Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein

To elucidate the function of keratins 8 and 18 (K8/18), major components of the intermediate filaments of simple epithelia, we searched for K8/18-binding proteins by screening a yeast two-hybrid library. We report here that human Mrj, a DnaJ/Hsp40 family protein, directly binds to K18. Among the interactions between DnaJ/Hsp40 family proteins and various intermediate filament proteins that we tested using two-hybrid methods, Mrj specifically interacted with K18. Immunostaining with anti-Mrj antibody showed that Mrj colocalized with K8/18 filaments in HeLa cells. Mrj was immunoprecipitated not only with K18, but also with the stress-induced and constitutively expressed heat shock protein Hsp/c70. Mrj bound to K18 through its C terminus and interacted with Hsp/c70 via its N terminus, which contains the J domain. Microinjection of anti-Mrj antibody resulted in the disorganization of K8/18 filaments, without effects on the organization of actin filaments and microtubules. Taken together, these results suggest that Mrj may play an important role in the regulation of K8/18 filament organization as a K18-specific co-chaperone working together with Hsp/c70.     

Monoclonal antibodies to epitopes on different regions of the 200 00 dalton neurofilament protein : Probes for the geometry of the filament

Neurofilaments in mammalian nervous tissues have three subunit proteins. These subunit proteins have apparent molecular masses of 200 (NF200), 150 (NF150) and 68 (NF68) kD. Biochemical assembly studies have indicated that the NF68 protein forms the core of the filament and that the other two proteins are associated proteins. Electron microscopy immunolocalization studies have been performed previously on isolated filaments and on filaments from neurons in culture, and have confirmed the localization of NF68 as a core filament protein and NF200 as a peripheral protein. We have raised two monoclonal antibodies to the NF200 components. Using immunogold labelled protein A, we have been able to localize these antibodies to tissue sections of adult cerebellum at the EM level. With this method, we have found that one of the monoclonal antibodies (NF2) shows a linear arrangement of gold particles directly on the filament, whereas the second monoclonal antibody (NF111) reacts with the filaments to give a periodic arrangement of gold particles. By immunoblotting against chymotryptic fragments of the NF200 protein, we have found that the mAB-NF111 reacts solely with a 160 kD piece, whereas the other monoclonal antibody reacts with both the 160 kD piece and the 40 kD piece. The latter piece was shown to be associated to the filament by binding studies with iodinated NF68. Thus the EM localization studies and the biochemical studies indicate that the two monoclonal antibodies react with different parts of the NF200 molecule, one binding to a part of the molecule which is located closer to the filament, and one to a more peripheral part of the molecule.     

Monoclonal antibodies to epitopes on different regions of the 200 000 dalton neurofilament protein. Probes for the geometry of the filament

Neurofilaments in mammalian nervous tissues have three subunit proteins. These subunit proteins have apparent molecular masses of 200 (NF200), 150 (NF150) and 68 (NF68) kD. Biochemical assembly studies have indicated that the NF68 protein forms the core of the filament and that the other two proteins are associated proteins. Electron microscopy immunolocalization studies have been performed previously on isolated filaments and on filaments from neurons in culture, and have confirmed the localization of NF68 as a core filament protein and NF200 as a peripheral protein. We have raised two monoclonal antibodies to the NF200 components. Using immunogold labelled protein A, we have been able to localize these antibodies to tissue sections of adult cerebellum at the EM level. With this method, we have found that one of the monoclonal antibodies (NF2) shows a linear arrangement of gold particles directly on the filament, whereas the second monoclonal antibody (NF111) reacts with the filaments to give a periodic arrangement of gold particles. By immunoblotting against chymotryptic fragments of the NF200 protein, we have found that the mAB-NF111 reacts solely with a 160 kD piece, whereas the other monoclonal antibody reacts with both the 160 kD piece and the 40 kD piece. The latter piece was shown to be associated to the filament by binding studies with iodinated NF68. Thus the EM localization studies and the biochemical studies indicate that the two monoclonal antibodies react with different parts of the NF200 molecule, one binding to a part of the molecule which is located closer to the filament, and one to a more peripheral part of the molecule.     

alpha-Internexin, a 66-kD intermediate filament-binding protein from mammalian central nervous tissues

In this paper we describe a 66-kD protein that co-purifies with intermediate filaments from rat optic nerve and spinal cord but can be separated further by ion-exchange chromatography. This protein is distinct from the 68-kD neurofilament subunit protein as judged by isoelectric focusing, immunoblotting, peptide mapping, and tests of polymerization competence. This protein is avidly recognized by the monoclonal anti-intermediate filament antigen antibody, previously demonstrated to recognize a common antigenic determinant in all five known classes of intermediate filaments. Also, when isolated this protein binds to various intermediate filament subunit proteins, which suggests an in vivo interaction with the intermediate filament cytoskeleton, and it appears to be axonally transported in the rat optic nerve. Because of this ability to bind to intermediate filaments in situ and in vitro we have named this protein alpha-internexin. A possible functional role for the protein in organizing filament assembly and distribution is discussed.     

Purification and characterization of an 82-kD membrane protein as a neurite outgrowth factor binding protein: possible involvement of NOF binding protein in axonal outgrowth in developing retina

Neurite outgrowth factor (NOF) is a glycoprotein isolated from an extract of gizzard that induces neurite outgrowth from cultured retinal or ciliary ganglionic (CG) neurons. We have reported that a glycoprotein of approximately 82 kD solubilized from gizzard muscles binds to NOF (ligand blotting) and inhibits the neurite promoting activity of NOF (inhibition assay). The 82-kD protein (NOF binding protein) was purified from gizzard muscle membranes as a doublet band on SDS-PAGE and a polyclonal antibody was raised against it. An NOF binding protein in developing retina exhibited the same physicochemical properties as that of the gizzard muscle. Quantitative decrease in NOF binding protein in embryonic retinas was observed after day 11 by the inhibition assay, ligand blotting, and immunoblotting, its decrease being parallel with reduction of NOF-induced neurite outgrowth of embryonic retinas. In an immunohistochemical study, the antibody stained only the optic fiber layers of the retinas of 8-d embryos, and this staining was no longer detectable in retinas of 18-d embryos. These results suggest that the 82-kD protein is a novel membrane protein that behaves as an NOF receptor and that the loss of neuritic response of the retinal neurons to NOF reflects a decrease in NOF receptor molecules.     

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.     

Cytokeratin, vimentin and E-cadherin immunodetection in the embryonic palate in two strains of mice with different susceptibility to glucocorticoid-induced clefting

An immunohistochemical study analyzing the pattern of distribution of some intermediate filament proteins, keratin and vimentin and, one adhesion molecule, cadherin in different stages of developing secondary palate in two strains of mice with different H-2 backgrounds was undertaken to investigate differences between a strain that is susceptible to glucocorticoid-induced cleft palate (A/Sn) and one that is resistant to glucocorticoid-induced cleft palate (C57/BL). The heads of embryos were processed by standard immunohistochemistry with antipancytokeratin (KAE1), antikeratins 18 (K18) and 19 (K19), antivimentin, and anti E-cadherin antibodies. Immunostaining with KAE1 antibody showed differences between the strains. The reaction was stronger in the medial edge epithelia of palatal processes in the A/Sn strain at all stages of palatogenesis. The C57/BL strain showed a weak immunostain to KAE1. Antivimentin antibody stained the mesenchymal cells of palatal processes and K18 and K19 showed no reaction in either strain of mice. Anti E-cadherin antibody was detected in the medial palatal epithelium of both strains of mice and in all stages of palate development. No differences were observed in E-cadherin and vimentin immunostain in palatal epithelium between the strains. The different expression of some cytokeratins in the embryonic palatal epithelium suggests that these intermediate filament proteins may be involved in different susceptibility to glucocorticoid-induced cleft palate in the mouse. The decreased immunoreaction of cytokeratins observed in the resistant strain would facilitate the disappearance of this molecule during the transformation from an epithelial to a mesenchymal phenotype that takes place during the development of the palate. These results may be related to the loss of cytokeratin expression observed during epithelial-mesenchymal transformation in the embryonic palate.     

The myosin Va head domain binds to the neurofilament-L rod and modulates endoplasmic reticulum (ER) content and distribution within axons

The neurofilament light subunit (NF-L) binds to myosin Va (Myo Va) in neurons but the sites of interaction and functional significance are not clear. We show by deletion analysis that motor domain of Myo Va binds to the NF-L rod domain that forms the NF backbone. Loss of NF-L and Myo Va binding from axons significantly reduces the axonal content of ER, and redistributes ER to the periphery of axon. Our data are consistent with a novel function for NFs as a scaffold in axons for maintaining the content and proper distribution of vesicular organelles, mediated in part by Myo Va. Based on observations that the Myo Va motor domain binds to intermediate filament (IF) proteins of several classes, Myo Va interactions with IFs may serve similar roles in organizing organelle topography in different cell types.     

A Conserved Rod Domain Phosphotyrosine That Is Targeted by the Phosphatase PTP1B Promotes Keratin 8 Protein Insolubility and Filament Organization

Post-translational modifications are important functional determinants for intermediate filament (IF) proteins. Phosphorylation of IF proteins regulates filament organization, solubility, and cell-protective functions. Most known IF protein phosphorylation sites are serines localized in the variable “head” and “tail” domain regions. By contrast, little is known about site-specific tyrosine phosphorylation or its implications on IF protein function. We used available proteomic data from large scale studies to narrow down potential phospho-tyrosine sites on the simple epithelial IF protein keratin 8 (K8). Validation of the predicted sites using a pan-phosphotyrosine and a site-specific antibody, which we generated, revealed that the highly conserved Tyr-267 in the K8 “rod” domain was basally phosphorylated. The charge at this site was critically important, as demonstrated by altered filament organization of site-directed mutants, Y267F and Y267D, the latter exhibiting significantly diminished solubility. Pharmacological inhibition of the protein-tyrosine phosphatase PTP1B increased K8 Tyr-267 phosphorylation, decreased solubility, and increased K8 filament bundling, whereas PTP1B overexpression had the opposite effects. Furthermore, there was significant co-localization between K8 and a “substrate-trapping” mutant of PTP1B (D181A). Because K8 Tyr-267 is conserved in many IFs (QYE motif), we tested the effect of the paralogous Tyr in glial fibrillary acidic protein (GFAP), which is mutated in Alexander disease (Y242D). Similar to K8, Y242D GFAP exhibited highly irregular filament organization and diminished solubility. Our results implicate the rod domain QYE motif tyrosine as an important determinant of IF assembly and solubility properties that can be dynamically modulated by phosphorylation.     

Lamins A, B and C share an epitope with the common domain of intermediate filament proteins

Peripheral lamina were isolated from rat liver nuclei and probed with a monoclonal antibody reactive against a common domain of all intermediate filament (IF) proteins. The antibody reacted against lamins A and C as expected from their known sequence homology with IF proteins. Lamin B was also recognized by the antibody. These results indicate that lamin B, despite important biochemical differences vs lamins A and C, also shares sequence homology with IF proteins.     

Cross-reactivity of antibodies specific for flagellar tektin and intermediate filament subunits

Monoclonal antibodies specific for each of the flagellar tektins were prepared and used to determine whether structures similar to tektin filaments are present in cells lacking cilia or flagella. This analysis was performed by double-label immunofluorescence microscopy of several cell lines and by immunoblots of protein fractions. Two of the four anti-tektin antibodies, the antibodies 3-7-1 and 3-10-1, which bind different epitopes of the C-tektin, label 3T3, HeLa, PtK2, and BHK-21 cells as well as myotubes. The antibody 3-7-1 stains intermediate filament structures in the cells and binds vimentin or desmin in preparations of cytoskeletal proteins; whereas the antibody 3-10-1 stains nuclear envelopes in the cells and binds lamin A and C in preparations of cytoskeletal proteins or nuclear lamina. Structural similarities between the C-tektin and intermediate filament proteins probably are extended to more than two epitopes because polyclonal antibodies anti-vimentin and anti-desmin bind to C-tektin. These polyclonal antibodies also bind to A-tektin. The cross-reaction of monoclonal and polyclonal antibodies binding to epitopes in tektin and intermediate filament components and the existence of a high content of alpha-helical structure in the tektin subunits (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22) indicate that tektin and intermediate filaments are homologous in several parts of their structure.     

Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal-Associated Intermediate Filament Protein Kinase Activity in Astrocytes

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Defective axonal transport of neurofilament proteins in neurons overexpressing peripherin

Peripherin is a type III neuronal intermediate filament detected in motor neuron inclusions of amyotrophic lateral sclerosis (ALS) patients. We previously reported that overexpression of peripherin provokes late-onset motor neuron dysfunction in transgenic mice. Here, we show that peripherin overexpression slows down axonal transport of neurofilament (NF) proteins, and that the transport defect precedes by several months the appearance of axonal spheroids in adult mice. Defective NF transport by peripherin up-regulation was further confirmed with dorsal root ganglia (DRG) neurons cultured from peripherin transgenic embryos. Immunofluorescence microscopy and western blotting revealed that excess peripherin provokes reduction in levels of hyperphosphorylated NF-H species in DRG neurites. Similarly the transport of a green fluorescent protein (GFP)-tagged NF-M, delivered by means of a lentiviral construct, was impaired in DRG neurites overexpressing peripherin. These results demonstrate that peripherin overexpression can cause defective transport of type IV NF proteins, a phenomenon that may account for the progressive formation of ALS-like spheroids in axons.     

Regulation of cell adhesion to collagen via β1 integrins is dependent on interactions of filamin A with vimentin and protein kinase C epsilon

Cell adhesion and spreading on collagen, which are essential processes for development and wound healing in mammals, are mediated by β1 integrins and the actin and intermediate filament cytoskeletons. The mechanisms by which these separate cytoskeletal systems interact to regulate β1 integrins and cell spreading are poorly defined. We previously reported that the actin cross-linking protein filamin A binds the intermediate filament protein vimentin and that these two proteins co-regulate cell spreading. Here we used deletional mutants of filamin A to define filamin A-vimentin interactions and the subsequent phosphorylation and re-distribution of vimentin during cell spreading on collagen. Imaging of fixed and live cell preparations showed that phosphorylated vimentin is translocated to the cell membrane during spreading. Knockdown of filamin A inhibited cell spreading and the phosphorylation and re-distribution of vimentin. Knockdown of filamin A and/or vimentin reduced the cell surface expression and activation of β1 integrins, as indicated by immunoblotting of plasma membrane-associated proteins and shear force assays. In vitro pull-down assays using filamin A mutants showed that both vimentin and protein kinase C? bind to repeats 1-8 of filamin A. Reconstitution of filamin-A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation, and the cell surface expression of β1 integrins. We conclude that the binding of filamin A to vimentin and protein kinase Cε is an essential regulatory step for the trafficking and activation of β1 integrins and cell spreading on collagen.     

α-internexin is the only neuronal intermediate filament expressed in developing cerebellar granule neurons

We have used immunocytochemistry and in situ hybridization to examine the distribution of neuronal intermediate filament proteins and their mRNAs in the developing mouse cerebellum. First, we demonstrate that α-internexin is abundantly expressed in the developing cerebellum and is the only neuronal intermediate filament protein expressed in developing, including migrating, granule neurons. Second, in granule neuron reaggregates in vitro, α-internexin is the only neuronal intermediate filament protein highly expressed in the processes of the cultured granule neurons. This in vitro observation is consistent with results from immunocytochemistry and in situ hybridization studies of developing granule neurons in vivo, which suggest that α-internexin is the major neuronal intermediate filament protein in developing granule neurons. Finally, the neurofilament triplet proteins are expressed later, and coexist with α-internexin in other cells, including Purkinje cells and interneurons in the mature mouse cerebellum. These changes in neuronal intermediate filament composition may regulate neuronal maturation and axonal stability in cerebellar development. Furthermore, α-internexin may play a key role in neurite outgrowth and the establishment of neuronal cytoarchitecture. © 1996 John Wiley & Sons, Inc.     

Investigation of general and cytoskeletal markers to estimate numbers and proportions of neurons in the human intestine

An important requirement in pathological diagnostics in the human enteric nervous system (ENS) is the estimation of the total numbers of neurons and of proportions of distinct subpopulations. In this study, we compared the suitability of two suggested panneuronal markers, cuprolinic blue (CB) and anti-Hu-protein (HU), for staining and counting human myenteric neurons in wholemounts, derived from small and large intestinal samples. Furthermore, the proportional expression of three cytoskeletal intermediate filaments, alpha-internexin (IN), neurofilament 200 (NF) and peripherin (PE), was correlated with both CB and HU. In 8 CB- and HU-stained wholemounts, 93.3% of all neurons were double labeled, 3.3% of neurons were stained only with CB whereas 3.3% were immuno-stained only for HU. Thus, both markers were comparably reliable in representing the putative total human myenteric neuron population in our material. The wholemounts double stained for IN/CB or IN/HU revealed between 56.2 and 71.5% of neurons to be IN-reactive. Between 42.8 and 50.9% of neurons were immunoreactive for NF whereas 53.9 to 62.4% of neurons were reactive for PE. Although our sample number was too small to allow final conclusions, we suggest that the variations in proportions of intermediate filament expression we observed may be due to individual circumstances rather than to correlation with age or region. The proportions of neurons positive for IN, NF or PE but unstained by CB histochemical or HU immunohistochemical techniques was between 0 and 2.2%. We conclude that both CB and HU techniques are suitable methods for representation of almost all myenteric neurons in the human gut and that the differential expression of the cytoskeletal proteins investigated has to be included in the classification of enteric neurons in pathological diagnostics of human gastrointestinal diseases.     

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.     

Two distinct regions of calponin share common binding sites on actin resulting in different modes of calponin–actin interaction

Calponins are a small family of proteins that alter the interaction between actin and myosin II and mediate signal transduction. These proteins bind F-actin in a complex manner that depends on a variety of parameters such as stoichiometry and ionic strength. Calponin binds G-actin and F-actin, bundling the latter primarily through two distinct and adjacent binding sites (ABS1 and ABS2). Calponin binds other proteins that bind F-actin and considerable disagreements exist as to how calponin is located on the filament, especially in the presence of other proteins. A study (Galkin, V.E., Orlova, A., Fattoum, A., Walsh, M.P. and Egelman, E.H. (2006) J. Mol. Biol. 359, 478–485.), using EM single-particle reconstruction has shown that there may be four modes of interaction, but how these occur is not yet known. We report that two distinct regions of calponin are capable of binding some of the same sites on actin (such as 18–28 and 360–372 in subdomain 1). This accounts for the finding that calponin binds the filament with different apparent geometries. We suggest that the four modes of filament binding account for differences in stoichiometry and that these, in turn, arise from differential binding of the two calponin regions to actin. It is likely that the modes of binding are reciprocally influenced by other actin-binding proteins since members of the α-actinin group also adopt different actin-binding positions and bind actin principally through a domain that is similar to calponin's ABS1.