Neuronal expression of peripherin,a type III intermediate filament protein,in the mouse hindbrain

Peripherin is a 57 kDa Type III intermediate filament protein associated with neurite extension, neuropathies such as amyotrophic lateral sclerosis, and cranial nerve and dorsal root projections. However, knowledge of peripherin expression in the CNS is limited. We have used immunoperoxidase histochemistry to characterise peripherin expression in the mouse hindbrain, including the inferior colliculus, pons, medulla and cerebellum. Peripherin immunolabelling was observed in the nerve fibres and nuclei that are associated with all cranial nerves [(CN) V–XII] in the hindbrain. Peripherin expression was prominent in the cell bodies and axons of the mesenchephalic trigeminal nucleus and the pars compacta region of nucleus ambiguus, and in the fibres that comprise the solitary tract, the descending spinal trigeminal tract and the trigeminal and facial nerves. A small proportion of peripherin positive fibres in CN VIII likely arise from cochlear type II spiral ganglion neurons. Peripherin positive fibres were also observed in the inferior cerebellar peduncle and folia in the intermediate zone of the cerebellum. Antibody specificity was confirmed by absence of labelling in hindbrain tissue from peripherin knockout mice. This study shows that in the adult mouse hindbrain, peripherin is expressed in discrete neuronal subpopulations that have sensory, motor and autonomic functions.     

Multiple mRNAs encode peripherin, a neuronal intermediate filament protein.

Three cDNA clones of 1.6 (3u), 1.2 (5g) and 0.6 (5b) kbp, specific for peripherin, a neuronal intermediate filament protein (IFP), have been isolated from a murine neuroblastoma cell lambda gt11 library by immunoscreening using peripherin antiserum. Antibodies eluted from the fusion proteins produced by clones 3u and 5g recognize the peripherin spots on immunoblots. Where they overlap the three cDNAs have identical sequences. cDNA 5g exhibits the closest homology to type III IFP cDNAs. cDNA 3u is identical to the corresponding region of cDNA 5g, except for the insertion of a 96 bp fragment at a position corresponding to the junction of exons 4 and 5 in type III IFP cDNAs. cDNA 5b is also identical to the corresponding region of cDNA 5g, except for the deletion of a 62 bp fragment at the junction of exons 8 and 9 in type III IFP cDNAs. S1 mapping experiments performed with probes covering the 3' end of the two unexpected regions show that three distinct mRNAs correspond to the three cDNAs. Moreover, three peripherin products, two minor 61 and 56 kd products in addition to the major 58 kd peripherin, are observed when poly(A)+ RNA is in vitro translated, the 61 kd peripherin being translated from the 3u-selected RNA. The three RNAs originate from alternative splicing of a unique peripherin gene, thus generating polymorphism of peripherin.     

Structure of the gene encoding peripherin, an NGF-regulated neuronal-specific type III intermediate filament protein

We have cloned the rat gene encoding peripherin, a neuronal-specific intermediate filament protein that is NGF-regulated. Determination of the complete sequence, including 821 nucleotides of the 5'-flanking region, allows us to make conclusions about the evolutionary origin of the peripherin gene, its homology with other intermediate filament proteins, and possible mechanisms of regulation of peripherin expression in neurons. The positions of the eight peripherin gene introns correspond to the intron patterns of desmin, vimentin, and GFAP, with one example of intron sliding. Together with protein sequence homologies, this conclusively demonstrates that peripherin is a type III intermediate filament protein. The peripherin promoter contains sequences homologous to regions of other NGF-regulated promoters, which may function in peripherin induction by NGF.     

Phosphorylation of the peripherin 58-kDa neuronal intermediate filament protein. Regulation by nerve growth factor and other agents

Peripherin, a recently described member of the intermediate filament multigene family, is present in peripheral and certain central nervous system neurons as well as in cultured neuron-like cell lines, including PC12 pheochromocytoma cells. In PC12 cells, peripherin appears to be the major intermediate filament protein and its relative levels and synthesis are specifically increased during nerve growth factor (NGF)-promoted neuronal differentiation. The present study examines the phosphorylation of peripherin and the regulation thereof by nerve growth factor and other agents in cultured PC12 cells. Immunoblotting experiments using a peripherin-specific antiserum show five distinct isoforms of this protein in whole cell and cytoskeletal extracts resolved by two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three of these isoforms incorporate detectable quantities of [32P]phosphate during metabolic radiolabeling. The small proportion (approximately 6%) of total cellular peripherin that is extractable with 1% Triton X-100, does not appear to incorporate phosphate. NGF increases peripherin phosphorylation by 2-3-fold within 1-2 h of treatment. Epidermal growth factor and insulin have no effect. The relative levels of phosphorylated peripherin are markedly elevated (17-fold) by long term NGF exposure, and peripherin becomes a major cytoskeletal phosphoprotein. Activators of protein kinases A and C and treatment with depolarizing levels of K+ also enhance peripherin phosphorylation by 2-3-fold, in cultures both with and without prior long term NGF treatment. Evidence is presented that NGF regulates peripherin phosphorylation by a mechanism independent of protein kinases A and C and of depolarization. The large increase in phosphorylated peripherin brought about by NGF treatment suggests that this neuronal filament protein may play a role in the elaboration and maintenance of neurites. The presence of multiple independent pathways that acutely enhance peripherin phosphorylation indicates that this role is subject to modulation by extrinsic signals.     

Mitosis-dependent protein expression in neuroblastoma cell line N1E-115

Systematic work on differential protein expression in mitosis is limited, and we therefore used neuroblastoma cells (N1E-115) incubated with either colcemid or nocodazole to arrest mitosis. Proteins were identified by MALDI-TOF/TOF and nano-LC-ESI-MS/MS with subsequent quantification of spot volumes with specific software. Immunoblotting was used for verification of selected proteins. Levels of 10 individual proteins were increased and levels of 6 proteins were decreased concordantly by both treatments. These proteins were constituents of heat shock and chaperone, cytoskeleton, proteasomal, heterochromatin, and DNA replication signaling as well as housekeeping and metabolic systems. Identification of mitosis-dependent proteins is of importance for the interpretation of previous work and for designing future experiments.     

A neuronal death model: overexpression of neuronal intermediate filament protein peripherin in PC12 cells

Background  

Abnormal accumulation of neuronal intermediate filament (IF) is a pathological indicator of some neurodegenerative disorders. However, the underlying neuropathological mechanisms of neuronal IF accumulation remain unclear. A stable clone established from PC12 cells overexpressing a GFP-Peripherin fusion protein (pEGFP-Peripherin) was constructed for determining the pathway involved in neurodegeneration by biochemical, cell biology, and electronic microscopy approaches. In addition, pharmacological approaches to preventing neuronal death were also examined.     

Comparative expression of 2 intermediate filament proteins, peripherin and the 68 kDa neurofilament protein, during embryonal development of the rat

Peripherin, an intermediate filament protein, was originally detected by biochemical methods in the neurons of the peripheral nervous system. We now studied its expression and cellular localization by immunocytochemical methods in the developing rat embryo, and compared them with the expression and localization of the 68 kDa neurofilament protein. It appears that peripherin is expressed not only in the neurons of the peripheral nervous system, but also in some well defined neuronal populations of the central nervous system. These results focus on the questions of the phylogenetic origin and of the function of peripherin.     

The intermediate filament protein, synemin, is an AKAP in the heart

Targeting of protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) contributes to high specificity of PKA signaling pathways. PKA phosphorylation of myofilament and cytoskeletal proteins may regulate myofibrillogenesis and myocyte remodeling during heart disease; however, known cardiac AKAPs do not localize to these regions. To identify novel AKAPs which target PKA to the cytoskeleton or myofilaments, a human heart cDNA library was screened and the intermediate filament (IF) protein, synemin, was identified as a putative RII (PKA regulatory subunit type II) binding protein. A predicted RII binding region was mutated and resulted in loss of RII binding. Furthermore, synemin co-localized with RII in SW13/cl.1-vim+ cells and co-immunoprecipitated with RII from adult rat cardiomyocytes. Synemin was localized at the level of Z-lines with RII and desmin in adult hearts, however, neonatal cardiomyocytes showed differential synemin and desmin localization. Quantitative Western blots also showed significantly more synemin was present in failing human hearts. We propose that synemin provides temporal and spatial targeting of PKA in adult and neonatal cardiac myocytes.     

Identification of functional cAMP-dependent protein kinase in a 'neurite minus' mouse neuroblastoma cell line

We have characterized and quantitated the level of cAMP-dependent protein kinase in the NS-20, N1E-115, N-18 and N1A-103 mouse neuroblastoma clonal cell lines, and we have correlated the occurrence of functional cAMP-dependent protein kinase with the dibutyryl cAMP-induced differentiated functions in these cells. Our results demonstrate the presence of functional cAMP-dependent protein kinase in extracts of all four cell lines examined, including the 'neurite minus' N1A-103 cell line. Dibutyryl cAMP induced neurite outgrowth and acetylcholinesterase activity in the NS-20, N1E-115 and N-18 neuroblastoma cell lines, but not in the N1A-103 cell line. However, dibutyryl cAMP caused a 2-3-fold increase in the R1 regulatory subunit protein and cAMP-phosphodiesterase activity in the 'neurite minus' N1A-103 cells in a manner similar to that of the other three 'neurite positive' cell lines. These results suggest that the biochemical lesion(s) subserving the neurite-minus phenotype of the N1A-103 cells may be distal to the activation of cAMP-dependent protein kinase and is in a biochemical pathway distinct from the induction of R1 regulatory subunit protein and cAMP-phosphodiesterase activity.     

Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line

Summary. No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.     

Differentiation-dependent expression of hypothetical proteins in the neuroblastoma cell line N1E-115

Several protein cascades, including signaling, cytoskeletal, chaperones, metabolic, and antioxidant proteins, have been shown to be involved in the process of neuronal differentiation (ND) of neuroblastoma cell lines. No systematic approach to detect hitherto unknown and unnamed proteins or structures that have been predicted upon nucleic acid sequences in ND has been published so far. We therefore decided to screen hypothetical protein (HP) expression by protein profiling. Two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF) identification was used for expression analysis of undifferentiated and dimethylsulfoxide-induced neuronally differentiated N1E-115 cells. We unambiguously identified six HPs: Q8C520, Q99LF4, Q9CXS1, Q9DAF8, Q91WT0, and Q8C5G2. A prefoldin domain in Q91WT0, a t-SNARE domain in Q9CXS1, and a bromodomain were observed in Q8C5G2. For the three remaining proteins, no putative function using Pfam, BLOCKS, PROSITE, PRINTS, InterPro, Superfamily, CoPS, and ExPASy could be assigned. While two proteins were present in both cell lines, Q9CXS1 was switched off (i.e., undetectably low) in differentiated cells only, and Q9DAF8, Q91WT0, and Q8C5G2 were switched on in differentiated cells exclusively. Herein, using a proteomic approach suitable for screening and identification of HP, we present HP structures that have been only predicted so far based upon nucleic acid sequences. The four differentially regulated HPs may play a putative role in the process of ND.     

Syncoilin is an intermediate filament protein in activated hepatic stellate cells

Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl₄- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, β-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.     

Subcellular localization of an intermediate filament protein and its mRNA in glial cells.

Eucaryotic mRNAs are generally localized in the cell body, where most protein synthesis occurs. We have found that mRNAs encoding the glial intermediate filament protein are spatially distributed in the glial cell cytoplasm close to the location of the glial filaments. Whereas the glial filament protein mRNA was located predominantly in the distal process, actin mRNA was found almost exclusively in the apical portion of the glial cell. This pattern of mRNA localization might provide a mechanism for synthesis of proteins in specific subcellular compartments by mRNA translation locally.     

Identification of an epithelial protein related to the desmosome and intermediate filament network

Using a mAb, referred to as 08L, we have identified a protein, of M(r) approximately 140,000, associated with desmosomes of epithelial cells. The 08L antibody stained the intracellular side of lateral cell margins of monolayer epithelial cells but did not stain cell margins free of cell contact. Immunoelectron microscopy revealed that the 08L antigen was localized to the cytosolic surface of the desmosomal plaque near points of intermediate filament convergence with apparently little staining of the desmosomal plaque proper. Western blots revealed the 08L antigen to be a protein, of M(r) approximately 140,000, found in the Triton-X 100 insoluble pellet. High salt-containing buffers extracted the 08L antigen from the insoluble material. Examination of the assembly of 08L to the desmosome complex, in cells grown in low confluent culture or in calcium-switch assays, by double immunofluorescence with 08L and anti-desmoplakin antibody, revealed that 08L was recruited to morphologically identifiable desmosomes. 08L antigen may exist in a cytosolic pool prior to assembly to the cell surface. The solubility of 08L in low calcium and normal calcium conditions, however, was similar. 08L association to the desmosome was correlated with increased organization of the intermediate filament network. We suggest that the 08L antigen may be involved in the organization and stabilization of the desmosome-IF complexes of epithelia.     

Identification and characterisation of arsenite (+3 Oxidation State) methyltransferase (AS3MT) in mouse neuroblastoma cell line N1E-115

Handling and detoxification of metals by enzymes is a major issue that is not in the focus of current biomedical research concepts. The finding of the presence of arsenic (+3 Oxidation State) methyltransferase (AS3MT) in neuroblastoma cells NE-115 as a high abundance protein made us investigate primary structure of AS3MT reflecting an example of metal-handling in eucaryotes. Proteins extracted from NE-115 cells were run on 2-DE followed by two different mass spectrometrical methods. High sequence coverage was obtained by multiple protease digestion and a sequence conflict was solved at arginine 335. These findings are important when future studies on this enzyme are designed at the protein level and in particular, when antibodies against this protein will be generated.     

Withaferin a alters intermediate filament organization, cell shape and behavior

Withaferin A (WFA) is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.