A protein that was immunologically related to the erythrocyte and brain alpha-240-subunit and to the brain beta-235-subunit of spectrin was characterized by immunoblotting and was detected by immunofluorescence in the apical part of ciliated cells from quail oviduct. After immunogold-labeling electron immunocytochemistry, spectrin was detected mainly in a fibrillar meshwork located between the proximal parts of the basal bodies. It was also observed to be in contact with the basal foot of basal bodies, but was not found to be associated with the apical plasma membrane. Cilia and microvilli were unlabeled. In contrast, spectrin was detected in close contact with the lateral plasma membrane of mature ciliated cells as well as in stem epithelial cells in unstimulated oviduct. During ciliogenesis induced by estrogen, spectrin gradually appeared at the apex of the cells as the apical cytoskeleton differentiated.
La spectrine a d'abord ete decrite et caracterisee dans les erythrocytes humains ou elle a, entre autres fonctions, de former un reseau sous-membranaires avec l'actine f, et d'ancrer ce reseau a la membrane par l'intermediaire de l'ankyrine. Mais la spectrine n'est pas uniquement trouvee dans les erythrocytes, puisqu'elle est repartie dans tout le regne animal, d'acanthamoeba jusqu'aux mammiferes. Si ces spectrines ont des fonctions communes (liaison a l'actine f); elles peuvent aussi presenter des fonctions propres. Nous avons montre que la spectrine de cerveau etait apte a se lier aux neurofilaments, par l'intermediaire de leur sous-unite de faible poids moleculaire nf-l (70 kda). Nous avons aussi montre que cette interaction etait regulee par la calmoduline, et qu'lle avait lieu dans les conditions physiologiques. Nous avons aussi montre que l'interaction de la spectrine de cerveau et des neurofilmanents se faisait surd la partie n-terminale du domaine central des neurofilaments. La spectrine, qui est composee de deux sous-unites alpa et beta, se lie aux neurofilaments par sa sous-unite beta, probablement par le domaine n-terminal de cette sous-unite. La spectrine de cerveau se lie aussi aux microfilaments d'actine par l'intermediaire de sa sous-unite beta, la sous-unite alpha ayant un role dans la stabilisation de la sous-unite beta, et facilitant son interaction avec les microfilaments. Il semble que le domaine par lequel la spectrine de cerveau se lie aux microfilaments presente des homologies avec le domaine de l'alpha-actinine qui se lie aux microfilaments. Nous recherchons si les domaines d'interaction de ces deux proteines sont communs
Abstract: Peripherin is a type III intermediate filament present in peripheral and certain CNS neurons. We report here that peripherin contains a phosphotyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specific for phosphotyrosine recognizes peripherin present in PC12 cells (with or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf‐9 cells and SW‐13 cl. 2 vim − cells. The identity of peripherin as a tyrosine‐phosphorylated protein in PC12 cells was confirmed by immunoprecipitation, two‐dimensional isoelectric focusing/sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phosphorylation, tyrosine phosphorylation of peripherin is not regulated by depolarization or nerve growth factor treatment. To identify the site of tyrosine phosphorylation, rat peripherin was mutated at several tyrosine residues and expressed in SW‐13 cl. 2 vim − cells. Tyrosine phosphorylation was selectively lost only for peripherin mutants in which the carboxy‐terminal tyrosine (Y474) was mutated. Indirect immunofluorescence staining indicated that both wild‐type peripherin and peripherin Y474F form a filamentous network in SW‐13 cl. 2 vim − cells. This indicates that tyrosine phosphorylation of the peripherin C‐terminal residue is not required for assembly and leaves open the possibility that this modification serves other functions.
Mutations in the gene for the microtubule associated protein, tau have been identified for fronto‐temporal dementia with Parkinsonism linked to chromosome 17 (FTDP‐17). In vitro data have shown that FTDP‐17 mutant tau proteins have a reduced ability to bind microtubules and to promote microtubule assembly. Using the baculovirus system we have examined the effect of the V337M mutation on the organization of the microtubules at the ultrastructural level. Our results show that the organization of the microtubules is disrupted in the presence of V337M tau with greater distances between the microtubules and fewer microtubules per process.
Tryptic digestion of brain spectrin generates a number of fragments from α and β subunits; when these fragments are incubated with F‐actin or neurofilament light subunit, four of them with molecular masses below 30 kDa sediment with the cytoskeleton structures. A selective purification of these fragments by ammonium sulfate fractionation and butyl‐Sepharose chromatography has been achieved. Two fragments with molecular masses of 28 and 23 kDa bind to F‐actin. Native brain spectrin causes half‐maximal inhibition of the association at a concentration of 3 μM. Protein sequencing indicates that the actin‐binding domain is contained in the β subunit, in a stretch of amino acids at the N terminus from Ala43 (28‐kDa fragment) or from Met104 (23‐kDa fragment) and terminate probably at the C‐terminal Lys288 or Lys284. Amino acids are numbered by reference to the sequence of the Drosophila β subunit. The 28‐kDa fragment also binds to the low‐molecular‐mass subunit of neurofilaments; brain spectrin heterodimer disrupts this binding. Hence, spectrin binds to F‐actin and to neurofilaments via a common binding domain.
During mitosis in higher eukaryotic cells, the nuclear envelope membranes break down into distinct populations of vesicles and the proteins of the nuclear lamina and the nuclear pore complexes disperse in the cytoplasm. Since phosphorylation can alter protein−protein interactions and membrane traffic, we have examined the cell cycle-dependent phosphorylation of nuclear pore complex proteins. Nonmembrane nucleoporins Nup153, Nup214, and Nup358 that are modified by O-linked N-acetylglucosamine and recognized by a monoclonal antibody were phosphorylated throughout the cell cycle and hyperphosphorylated during M phase. Pore membrane glycoprotein gp210, that has a cytoplasmic, carboxyl-terminal domain facing the pore, was not phosphorylated in interphase but specifically phosphorylated in mitosis. Mutant and wild-type fusion proteins containing the cytoplasmic domain of gp210 were phosphorylated in vitro and their phosphopeptide maps compared to that of mitotic gp210. This analysis showed that Ser1880 of gp210 was phosphorylated in mitosis, possibly by cyclin B-p34cdc2 or a related kinase. Several nuclear pore complex proteins are therefore differentially phosphorylated during mitosis when pore complexes disassemble and reassemble.
beta-amyloid (Abeta) has been proposed to play a role in the pathogenesis of Alzheimer's disease (AD). Deposits of insoluble Abeta are found in the brains of patients with AD and are one of the pathological hallmarks of the disease. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. In our current study, treatment with nitric oxide generators protected against Abeta-induced death, whereas inhibition of nitric oxide synthase afforded no protection, suggesting that formation of peroxynitrite is not critical for Abeta-mediated death. Previous studies have shown that aggregated Abeta can induce caspase-dependent apoptosis in cultured neurons. In all of the neuronal populations studied here (hippocampal neurons, sympathetic neurons, and PC12 cells), cell death was blocked by the broad spectrum caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone and more specifically by the downregulation of caspase-2 with antisense oligonucleotides. In contrast, downregulation of caspase-1 or caspase-3 did not block Abeta(1-42)-induced death. Neurons from caspase-2 null mice were totally resistant to Abeta(1-42) toxicity, confirming the importance of this caspase in Abeta-induced death. The results indicate that caspase-2 is necessary for Abeta(1-42)-induced apoptosis in vitro.
The stability of proteins that constitute the neurofibrillary tangles and senile plaques of Alzheimer disease suggests that they would be ideal substrates for nonenzymatic glycation, a process that occurs over long times, even at normal levels of glucose, ultimately resulting in the formation of advanced glycation end products (AGEs). AGE-modified proteins aggregate, and they generate reactive oxygen intermediates. Using monospecific antibody to AGEs, we have colocalized these AGEs with paired helical filament tau in neurofibrillary tangles in sporadic Alzheimer disease. Such neurons also exhibited evidence of oxidant stress: induction of malondialdehyde epitopes and heme oxygenase 1 antigen. AGE-recombinant tau generated reactive oxygen intermediates and, when introduced into the cytoplasm of SH-SY5Y neuroblastoma cells, induced oxidant stress. We propose that in Alzheimer disease, AGEs in paired helical filament tau can induce oxidant stress, thereby promoting neuronal dysfunction.
We have previously demonstrated that brain spectrin binds to the low-molecular-mass subunit of neurofilaments (NF-L) [Frappier, Regnouf & Pradel (1987) Eur. J. Biochem. 169, 651-657]. In the present study, we seek to locate their respective binding domains. In the first part we demonstrate that brain spectrin binds to a 20 kDa domain of NF-L. This domain is part of the rod domain of neurofilaments and plays a role in the polymerization process. However, the polymerization state does not seem to have any influence on the interaction. In the second part, we provide evidence that NF-L binds to the beta-subunit of not only brain spectrin but also human and avian erythrocyte spectrins. The microtubule-associated protein, MAP2, which has also been shown to bind to microfilaments and neurofilaments, binds to the same domain of NF-L as spectrin does. Finally, among the tryptic peptides of brain spectrin, we show that some peptides of low molecular mass (35, 25, 20 and 18 kDa) co-sediment with either NF-L or F-actin.
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