Immunocytochemical study of the distribution of cytoskeletal proteins in neurons from mouse embryo spinal cord in culture

Monoclonal FITC and Rhodamine-labeled antibodies were used to investigate distribution of tubulin, actin, and neurofilament protein with a molecular weight of 160 kDa in neurons from mouse embryo spinal cord cultivated in a monolayer. It was found that migration of tubulin from the neuronal soma occurs during the course of nerve cell development. The extent to which neuronal processes became filled with tubulin varied according to the stage of cell differentiation. Neuronal actin concentration was negligible and was found at points along the processes close to focal contacts with lining cells. The main body of neurofilament protein is concentrated in the neuronal soma and proximal portions of neurites.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 5, pp. 618–623, 1988.     

Immunocytochemical studies of intermediate filament aggregates and their relationship to microtubules in cultured skin fibroblasts from patients with giant axonal neuropathy

Giant axonal neuropathy (GAN) is a severe autosomal recessive disease affecting both the peripheral and central nervous systems. It is characterized by segmental axonal ballooning due to large neurofilamentous masses and abnormal aggregation of filaments in other cell types including glial cells. Coomassie blue staining of the detergent-resistant cytoskeleton of cultured skin fibroblasts from three patients with GAN revealed the presence of large cytoplasmic filamentous aggregates in the great majority of cells. The aggregates were birefringent when viewed under polarization microscopy and electron microscopy showed that they were composed of aggregates of 8 to 10 nm intermediate filaments. The aggregates stained with antisera specific for vimentin but did not stain with antibodies to actin, tubulin, or the high molecular weight (HMW) microtubule associated protein. Examination of the fibroblasts containing the vimentin aggregates with antibodies to tubulin and the HMW protein showed that they had a normal distribution of microtubules and that the microtubules present were normally associated with the HMW protein. The results suggest that giant axonal neuropathy is a generalized inborn error of organization of intermediate filaments and that a defect in microtubules or their association with HMW protein is not responsible for the observed aggregation of intermediate filaments in this disease. Further study of GAN may be useful in understanding the function of intermediate filaments.     

Changes in Organization and Axonal Transport of Cytoskeletal Proteins During Regeneration

Changes in solubility and transport rate of cytoskeletal proteins during regeneration were studied in the motor fibers of the rat sciatic nerve. Nerves were injured by freezing at the midthigh level either 1-2 weeks before (experiment I) or 1 week after radioactive labeling of the spinal cord with L-[35S]methionine (experiment II). Labeled proteins in 6-mm consecutive segments of the nerve 2 weeks after labeling were analyzed following fractionation into soluble and insoluble populations with 1% Triton at 4 degrees C. When axonal transport of newly synthesized cytoskeleton was examined in the regenerating nerve in experiment I, a new faster component enriched in soluble tubulin and actin was observed that was not present in the control nerve. The rate of the slower main component containing most of the insoluble tubulin and actin together with neurofilament proteins was not affected. A smaller but significant peak of radioactivity enriched in soluble tubulin and actin was also detected ahead of the main peak when the response of the preexisting cytoskeleton was examined in experiment II. It is thus concluded that during regeneration changes in the organization take place in both the newly synthesized and the preexisting axonal cytoskeleton, resulting in a selective acceleration in rate of transport of soluble tubulin and actin.     

Okadaic Acid Induces Early Changes in Microtubule-Associated Protein 2 and γ Phosphorylation Prior to Neurodegeneration in Cultured Cortical Neurons

Abstract: Microtubules and their associated proteins play a prominent role in many physiological and morphological aspects of brain function. Abnormal deposition of the microtubule-associated proteins (MAPs), MAP2 and γ , is a prominent aspect of Alzheimer's disease. MAP2 and γ are heat-stable phosphoproteins subject to high rates of phosphorylation/dephosphorylation. The phosphorylation state of these proteins modulates their affinity for tubulin and thereby affects the structure of the neuronal cytoskeleton. The dinoflagellate toxin okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A. In cultured rat cortical neurons and a human neuroblastoma cell line (MSN), okadaic acid induces increased phosphorylation of MAP2 and γ concomitant with early changes in the neuronal cytoskeleton and ultimately leads to cell death. These results suggest that the diminished rate of MAP2 and γ dephosphorylation affects the stability of the neuronal cytoskeleton. The effect of okadaic acid was not restricted to neurons. Astrocytes stained with antibodies to glial fibrillary acidic protein (GFAP) showed increased GFAP staining and changes in astrocyte morphology from a flat shape to a stellate appearance with long processes.     

A calcium-activated protease which preferentially degrades the 160-kDa component of the neurofilament triplet

A calcium-dependent protease fully active with 0.2 mM Ca2+ was found associated with the neurofilament-enriched cytoskeleton of the rat spinal cord prepared by the treatment with Triton X-100. The enzyme preferentially degrades the 160-kDa component of the neurofilament triplet. In addition, a soluble calcium-dependent protease activity was found in the supernatant from the spinal cord, which degraded a variety of cytoskeletal proteins including the neurofilament triplet, glial fibrillary acidic (GFA) protein, actin, tubulin, and a high molecular weight protein associated with microtubules. The possibility that the cytoskeleton-bound activity is an artefactual association of the soluble enzyme to the cytoskeleton seems to be negated on the basis of the following dissociation and reassociation experiments. The protease activity remained associated with the cytoskeleton in the physiological ionic strength, and was not completely dissociated from it until the KC1 concentration was raised to 0.6 M. When the 0.6 M KCl-extract was dialysed against salt-free buffer to remove KC1, and added back to the protease-free cytoskeletal pellet, proteolytic activity was partially restored. Full activity returned only when the extract and the protease-free cytoskeletal pellet were first combined in the presence of 0.6 M KC1, and then slowly reassociated by dialysis against salt-free buffer. Dissociated enzyme was rapidly inactivated at 37 degrees C in the presence of Ca2+. These results suggest the structural association of the protease with the cytoskeleton under the physiological condition.     

In vivo and in vitro characterization of novel neuronal plasticity factors identified following spinal cord injury

Following spinal cord injury, there are numerous changes in gene expression that appear to contribute to either neurodegeneration or reparative processes. We utilized high density oligonucleotide microarrays to examine temporal gene profile changes after spinal cord injury in rats with the goal of identifying novel factors involved in neural plasticity. By comparing mRNA changes that were coordinately regulated over time with genes previously implicated in nerve regeneration or plasticity, we found a gene cluster whose members are involved in cell adhesion processes, synaptic plasticity, and/or cytoskeleton remodeling. This group, which included the small GTPase Rab13 and actin-binding protein Coronin 1b, showed significantly increased mRNA expression from 7-28 days after trauma. Overexpression in vitro using PC-12, neuroblastoma, and DRG neurons demonstrated that these genes enhance neurite outgrowth. Moreover, RNAi gene silencing for Coronin 1b or Rab13 in NGF-treated PC-12 cells markedly reduced neurite outgrowth. Coronin 1b and Rab13 proteins were expressed in cultured DRG neurons at the cortical cytoskeleton, and at growth cones along with the pro-plasticity/regeneration protein GAP-43. Finally, Coronin 1b and Rab13 were induced in the injured spinal cord, where they were also co-expressed with GAP-43 in neurons and axons. Modulation of these proteins may provide novel targets for facilitating restorative processes after spinal cord injury.     

Nerve fibers in culture and their interactions with non-neural cells visualized by immunofluorescence

Cultures of embryonic mouse spinal cord explants, alone or in combination with rat myotubes, were stained by indirect immunofluorescence using antibodies against three structural proteins to: (a) reveal the distribution of these proteins among different cell types, and (b) test the usefulness of antibody staining to reveal the gross morphology of the neurite network in complex cultures. Affinity column purified antibodies were used against chicken gizzard actin, porcine brain tubulin, and skeletal muscle alpha-actinin. Neurites were stained intensely by anti-actin as was the stress fiber pattern of underlying fibroblasts. With anti-tubulin, the staining of neurites was an order of magnitude more intense than the staining of the microtubule pattern of background fibroblasts. Neurite cell bodies and astrocyte-like glia cells were stained with anti-tubulin and their nuclei remained unstained. Anti-tubulin could thus be used to trace even the finest extensions of nerve processes in spinal cord and spinal cord-muscle cultures. Furthermore, it could be combined with the histochemical reaction for acetylcholinesterase (AChE, EC 3.1.1.7) to demonstrate AChE-positive neurons and specialized nerve-muscle contact sites. The staining of neural elements with anti-alpha-actinin was generally much weaker than with anti-actin and anti-tubulin. Neurites were stained only moderately in comparison to myotube Z lines in the same culture. However, a distinct staining of the periphery of dorsal root ganglion cells was observed. Thus, a protein immunologically related to muscle alpha-actinin is present in the nervous system. In myotubes, Z lines were stained intensely with anti-alpha-actinin while I bands were only faintly stained with anti-actin. In isolated myofibrils, both structures were stained intensely with the same antibody preparations.     

Effects of abscisic acid,low temperature,and plant age on cytoskeleton and phosphorylated proteins

The effects of exogenous abscisic acid (ABA), low temperature, and seedling age on the content of tubulin, actin, and phosphorylated proteins and the structural organization of microtubules (MTs) in cells of different tissues and organs of winter wheat cultivars contrasting in cold hardiness were studied by immunocytochemical methods using monoclonal (against - and -tubulin and actin) and polyclonal (phosphothreonine) antibodies. The leaves and roots of five- and nine- day-old seedlings of three cultivars were characterized by unequal proportion of actin/tubulin proteins. ABA decreased the content of the cytoskeleton and the 60-kD phosphorylated proteins, thus promoting a decrease in the number of MTs and occurrence of a less branched network of weakly fluorescent tubulin components in the cells of the root differentiating zone (which is most responsible for the development of cold hardiness in wheat). Although the cold acclimation of plants (3°C, 7 days) did not change the level of tubulin and actin proteins, it evoked the spatial aggregation of MT, leading to formation of a dense network of tubulin cytoskeleton comprised of thick bundles of intensively fluorescent MTs. In the case of a combined action of the studied factors, low temperatures abolished the hormone effect described above, evoking an increase in the content of the cytoskeletal and 60-kD phosphorylated proteins and MT structures. We suggest that the ABA-induced decrease in the levels of proteins and MTs occurs at the initial stages of plant cold acclimation (3°C, 2-3 days). It may be the signal that triggers the processes of low-temperature adaptation. As the duration of cold acclimation increased (3°C, 7 days), the role of ABA in the formation of plant tolerance decreased. Apparently, in this case other hormone-independent mechanisms of frost hardiness development are triggered, in which the role of the cytoskeleton components and cytoskeleton-associated proteins increases.     

Effects of methylmercury on retinoic acid-induced neuroectodermal derivatives of embryonal carcinoma cells

Immunofluorescence staining with antibodies to tubulin, neurofilaments and glial filaments was used to study the effects of methylmercury on the differentiation of retinoic acid-induced embryonal carcinoma cells into neurons and astroglia and on the cytoskeleton of these neuroectodermal derivatives. Methylmercury did not prevent undifferentiated embryonal carcinoma cells from developing into neurons and glia. Treatment of committed embryonal carcinoma cells with methylmercury doses exceeding 1 M resulted in the formation of neurons with abnormal morphologies. In differentiated cultures, microtubules were the first cytoskeletal element to be affected. Their disassembly was time- and concentration-dependent. Microtubules in glial cells and in neuronal perikarya were more sensitive than those in neuronal processes. Neurofilaments and glial filaments appeared relatively insensitive to methylmercury treatment but showed reorganization after complete disassembly of the microtubules. The data demonstrate 1) the sensitivity of microtubules of both neurons and glia to methylmercury-induced depolymerization, and 2) the heterogeneous response of neuronalAbbreviations -MEM alpha minimal essential medium - EC embryonal carcinoma cells - FCS fetal calf serum - MAP microtubule-associated protein - MeHg methylmercury - RA retinoic acid     

Polarized distribution of Mr 210,000 and 190,000 analogs of erythrocyte ankyrin along the plasma membrane of transporting epithelia, neurons and photoreceptors

In the present study we have examined several types of nucleated cells with respect to the occurrence and subcellular distribution of ankyrin. In red blood cells ankyrin links and integral membrane protein, the anion channel (band 3), to the subplasmalemmal cytoskeleton which is comprised largely of spectrin and actin. Since nucleated cells also contain spectrin and other constituents of the erythrocyte membrane skeleton it is possible that in nonerythroid cells ankyrin is also important for connecting membrane proteins to the cytoskeleton. We show here that membrane fractions of rat brain and various types of rat epithelial cells contain analogs of ankyrin at Mr 210,000 and 190,000 that are immunologically related to human erythrocyte ankyrin. In transporting epithelial cells, such as epithelia of the intestine, pancreas, prostate or kidney (various species) the analogs of ankyrin are confined to the basolateral plasma membrane and are absent from the apical membrane. In neurons of the central and peripheral nervous system and in photoreceptors of the retina, ankyrin was found restricted to the membrane of the cell body and axons and was not detected by immunostaining along the afferent processes (dendrites, photoreceptor inner and outer segments). Linkage of integral membrane proteins via ankyrin to the spectrin-based membrane cytoskeleton may provide a molecular basis for restricting the lateral mobility of certain membrane proteins and localizing them in a nonrandom or polarized fashion at specialized domains of the plasma membrane.     

The Cytoskeleton of Primary Astrocytes in Culture Contains Actin, Glial Fibrillary Acidic Protein, and the Fibroblast-Type Filament Protein, Vimentin

Abstract: Primary astrocytes were cultured from the forebrains of 1-day-old rats. Immunofluorescence microscopy showed that approximately 80% of the cells were positive for glial fibrillary acidic protein (GFAP) and >80% were stained with an antiserum to the molecular weight 58,000 fibroblast intermediate filament protein (vimentin). Gel electrophoresis of Triton-insoluble cytoskeleton preparations from these cultures revealed three major bands having molecular weights of 58,000, 51,000, and 42,000, together with some prominent lower-molecular-weight species. The protein of molecular weight 51,000 was not present in preparations from fibroblasts. Each of the three major astrocyte proteins was subjected to limited proteolysis, while two of the proteins were cleaved by cyanogen bromide. The electrophoretic peptide patterns of the 58,000 protein were similar to those of vimentin isolated from NIL-8 fibroblasts, and the patterns of the 51,000 protein were similar to those of GFAP isolated from rat spinal cord. The patterns of the protein of molecular weight 42,000 resembled those of muscle actin. Rocket immunoelectrophoresis showed that the 51,000 astrocyte protein reacted with an antiserum to bovine GFAP, but the 58,000 and 42,000 proteins failed to react. We conclude that the major proteins of cytoskeleton preparations from cultured primary astrocytes are vimentin (58,000), GFAP (51,000), and actin (42,000), and that our data show no obvious structural relationship among them.     

Subpopulations of tau interact with microtubules and actin filaments in various cell types

It has been demonstrated that microtubule-associated proteins (MAPs) interact with tubulin in vitro and in vivo. However, there is no clear evidence on the possible roles of the interactions of MAPs in vivo with other cytoskeletal components in maintaining the integrity of the cell architecture. To address this question we extracted the neuronal cytoskeleton from brain cells and studied the selective dissociation of specific molecular isospecies of tau protein under various experimental conditions. Tau, and in some cases MPA-2, were analysed by the use of anti-idiotypic antibodies that recognize epitopes on their tubulin binding sites. Fractions of microtubule-bound tau isoforms were extracted with 0.35 M NaCl or after the addition of nocodazole to allow microtubule depolymerization. Protein eluted with this inhibitor contained most of the assembled tubulin dimer pool and part of the remaining tau and MAP-2. When the remaining cytoskeletal pellet was treated with cytochalasin D to allow depolymerization of actin filaments, only tau isoforms were extracted. Immunoprecipitation studies along with immunolocalization experiments in cell lines containing tau-like components supported the findings on the roles of tau isospecies as linkers between tubulin in the microtubular structure with actin filaments. Interestingly, in certain types of cells, antibody-reactive tau isospecies were detected by immunofluorescence with a discrete distribution pattern along actin filaments, which was affected by cytochalasin disruption of the actin filament network. These results suggest the possible in vivo roles of subsets of tau protein in modulating the interactions between microtubules and actin filaments.     

Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8

Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1) and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.     

In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.     

58,000 Dalton Intermediate Filament Proteins of Neuronal and Nonneuronal Origin in the Goldfish Visual Pathway

A group of proteins in the goldfish optic nerve with a molecular weight of 58K daltons was analyzed by two-dimensional gel electrophoresis. Results show that the proteins are differentially phosphorylated and found exclusively in a cytoskeletal-enriched fraction. The proteins from this fraction can be reconstituted into typical intermediate filament structures, as shown by electron microscopy. Two components which are of neuronal origin are transported within the slow phase of transport. The 58K proteins are the most abundant proteins in the optic nerve, and they are distinct from actin and tubulin. It was concluded that they are intermediate filament proteins. Cytoskeletal preparations of rat spinal cord, rat optic nerve, and goldfish optic nerve were compared by one-dimensional gel electrophoresis. The rat spinal cord contains glial fibrillary acidic protein (GFAP), and the rat optic nerve contains vimentin and GFAP, in addition to the neurofilament triplet. A typical mammalian neurofilament triplet is not detected in the goldfish optic nerve, while the major cytoskeletal constituent is a 58K band which coelectrophoreses with vimentin in the rat optic nerve by one-dimensional gel electrophoresis.     

Keratin 8 of simple epithelia is expressed in glia of the goldfish nervous system

The intermediate filament protein composition in glial cells of goldfish optic nerve differs from that found in glial cells of the goldfish spinal cord and brain. Brain and spinal cord glial cells contain glial fibrillary acidic protein (GFAP), whereas glial cells in the optic nerve contain ON3. The ON3 protein of the goldfish optic nerve was recently identified as the goldfish equivalent to the mammalian type II keratin 8 protein. In addition to the ON3 protein, the goldfish optic nerve also contains a 48-kDa protein. Immunoblotting experiments suggest that this protein is equivalent to the mammalian type I keratin 18 protein, which typically pairs with keratin 8 to form filaments. We show that these proteins are not specific to the optic nerve. The ON3 and 48-kDa proteins of the goldfish optic nerve share common antigenic properties with the predominant keratin pair expressed in the goldfish liver. These proteins are also expressed at low levels in the goldfish brain and spinal cord. In addition RNase protection assays and Northern blots indicate that the mRNA for the ON3 protein in optic nerve is identical to the message found in other goldfish tissues. The expression of ON3 was also examined in cultured glial cells from goldfish spinal cord and optic nerve and cultured fibroblast cells. Analysis of intermediate filament protein expression in cultured glial cells taken from goldfish spinal cord demonstrated the absence of GFAP in these cells and the expression of ON3. This protein was also the predominant intermediate filament protein of cultured optic nerve glial cells and fibroblasts. The differences in the expression of intermediate filament proteins in mammals and lower vertebrates are discussed. In addition, we discuss how the expression of a simple epithelial keratin pair in glial cells of the goldfish optic nerve may be associated with this system's capacity for continuous growth and regeneration.     

ABRA在不同年龄大鼠腰段脊髓中的表达变化

目的检测Actin binding Rho activator（ABRA）在不同年龄大鼠腰段脊髓中的表达变化。方法采用Western blot定量检测不同年龄大鼠腰段脊髓中ABRA蛋白水平表达变化,采用免疫荧光染色显示不同年龄大鼠腰髓中ABRA细胞定位。结果Western blot显示ABRA在新生鼠腰段脊髓中表达显著高于成年鼠及老年鼠。免疫荧光染色显示ABRA广泛表达于神经元的胞核、胞浆和突起,在腰髓前角,与前角运动神经元存在共定位,在腰髓后角,与小的NeuN阳性感觉神经元存在共定位。腰髓前角、后角的阳性细胞计数均显示新生鼠ABRA＋NeuN双阳性细胞占总ABRA阳性细胞百分比显著低于成年鼠及老年鼠。结论ABRA广泛表达于腰髓中的神经元,ABRA在新生鼠腰髓中表达最强,随年龄的增长呈现明显的时相变化,提示ABRA可能参与了腰髓中神经元的发育和成熟。     

Electrophoretic pattern and distribution of cytoskeletal proteins in flat-epitheloid and stellate process-bearing astrocytes in primary culture

One- and two-dimensional electrophoresis patterns and distribution of major cytoskeletal proteins were studied in primary astrocytes with either flat-epitheloid or stellate appearance. No major differences in the electrophoretic patterns of actin, tubulin, glial fibrillary acidic protein (GFAP) and vimentin were detected between flat-epitheloid and stellate process-bearing astrocytes produced by the exposure of cultures to dibutyryl cyclic AMP (dBcAMP). However the morphological changes of astrocytes were accompanied by marked changes in the quantitative distribution of cytoskeletal proteins. The most prominent change was a large and specific decrease in the amount of actin, detected by [³⁵S]methionine incorporation, densitometric scanning of one-dimensional gels and DNase inhibition assay. In stellate astrocytes produced by a 4 day treatment with dibutyryl cyclic AMP, the amount of actin decreased by 50%. This decrease was not apparently related to the depolymerization of actin.