-mass neurofilament subunit during nerve regeneration

A reduction in neurofilament (NF) protein synthesis and changes in their phosphorylation state are observed during nerve regeneration. To investigate how such metabolic changes are involved in the reorganization of the axonal cytoskeleton, we studied the injury-induced changes in the solubility and axonal transport of NF proteins as well as their phosphorylation states in the rat sciatic nerve. In the control nerve, 15-25% of high-molecular-mass NF subunit (NF-H) was recovered in the 1% Triton-soluble fraction when fractionated in the presence of phosphatase inhibitors. After a complete loss of NF proteins distal to the injury site (70-75 mm from the spinal cord) 1 week after injury, NF-H detected in the regenerating sprouts at 2 weeks or later exhibited higher solubility (>50%) and lower C-terminal phosphorylation level than NF-H in the control nerve. Solubility increase was also apparent with L-[35S]methionine-labeled NF-H that was in transit in the proximal axon at the time of injury. The low-molecular-mass subunit remained in the insoluble fraction in both the normal and the regenerating nerves, indicating that selective solubilization of NF-H rather than total filament disassembly occurs during regeneration.     

Simultaneous Separation and Purification of Neurofilament and Glial Filament Proteins from Brain

Neurofilaments (NF) and glial filaments (GF) were purified from bovine brain by the axonal flotation method, followed by hydroxylapatite chromatography in 8 M-urea. The proteins were shown to be competent to reassemble into intermediate filaments with removal of the denaturant, and reassembly was used as the final step in the purification of the filament proteins. The reassembly was found to be dependent on ionic strength and pH. This dependence was greater for neurofilaments than for the glial filaments. The NF and GF preparations were found not to be contaminated with each other by their gel electrophoretic profile and their immunological distinctness. The filament proteins can be obtained in high yield, and remain in solution if the urea is removed by dialysis against a low-ionic-strength buffer. Hence, they can provide a source for further biochemical studies.     

Dynamics of Phosphorylation and Assembly of the High Molecular Weight Neurofilament Subunit in NB2a/d1 Neuroblastoma

In neuronal systems thus far studied, newly synthesized neurofilament subunits rapidly associate with the Triton-insoluble cytoskeleton and subsequently undergo extensive phosphorylation. However, in the present study we demonstrate by biochemical and immunological criteria that NB2a/d1 neuroblastoma cells also contain Triton-soluble, extensively phosphorylated 200-kDa high molecular weight neurofilament subunits (NF-H). High-speed centrifugation (100,000 g) of the Triton-soluble fraction for 1 h sedimented some, but not all, soluble NF-H subunits; immunoelectron microscopic analyses of the resulting pellet indicated that a portion of the NF-H subunits in this fraction are assembled into (Triton-soluble) neurofilaments. When cells were pulse labeled for 15 min with [35S]methionine, radiolabel was first associated with the Triton-soluble 200-kDa NF-H variants. Because only extensively phosphorylated NF-H subunits migrate at 200 kDa, whereas hypophosphorylated subunits migrate instead at 160 kDa, these findings suggest that some newly synthesized subunits were phosphorylated before they polymerized. In pulse-chase analyses, radiolabeled 200-kDa NF-H migrated into the 100,000 g particulate fraction of Triton-soluble extracts before its arrival in the Triton-insoluble cytoskeleton. Undifferentiated cells, which do not possess axonal neurites and lack a significant amount of Triton-insoluble, extensively phosphorylated NF-H, contain a sizeable pool of Triton-soluble extensively phosphorylated NF-H subunits and polymers. We interpret these data to indicate that the integration of newly synthesized NF-H into the cytoskeleton occurs in a progression of distinct stages, and that assembly of NF-H into neurofilaments and integration into the Triton-insoluble cytoskeleton are not prerequisites for the incorporation of certain phosphate groups on these polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of Sites in the Tail Domain of the Middle Molecular Mass Neurofilament Subunit Phosphorylated by a Neurofilament-Associated Kinase and by Casein Kinase I

Abstract: We have shown previously that a neurofilament (NF)-associated kinase (NFAK) extracted from chicken NF preparations phosphorylates selectively the middle molecular mass NF subunit (NF-M). Here we show that the major kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKI) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKI inhibitor CKI-7, (2) the similarity in substrate specificity of NFAK and authentic CKI, (3) the correspondence of two-dimensional phosphopeptide maps of NF-M phosphorylated in vitro by NFAK with those generated by CKI under similar conditions, and (4) immunological cross-reactivity of NFAK with an antibody raised against CKI. We have also identified Ser⁵⁰², Ser⁵²⁸, and Ser⁵³⁶ as phosphorylation sites by NFAK/CKI in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKI phosphorylation consensus sequences, and Ser⁵²⁸ and Ser⁵³⁶ and flanking amino acids are highly conserved in higher vertebrate NF-M sequences. Neither Ser⁵⁰² nor Ser⁵³⁶ has been identified previously as NF-M phosphorylation sites.     

Calcium-Activated Proteolysis of Neurofilament Proteins in the Squid Giant Neuron

The phosphorylation and proteolysis of squid neurofilament proteins by endogenous kinase and calcium-activated protease activities, respectively, were studied. When axoplasm was incubated in the presence of [gamma-32P]ATP, most of the phosphate was incorporated into two neurofilament proteins: a 220-kilodalton (NF-220) and a high-molecular-weight (HMW) protein. When this phosphorylated axoplasm was subjected to endogenous calcium-activated proteolysis, two significant phosphorylated fragments were generated, i.e., a soluble 110K fragment and a pelletable 100K fragment. Immunochemical and other analyses suggest that the pelletable 100K fragment contains the common helical neurofilament rod region and that the soluble 110K protein is the putative side arm of the NF-220. In contrast, neither the HMW or the NF-220 was detected in the region of the stellate ganglion which contains the cell bodies of the giant axon. However, this region did contain a number of proteins that were sensitive to calcium-activated proteolysis and reacted with a monoclonal intermediate filament antibody. This intermediate filament antibody reacts with most of the axoplasmic proteins that copurify with neurofilaments, i.e., in the order of their intermediate filament antibody staining intensity, a 60K, 65K, 220K, and 74K protein. In the cell body preparation, the intermediate filament antibody labeled, in order of their staining intensity, a 65K, 60K, 74K, and 180K protein. In both the axoplasmic and cell body preparations, endogenous calcium-activated proteolysis generated characteristic fragments that could be labeled with the anti-intermediate filament antibody.     

Intermediate filaments,microtubules and microfilaments in epidermis of sea urchin tube foot

Summary Tube foot epidermal cells of the sea urchin Strongylocentrotus purpuratus were examined by transmission electron microscopy and fluorescence microscopy to identify the chemical nature of prominent bundles of cytoplasmic filaments. Cross sections revealed filaments of roughly 7–8 nm in diameter closely packed into dense bundles. These bundles, in turn, were each surrounded by a loose sheath of microtubules. The filament size and negative reaction with the fluorescent F-actin binding drug NBD-phallacidin indicated that they were not actin. Indirect immunofluorescence microscopy of whole tissues and frozen sections revealed a strong reaction of the filaments with a monoclonal antibody prepared against porcine stomach desmin. In SDS-polyacrylamide gels of whole tube foot protein, a band of apparent molecular weight around 50 000 daltons reacted with the anti-desmin monoclonal antibody. The combined data provide evidence that the epidermal filament bundles are related to vertebrate intermediate filaments, but further biochemical studies will be necessary to assign them to a particular class of filament proteins.     

Two 68-kDa Proteins in Slow Axonal Transport Belong to the 70-kDa Heat Shock Protein Family and the Annexin Family

The major 68-kDa protein found selectively in the faster of the two subcomponents of slow axonal transport [group IV or slow component b (SCb)] in the rat sciatic nerve has been characterized. It was found to contain two distinct classes of proteins, S1 and S2, both of which have isoelectric points of 5.7, but differ in their solubility in the presence of calcium. The S1 protein, which contributes up to 70% of the 68-kDa component, was soluble in the presence or absence of calcium, whereas the S2 protein was bound to the cytoskeleton in a calcium-dependent manner. Further characterization of the two proteins by peptide mapping and immunological methods revealed that the S1 protein belonged to a family of proteins related to the 70-kDa heat shock protein, whereas the S2 protein was identical to 68-kDa calelectrin (annexin VI). Selective occurrence in SCb of these proteins with potential abilities to regulate protein-protein or protein-membrane interactions suggests that they may play important roles in the control of cytoskeletal organization in the axon, because SCb contains mainly cytoskeletal proteins in a more dynamic form compared with the slowest rate component, slow component a, which is enriched in the stably polymerized form of these proteins.     

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.