Metabolic studies in vitro of the CNS cytoskeletal proteins: Synthesis and degradation

General aspects of metabolic features of the most prominent CNS intermediate filament proteins, the 200,000 (200K), 150,000 (150K), and 70,000 (70K) dalton proteins of the neuron, and the glial fibrillary acidic protein (GFAP) have been explored using the incubated spinal cord slice from the rat. Measurement of shortterm uptake of³H-labeled amino acids into the individual proteins separated on polyacrylamide gels revealed that of the three neurofilament proteins, 200K was most metabolically active, 150K was less active, and 70K contained very little incorporated radioactivity. Glial fibrillary acidic protein based on Coomassie blue stain affinity showed less metabolic activity than any of the neurofilament proteins. Those relationships were constant at all ages, but the metabolic activity of all CNS intermediate filaments decreased with age. When Ca²⁺ was present in the medium of the incubated slices, the intermediate filaments were rapidly destroyed, but GFAP was more resistant to degradation than the neurofilament proteins. GFAP and probably the neurofilament proteins also were relatively resistant to Ca²⁺-activated degradative mechanisms in spinal cords of rats at younger ages (15 day) than in those of older animals (10–18 months). It is likely that the Ca²⁺ activated protease is less active in developing animals in which the nerve tracts are still elongating, than in adults. These results suggest that GFAP is less active metabolically and more resistant to degradation than the neurofilament proteins at all stages of maturation, but that metabolic activity of all CNS intermediate filaments decreases with age while the susceptibility to degradation increases.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

Phosphorylation Modulates Calpain-Mediated Proteolysis and Calmodulin Binding of the 200-kDa and 160-kDa Neurofilament Proteins

Abstract: The effects of enzymatic dephosphorylation on neurofilament interaction with two calcium-binding proteins, calpain and calmodulin, were examined. Dephosphorylation increased the rate and extent of 200-kDa neurofilament protein proteolysis by calpain. In contrast, dephosphorylation of the 160-kDa neurofilament protein did not alter the rate or extent of calpain proteolysis. However, the calpain-induced breakdown products of native and dephosphorylated 160-kDa neurofilament protein were different. Dephosphorylation did not change the proteolytic rate, extent, or breakdown products of the 68-kDa neurofilament protein. Calmodulin binding to the purified individual 160- and 200-kDa neurofilament proteins was increased following dephosphorylation. These results suggest that phosphorylation may regulate the metabolism and function of neurofilaments by modulating interactions with the calcium-activated proteins calpain and calmodulin.     

"Buttonin," a unique button-shaped microtubule-associated protein (75 kD) that decorates spindle microtubule surface hexagonally

A 75-kD protein was purified from sea urchin egg microtubule proteins through gel filtration. It enhanced the polymerization of porcine brain tubulin, but was not heat-stable and did not bind to calmodulin in the presence of calcium as demonstrated by calmodulin affinity column chromatography. Rotary shadowing of the freeze-etched 75-kD protein adsorbed on mica revealed the protein to be a spherical molecule (approximately 9 nm in diameter). Quick-freeze deep-etch electron microscopy revealed that the surface of microtubules polymerized with 75-kD protein was entirely covered with hexagonally packed, round, button-like structures that were quite uniform in shape and size (approximately 9 nm) and similar to the buttons observed on microtubules of mitotic spindles in vivo or microtubules isolated from mitotic spindles. Judging from calibration studies of molecular mass by gel filtration, the 75-kD protein probably exists in a dimeric form (approximately 150 kD) in its native condition. The stoichiometry of tubulin (dimer) versus 75-kD protein (dimer) in the polymerized pellet was 3-3.4:1. Hence, we concluded that the 75-kD protein was a unique microtubule-associated protein that formed the microtubule button in vivo and in vitro. We propose to name this protein "buttonin".     

Proteolysis of filament proteins in glial and neuronal cells afterIn vivo stimulation of hippocampal NMDA receptors

An intrahippocampal injection of N-methyl-D-aspartate induced the appearance of degradation products of both the 68 kiloDalton neurofilament protein and the glial fibrillary acidic protein, as revealed by immunoblot techniques. The degradation of these two filament proteins was maximal at 10 days after the lession. The degradation patterns were similar to those induced with calpains or calcium in vitro. There were no degradation effects on the 200 kD neurofilament protein as tested with both mono- and polyclonal antibodies. Consequently, the neuronal degeneration after excessive activation of NMDA receptors appears to involve calcium activation of proteolytic enzymes. The effects on the glial proteins are probably secondary to neuronal damage but could be related to calcium dependent processes.     

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.     

Identification by monoclonal antibodies and characterization of human platelet caldesmon

Actin-based gels were prepared from clarified high-salt extracts of human platelets by dialysis against physiological salt buffers. The gel was partially solubilized with 0.3 M KCl. Mice were immunized with the 0.3 M KCl extract of the actin gel, and hybridomas were produced by fusion of spleen cells with myeloma cells. Three hybridomas were generated that secrete antibodies against an 80-kD protein. These monoclonal antibodies stained stress fibers in cultured cells and cross-reacted with proteins in several tissue types, including smooth muscle. The cross-reacting protein in chicken gizzard smooth muscle had an apparent molecular weight of 140,000 and was demonstrated to be caldesmon, a calmodulin and actin-binding protein (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, Proc. Natl. Acad. Sci. USA, 78:5652-5655). No proteins of molecular weight greater than 80 kD were detectable in platelets by immunoblotting using the monoclonal antibodies. The 80-kD protein is heat stable and was purified using modifications of the procedure reported by Bretscher for the rapid purification of smooth muscle caldesmon (Bretscher, A., 1985, J. Biol. Chem., 259:12873-12880). The 80-kD protein bound to calmodulin-Sepharose in a Ca++-dependent manner and sedimented with actin filaments, but did not greatly increase the viscosity of F-actin solutions. The actin-binding activity was inhibited by calmodulin in the presence of calcium. Except for the molecular weight difference, the 80-kD platelet protein appears functionally similar to 140-kD smooth muscle caldesmon. We propose that the 80-kD protein is platelet caldesmon.     

Multiple fates of newly synthesized neurofilament proteins: evidence for a stationary neurofilament network distributed nonuniformly along axons of retinal ganglion cell neurons

We have studied the fate of neurofilament proteins (NFPs) in mouse retinal ganglion cell (RGC) neurons from 1 to 180 d after synthesis and examined the proximal-to-distal distribution of the newly synthesized 70-, 140-, and 200-kD subunits along RGC axons relative to the distribution of neurofilaments. Improved methodology for intravitreal delivery of [3H]proline enabled us to quantitate changes in the accumulation and subsequent decline of radiolabeled NFP subunits at various postinjection intervals and, for the first time, to estimate the steady state levels of NFPs in different pools within axons. Two pools of newly synthesized triplet NFPs were distinguished based on their kinetics of disappearance from a 9-mm "axonal window" comprising the optic nerve and tract and their temporal-spatial distribution pattern along axons. The first pool disappeared exponentially between 17 and 45 d after injection with a half-life of 20 d. Its radiolabeled wavefront advanced along axons at 0.5-0.7 mm/d before reaching the distal end of the axonal window at 17 d, indicating that this loss represented the exit of neurofilament proteins composing the slowest phase of axoplasmic transport (SCa or group V) from axons. About 32% of the total pool of radiolabeled neurofilament proteins, however, remained in axons after 45 d and disappeared exponentially at a much slower rate (t 1/2 = 55 d). This second NFP pool assumed a nonuniform distribution along axons that was characterized proximally to distally by a 2.5-fold gradient of increasing radioactivity. This distribution pattern did not change between 45 and 180 d indicating that neurofilament proteins in the second pool constitute a relatively stationary structure in axons. Based on the relative radioactivities and residence time (or turnover) of each neurofilament pool in axons, we estimate that, in the steady state, more neurofilament proteins in mouse RGC axons may be stationary than are undergoing continuous slow axoplasmic transport. This conclusion was supported by biochemical analyses of total NFP content and by electron microscopic morphometric studies of neurofilament distribution along RGC axons. The 70-, 140-, and 200-kD subunits displayed a 2.5-fold proximal to distal gradient of increasing content along RGC axons. Neurofilaments were more numerous at distal axonal levels, paralleling the increased content of NFP.(ABSTRACT TRUNCATED AT 400 WORDS)     

An isoelectric variant of the 150,000-dalton neurofilament polypeptide. Evidence that phosphorylation state affects its association with the filament

A soluble isoelectric variant of the 150,000-dalton neurofilament protein was isolated from bovine brain by treating a partially purified filament preparation with a low-ionic-strength high-pH buffer. The protein (S150) had similar peptide maps to the neurofilament component of the same molecular weight (NF150) and was recognized by a polyclonal antibody made against the NF150 polypeptide. However, only half the anti-NF150 activity could be removed with the S150 protein. In addition, the S150 protein had a higher isoelectric point than the NF150 protein. Phosphate analysis indicated that the S150 protein was considerably lessened in phosphate content, which could account for the higher isoelectric point of the protein. It appears, therefore, that the S150 protein may be a precursor of NF150 or the result of phosphatase activity during the isolation procedure. Assembly studies showed that the S150 protein, unlike the NF150 protein, could not assemble with the 70-kDa neurofilament protein, indicating that the phosphate groups which were removed are important in the association of this protein to the neurofilament. When filaments containing all three triplet neurofilament polypeptides or those composed of the 70- and 150-kDa neurofilament proteins were subjected to acid phosphatase, a soluble fraction was obtained, which contained isoelectric variants with higher pI values than the NF150 polypeptide. Only unmodified NF150 protein was found in the insoluble fraction. These results support the argument that removal of phosphate groups results in the dissociation of this protein from the filament.     

Calcium Calmodulin Dependent Phosphorylation of Proteins: Fetal Cortical Neurons and Adult Cortex

In the present investigation, changes in the calcium calmodulin-dependent phosphorylation of proteins have been examined in murine fetal cortical neurons and adult cortex. An approximately 80-kD protein in the fetal neurons was not phosphorylated/dephosphorylated in a calmodulin-dependent manner. However, this protein was phosphorylated by PMA both in the presence and absence of calcium. These data suggest that calmodulin inhibits the phosphorylation of a approximately 80-kD protein by inhibiting PKC in murine fetal cortical neurons but not in the adult cortex. More importantly, we demonstrate that the calmodulin-mediated inhibition of phosphorylation was restored by preincubating the cortical neurons with KN-62, a CaM kinase inhibitor.     

The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve

The ontogenetic appearance of the individual triplet polypeptides that comprise mammalian neurofilaments was studied in the developing rat optic nerve. Triton-insoluble cytoskeletal preparations from the optic nerves of rats of postnatal ages 1 Day (P1), 6 days (P6), 10 days (P10), 20 days (P20), and 3 months (adult) were analyzed for protein composition by one and two-dimensional gel electrophoresis. Results indicate that at P1, both the 150- and 68-kDa neurofilament subunit proteins are present. The 200-kDa subunit first becomes discernible at P20, but, at this age, it is still present in considerably less quantity than in the adult. Immunocytochemical verification of the presence of neurofilament protein was accomplished by staining tissue sections with specific antibodies against the 150- and the 68-kDa neurofilament subunits using the peroxidase-antiperoxidase technique. Results of the morphological analyses have shown that neurofilaments are not present in quantity until P10, which coincides with the time when the 68-kDa subunit increases in quantity by one dimensional gel analysis. Thus, the 150- and 68-kDa subunits can be detected prior to the appearance of neurofilaments, and the 200-kDa protein is not observed until sometime later. The potential physiological significance of the differential subunit transport is discussed with respect to neuronal differentiation in the developing mammalian CNS.     

Purification and characterization of a basal body-associated Ca2+- binding protein

Isolated basal body complexes from the unicellular alga, Chlamydomonas reinhardtii were found to contain a low molecular mass acidic polypeptide, distinct from calmodulin, but with biochemical features in common with members of the calmodulin family of calcium-binding proteins. These common characteristics included a relative low molecular mass of 20 kD, an experimentally determined acidic pI of 5.3, an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of added calcium, and a calcium-dependent binding to the hydrophobic ligand phenyl-Sepharose which allowed its purification by affinity chromatography. The relatedness of the basal body-associated 20-kD calcium-binding protein (CaBP) to calmodulin was confirmed by amino acid compositional analysis and partial peptide sequencing of the isolated protein. A rabbit antibody specific for the 20-kD CaBP was raised and used to determine by indirect immunofluorescence the cellular localization of the protein in Chlamydomonas cells. In interphase cells the antibody stained intensely the region between the paired basal bodies, two fibers extending between the basal bodies and the underlying nucleus, and an array of longitudinal filaments surrounding the nucleus. The two basal body-nuclear connecting fibers were identified in thin-section electron micrographs to be narrow striated fiber roots. In mitotic cells the 20-kD CaBP was specifically associated with the poles of the mitotic spindle at the sites of the duplicated basal body complexes.     

Binding of microtubule-associated protein 2 and tau to the intermediate filament reassembled from neurofilament 70-kDa subunit protein. Its regulation by calmodulin

Two major brain microtubule-associated proteins (MAPs), MAP2 and tau, were found to bind to the intermediate filaments reassembled from neurofilament 70-kDa subunit protein (= 70-kDa filaments). The binding was saturable. The apparent dissociation constant (KD) for the binding of MAP2 to the 70-kDa filaments was estimated to be 4.8 X 10(-7) M, and the maximum binding reached 1 mol of MAP2/approximately 30 mol of 70-kDa protein. The apparent KD for the tau binding was 1.6 X 10(-6) M, and the maximum binding was 1 mol of tau/approximately 3 mol of 70-kDa protein. It was also found that MAP2 and tau did not compete with each other for binding to the 70-kDa filaments. Most interestingly, calmodulin, a ubiquitous Ca2+-binding protein in eukaryotic cells, was found to inhibit the binding of MAP2 and tau to the 70-kDa filaments. The inhibition by calmodulin was regulated by changes in Ca2+ concentration around 10(-6) M, and was canceled by trifluoperazine, a calmodulin inhibitor.     

Ca2+-mediated phosphorylation and proteolysis activity associated with the cytoskeletal fraction from cerebral cortex of rats

We describe a Triton-insoluble cytoskeletal fraction extracted from cerebral cortex of young rats retaining an endogenous Ca²⁺-mediated mechanism acting in vitro on Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII) activity and on phosphorylation and proteolysis of the 150 kDa neurofilament subunit (NF-M), α and β tubulin. Exogenous Ca²⁺ induced a 70% decrease in the in vitro phosphorylation of the NF-M and tubulins and a 30–50% decrease in the total amount of these proteins. However, when calpastatin was added basal phosphorylation and NF-M and tubulin content were recovered. Furthermore, exogenous Ca²⁺/calmodulin induced increased in vitro phosphorylation of the cytoskeletal proteins and CaM-KII activity only in the presence of calpastatin, suggesting the presence of Ca²⁺-induced calpain-mediated proteolysis. This fraction could be an interesting model to further studies concerning the in vitro effects of Ca²⁺-mediated protein kinases and proteases associated with the cytoskeletal fraction.     

Control by the extracellular environment of differentiation pathways in 1003 embryonal carcinoma cells: study at the level of specific intermediate filaments.

1003 is a multipotential embryonal carcinoma (EC) clonal cell line which can be induced to follow different developmental pathways by altering the composition of the culture medium. When grown in serum-containing medium the great majority of 1003 cells remain undifferentiated; they express the ECMA 7 cell-surface embryonic antigen and very low amounts of vimentin. In serum-free medium, most 1003 cells differentiate into neuroepithelial cells. The majority of these cells are still labelled with ECMA 7 antibodies. They contain higher amounts of vimentin than EC cells, but no neurofilament proteins. Neuroepithelial cells then differentiate into neurons through a stage of preneurons containing both vimentin and the 70-K neurofilament protein. Fully differentiated neurons contain 70-K neurofilament protein but no vimentin. The 200-K neurofilament protein is detected later in the neurons. Mesenchymal cells (induced by re-adding serum) express high amounts of vimentin organized in networks. Preneurons , neurons, and mesenchymal cells do not express ECMA 7 antigen.     

Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain.

The degradation of phosphorylated and dephosphorylated neurofilament proteins by the Ca2+-activated neutral proteinase calpain was studied. Neurofilaments were isolated from bovine spinal cord, dephosphorylated by alkaline phosphatase (from Escherichia coli) and radioiodinated with [125I]-Bolton-Hunter reagent. The radioiodinated neurofilament proteins (untreated and dephosphorylated) were incubated in the presence and absence of calpain from rabbit skeletal muscle, and the degradation rates of large (NF-H), mid-sized (NF-M) and small (NF-L) neurofilament polypeptides were analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. The degradation of dephosphorylated neurofilament proteins occurred at a higher rate, and to a greater extent, than did that of the phosphorylated (untreated) neurofilament proteins. The dephosphorylated high-molecular-mass neurofilament (NF-HD) was proteolyzed 6 times more quickly than the untreated NF-H. The degradation rate of the NF-M and NF-L neurofilament proteins was also enhanced after dephosphorylation, but less than that of NF-H. This indicates that the dephosphorylation of neurofilament proteins can increase their sensitivity to calpain degradation.     

Production of a unique antibody specific for membrane ruffles and its use to characterize the behavior of two distinct types of ruffles

I have produced a new monoclonal antibody, YF-169, against membrane ruffle specific 55-kD protein. YF-169 stained membrane ruffles of chick embryo fibroblasts so definitely that it enabled clear and reliable analyses of membrane ruffles. Fibroblasts organized two distinct types of membrane ruffles. One type of the ruffles were transiently formed in serum-starved cells (Type I) when stimulated by serum or platelet- derived growth factor. After spontaneous degradation of Type I ruffles, the other type of ruffles containing many microspikes were gradually organized at leading edges (Type II). The formation of Type I ruffles was not affected by either nocodazole, a microtubule-disrupting drug, or taxol, a microtubule-stabilizing reagent. However, Type II ruffles were entirely destroyed not only by nocodazole but also by taxol, suggesting that regulated organization of microtubule network is important to maintain Type II ruffles. H8, a protein kinase inhibitor prevented the spontaneous degradation of Type I ruffles and also reduced the destructive effect of nocodazole on Type II ruffles without affecting microtubule-disrupting activity. Protein kinases may be involved in the degradation processes of both types of ruffles. W7, a calmodulin antagonist, strongly inhibited Type I ruffle formation and completely destroyed Type II ruffles. W7 was also found to induce a remarkable change of 55-kD protein localization. After degradation of Type II ruffles, most of 55-kD protein was incorporated into newly formed unusual thick fibers. These results suggest that regulated organization of microtubule network is not necessary to form Type I ruffles but is important to maintain Type II ruffles, while calmodulin function is essential for both types of membrane ruffles.     

Identification of a novel member of yeast mitochondrial Hsp70-associated motor and chaperone proteins that facilitates protein translocation across the inner membrane

Here, we report the identification of yeast 15-kD Tim15/Zim17, a new member of mitochondrial Hsp70 (mtHsp70)-associated motor and chaperone (MMC) proteins. The 15-kD MMC protein is a peripheral inner membrane protein with a zinc-finger motif. Depletion of the 15-kD protein led to impaired import of presequence-containing proteins into the matrix in vivo and in vitro. Overexpression of the 15-kD protein rescued the functional defects of mtHsp70 in ssc1-3 cells, and a fusion protein containing the 15-kD protein physically interacts with purified mtHsp70. Tim15/Zim17 therefore cooperates with mtHsp70 to facilitate import of presequence-containing proteins into the matrix.     

Immunohistochemical differences between neurofilaments in perikarya, dendrites and axons : Immunofluorescence study with antisera raised to neurofilament polypeptides (200K, 150K, 70K) isolated by anion exchange chromatography

Neurofilament (NF) proteins (70K, 150K and 200K D) were isolated from 2 M urea extracts of bovine spinal cord by anion exchange chromatography. Antisera to the individual NF polypeptides were produced in rabbits and affinity-purified on Sepharose columns prepared with their own antigen. The NF antisera were completely absorbed by their own antigen at protein concentrations that did not decrease the staining when the absorption was conducted with the heterologous NF antigens. Partial absorption (decrease in immunofluorescence titer) occurred at higher concentrations of the heterologous antigens. Cross-reactivity between the polypeptides of the NF triplet could not be detected by double immunodiffusion. The antisera formed immunoprecipitin lines only when reacted with their own antigen. Conversely, cross-reactivity was demonstrated by the immune blotting procedure. Anti-70K stained all three NF polypeptides. Anti-200K and anti-150K stained both 200K and 150K but not 70K, the main reaction being with their own antigen. The antisera were rendered monospecific by adsorption of the common antigenic determinants on Sepharose columns prepared with the heterologous NF antigens. The localization of the NF proteins was studied by immunofluorescence on cryostat sections of rat brain, cerebellum, spinal cord and posterior root ganglia. All NF antisera (anti-70K, anti-150K and anti-200K) stained axons including Purkinje cell baskets with identical pattern. Spinal cord motor neurons, posterior root ganglia neurons and pyramidal neurons in the cerebral cortex stained with anti-70K and anti-200K. No staining of neuronal perikarya and dendrites was observed with anti-150K. Aluminium-induced neurofibrillary tangles in rabbit spinal cord stained with anti-70K and anti-200K. The tangles were not decorated by anti-150K. It is concluded that a marked difference exists in the concentration of 150K depending on the location, i.e., cell body or axon; or, alternatively, that 150K undergoes modification of antigenic sites within the axon so that it may not be recognized immunologically as a component of the neurofilament within perikarya and dendrites.     

The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state

Treatment of freshly isolated, bovine neurofilaments with Escherichia coli alkaline phosphatase removes over 90% of the phosphate groups from serine residues of the Mr 200,000 and 150,000 polypeptide components (NF200 and NF150). Dephosphorylated NF200 and NF150 remain associated with filaments, but migrate in sodium dodecyl sulfate gels with reduced apparent molecular weights. Unusual migration appears to be due to modification at regions of these polypeptides that are peripheral to the neurofilament backbone as defined by limited chymotryptic digestion. Over 90 monoclonal antibodies recognizing epitopes located within the peripheral domain of native NF200 all show reduced affinity for dephosphorylated NF200. A single monoclonal antibody binds within the filament-associated domain of NF200 and its recognition of NF200 is unaffected upon treatment of neurofilaments with phosphatase. Around 50% of our monoclonal antibodies that bind NF150 monospecifically and at epitopes within its peripheral domain have reduced affinities for NF150 from phosphatase-treated filaments, while the remaining 50% bind native and dephosphorylated NF150 equally well. The smallest neurofilament component (NF70) contains few phosphate groups, most of which remain after treatment of neurofilaments with phosphatase. The resulting form of NF70 migrates normally in gels and its recognition by antibodies is unchanged. We conclude that phosphorylation modifies the structure of the two larger neurofilament polypeptides along domains that are peripheral to the filamentous backbone and that these effects are more pronounced for NF200 than for NF150.