Study of the 10-nm-filament fraction isolated during the standard microtubule preparation.

The cold non-depolymerizable fractions obtained during the standard procedure for the isolation of microtubules from ox brain stem-cerebral hemispheres and spinal cord have been studied. The cerebral-hemisphere preparation was composed of 10-nm filaments but also contained large amounts of membranes. The polypeptide content included tubulin, microtubule-associated proteins and minor proteins corresponding to the neurofilament triplet of proteins of mol.wt. 210 000, 160 000 and 70 000 respectively. The brain-stem preparation contained more 10-nm filaments than membranes. The polypeptide content consisted of the neurofilament triplet (35%), tubulin (30%) and minor proteins. In contrast, the spinal-cord preparation was mainly composed of 10-nm filaments, free of membranes and containing essentially the neurofilament protein triplet (64%). These filaments appeared very similar to the peripheral-nervous-system neurofilaments described by several authors. Since the best neurofilament from the central nervous system often contained less than 15% of the neurofilament protein triplet, our spinal-cord preparation is an improvement on the usual neurofilament preparation. This simple and rapid method gave large amounts of 10-nm filaments (100 mg per 100 g of spinal cord) characterized by the absence of membranous material, a low content of tubulin and the 50 000-mol.wt.-protein component, and a high content of neurofilament peptides. Thus, the presence of tubulin in 10-nm filament preparations seems to be related to the contaminant membranous material and not to be linked to the interaction in vitro of tubulin or microtubules with neurofilaments, as has been suggested previously.     

The structure of postsynaptic densities isolated from dog cerebral cortex: I. overall morphology and protein composition

A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of methods of Davis and Bloom (21, 22) and Cotman and Taylor (20), using Triton X-100. The fraction has been visualized by thin-section, replica, and negative (phosphotungstic acid) staining electron microscopy and its proteins separated by high-resoltuion SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nuclei acids, and very little phospholipids being detectable. The fraction had no ATPase or GTPase activity, but it did have a very small amount of cytochrome c oxidase activity (of a specific activity less than 0.5 percent that of a mitochondrial fraction) and a small amount of 5'- nucleotidase activity (of a specific activity between 6 and 7 percent that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures approximately 400nm long and approximately 40nm wide, made up of apparent particles 13-28nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter approximately 400 nm, in which filaments are seen to extend from the central part of the density. High resolution gel electrophoresis studies indicated some 15 major proteins and perhaps 10 or more minor ones; the predominant protein had a mol wt of 51,000, followed by ones at 45,000, 40,000, 31,000, 26,000, and several at 100,000. A comparison by gel electrophoresis of density fraction proteins with those of a lysed synaptosomal membrane fraction containing some adherent densities indicated some comigrating proteins, but the major membrane fraction protein, mol wt 52,000, was not found in the density fraction. Antibodies raised against the density fraction reacted with a preparation of solubilized synaptic membrane proteins. By both these criteria, it was considered that the density and the synaptic membrane have some proteins in common. By separately mixing (125)I-labeled myelin, synaptic vesicle, and mitochondrial fraction proteins with synaptosomes, and then isolating the density fraction from the mixture, it was concluded that a major 26,000 mol wt density fraction protein was common to both mitochondria and density, that none of the proteins of the density were contaminants from the mitochondrial fraction, that a minor approximately 150,000 band was a contaminant from the synaptic vesicle fraction, and that the moderately staining PSD fraction protein of 17,000 mol wt band was the result of contamination by the major basic protein of myelin. On the basis of the marker enzymatic assays and the mixing experiments, it is considered that the density fraction is moderately pure biochemically, and that its protein composition, aside from a few exceptions noted above, reflects its in situ character.     

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.     

Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells

The bundle of filaments within microvilli of intestinal epithelial cells contains five major proteins including actin, calmodulin, and subunits of 105-, 95-, and 70-kdaltons. It has been previously shown (Howe, C. L., M. S. Mooseker, and T. A. Graves. 1980. Brush-border calmodulin: a major component of the isolated microvillus core. J. Cell Biol. 85: 916-923) that the addition of Ca++ (> 10(-6) M) to microvillus cores causes a rapid, drastic, but at least partially reversible disruption of this actin filament bundle. High-speed centrifugation of microvillus cores treated with Ca++ indicates that several core proteins are solubilized, including 30-50% of the actin and calmodulin, along with much of the 95- and 70-kdalton subunits. Gel filtration of such Ca++ extracts in the presence and absence of Ca++ indicates that microvillar actin "solated" by Ca++ is in an oligomeric state probably complexed with the 95-kdalton subunit. Removal of Ca++ results in the reassembly of F-actin, probably still complexed with 95- kdalton subunit, as determined by gel filtration, cosedimentation, viscometry, and electron microscopy. The 95-kdalton subunit (95K) was purified from Ca++ extracts by DEAE-Sephadex chromatography and its interaction with actin characterized by viscometry, cosedimentation, and EM in the presence and absence of Ca++. In the presence, but not absence, of Ca++, 95K inhibits actin assembly (50% inhibition at 1:50- 60 95K to actin) and also reduces the viscosity of F-actin solutions. Similarly, sedimentation of actin is inhibited by 95K, but a small, presumably oligomeric actin- 95K complex formed in the presence of Ca++ is pelletable after long-term centrifugation. In the absence of Ca++, 95K cosediments with F-actin. EM of 95K-actin mixtures reveals that 95K "breaks" actin into small, filamentous fragments in the presence of Ca++. Reassembly of filaments occurs once Ca++ is removed. In the absence of Ca++, 95K has no effect on filament structure and, at relatively high ratios (1:2-6) of 95K to actin, this core protein will aggregate actin filaments into bundles.     

Intermediate filaments in nervous tissues

Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics.     

Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments

When Limulus sperm are induced to undergo the acrosomal reaction, a process, 50 mum in length, is generated in a few seconds. This process rotates as it elongates; thus the acrosomal process literally screws through the jelly of the egg. Within the process is a bundle of filaments which before induction are coiled up inside the sperm. The filament bundle exists in three stable states in the sperm. One of the states can be isolated in pure form. It is composed of only three proteins whose molecular weights (mol wt) are 43,000, 55,000, and 95,000. The 43,000 mol wt protein is actin, based on its molecular weight, net charge, morphology, G-F transformation, and heavy meromyosin (HMM) binding. The 55,000 mol wt protein is in equimolar ratio to actin and is not tubulin, binds tenaciously to actin, and inhibits HMM binding. Evidence is presented that both the 55,000 mol wt protein and the 95,000 mol wt protein (possibly alpha-actinin) are also present in Limulus muscle. Presumably these proteins function in the sperm in holding the actin filaments together. Before the acrosomal reaction, the actin filaments are twisted over one another in a supercoil; when the reaction is completed, the filaments lie parallel to each other and form an actin paracrystal. This change in their packing appears to give rise to the motion of the acrosomal process and is under the control of the 55,000 mol wt protein and the 95,000 mol wt protein.     

The Postsynaptic Density: Constituent and Associated Proteins Characterized by Electrophoresis, Immunoblotting, and Peptide Sequencing

The proteins of the postsynaptic density (PSD) fraction of cerebral cortex were resolved by two-dimensional electrophoresis (2DE) and more than 30 proteins identified by characteristic 2DE mobility, immunoblotting with specific antibodies, and N-terminal and peptide sequencing. The PSD fraction is enriched for spectrin, actin, tublin and microtubule associated protein II, myosin, enzymes of glycolysis, creatine kinase, elongation factor 1 alpha, and receptor protein. The three neurofilament proteins are detected but a 58-kDa protein is prominent and is, by peptide sequencing, the bovine homolog of the recently cloned 66-kDa neurofilament protein; in contrast to the latter, however, it is enriched in cerebrum compared with spinal cord. A 68-kDa protein is identified as a member of the hsp70/BiP family of proteins. A protein, designated dynamin, indicating its putative role as a microtubule motor, is identified as a major protein, is found, however, greatly enriched in the particulate fraction, and is significantly denaturant and detergent insoluble. A protein designated N-ethylmaleimide-sensitive factor is also detected. Thus, two proteins implicated in vesicular transport are present in the PSD fraction. Seven polyclonal antibodies were produced to 2DE separated and electroeluted proteins of the PSD and were identified by peptide sequence analysis and 2DE profile as the hsp70/BiP homologous protein, the novel neurofilament protein synapsin IIa, pyruvate kinase, dynamin, aconitase and an unknown contaminating protein, and a 115-kDa protein that by subcellular fractionation and immunoblotting is a diagnostic PSD molecule. In addition, peptide sequences are obtained for four additional higher molecular weight proteins of the PSD that are not related at the level of primary structure to any known proteins.     

Comparison of Ca++-regulated events in the intestinal brush border

The intestinal epithelial cell and specifically the cytoskeleton of the brush border are thought to be controlled by micromolar levels of free calcium. Calcium-binding proteins of this system include intestinal calcium binding protein (CaBP), calmodulin (CaM), villin, and a 36,000-mol-wt protein substrate of tyrosine kinases. To assess the sequence of events as the intracellular Ca++ level rises, we determined the amount of CaM and CaBP in the intestinal epithelium by western blotting and tested the Ca++ binding of CaM and CaBP by equilibrium dialysis. The Ca++-dependent actin severing activity of villin was analyzed in the presence of physiological CaM levels and increasing calcium concentrations. In addition, we analyzed the Ca++ levels required for interaction between CaM and the microvillus 110,000-mol-wt protein as well as fodrin and the interaction between a polypeptide of 36,000 mol wt (P-36) and actin. The results suggest that CaBP serves as the predominant Ca++ buffer in the cell, but CaM can effectively buffer ionic calcium in the microvillus and thus protect actin from the severing activity of villin. CaM binds to its cytoskeletal receptors, 110,000-mol-wt protein and fodrin differently, governed by the free Ca++ and pH. The interaction between P-36 and actin, however, appears to require an unphysiologically high calcium concentration (10(-4) to 10(-3) M) to be meaningful. The results provide a coherent picture of the different Ca++ regulated events occurring when the free calcium rises into the micromolar level in this unique system. This study would suggest that as the Ca++ rises in the intestinal epithelial cell an ordered sequence of Ca++ saturation of intracellular receptors occurs with the order from the lowest to highest Ca++ requirements being CaBP less than CaM less than villin less than P-36.     

Isolation of calcium-dependent platelet proteins that interact with actin

Low Ca2+ extracts of platelets rapidly form an actin gel when warmed to 25 degrees C. The addition of Ca2+ has three effects. At Ca/EGTA = 0.4, the gel begins to contract. Increasing the Ca2+ concentration increases the rate of contraction and reduces the amount of actomyosin gel. Between Ca/EGTA = 0.4 and 0.5, a protease is activated that selectively degrades polypeptides with molecular weight greater than the myosin heavy chain. At Ca/EGTA = 1, about 70% of the total actin is nonsedimentable. Addition of excess EGTA produces the rapid formation of an actomyosin gel, which is not readily solubilized by re-addition of calcium. Using DNAase l-Sepharose chromatography, we have isolated a protein fraction whose binding to actin is Ca2+ -dependent. This fraction contains a major polypeptide with a molecular weight of 90,000. This fraction increases the rate of development of high sheer viscosity, but lowers the final value if Ca2+ is present. This decrease in viscosity is due to the generation of shorter filaments. In the presence of Ca2+, this protein(s) selectively blocks the addition of actin monomers to the barbed end of glutaraldehyde-fixed S1-decorated actin fragments and will nucleate assembly of filaments. We speculate that this protein(s) may serve as a Ca2+ -dependent nucleation site in situ.     

Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density

A method has been developed for binding calmodulin, radioiodinated by the lactoperoxidase method, to denaturing gels and has been used to attempt to identify the calmodulin-binding proteins of cerebral cortex postsynaptic densities (PSDs). Calmodulin primarily bound to the major 51,000 Mr protein in a saturatable manner; secondarily bound to the 60,000 Mr region, 140,000 Mr region, and 230,000 Mr protein; and bound in lesser amounts to a number of other proteins. The major 51,000 Mr calmodulin-binding protein is one of unknown identity. Binding of iodinated calmodulin to these proteins was blocked by EDTA, EGTA, chlorpromazine, and preincubation with unlabeled calmodulin. Calmodulin iodinated by the chloramine-T method, which inactivates calmodulin did not bind to the PSD but bound nonspecifically to histone. Calmodulin did not bind to proteins from a variety of sources for which calmodulin interactions have not been found. Except for three proteins, all of the proteins of synaptic membranes that bind calmodulin could be accounted for by proteins of the PSD which are a part of the synaptic membrane fraction. The major 51,000 M, protein and the corresponding iodinated calmodulin binding were greatly reduced in cerebellar PSDs and this difference between cerebral cortex and cerebellar PSDs is discussed in light of the possible function of calmodulin in synaptic excitatory responses.     

The structure of divalent cation-induced aggregates of PIP2 and their alteration by gelsolin and tau.

Phosphatidylinositol bisphosphate (PIP2) serves as a precursor for diacylglycerol and inositol trisphosphate in signal transduction cascades and regulates the activities of several actin binding proteins that influence the organization of the actin cytoskeleton. Molecules of PIP2 form 6-nm diameter micelles in water, but aggregate into larger, multilamellar structures in physiological concentrations of divalent cations. Electron microscopic analysis of these aggregates reveals that they are clusters of striated filaments, suggesting that PIP2 aggregates form stacks of discoid micelles rather than multilamellar vesicles or inverted hexagonal arrays as previously inferred from indirect observations. The distance between striations within the filaments varies from 4.2 to 5.4 nm and the diameter of the filaments depends on the dehydrated ionic radius of the divalent cation, with average diameters of 19, 12, and 10 nm for filaments formed by Mg2+, Ca2+, and Ba2+, respectively. The structure of the divalent cation-induced aggregates can be altered by PIP2 binding proteins. Gelsolin and the microtubule associated protein tau both affect the formation of aggregates, indicating that tau acts as a PIP2 binding protein in a manner similar to gelsolin. In contrast, another PIP2 binding protein, profilin, does not modify the aggregates.     

A calcium- and pH-regulated protein from Dictyostelium discoideum that cross-links actin filaments

We have purified an actin binding protein from amebas of Dictyostelium discoideum which we call 95,000-dalton protein (95K). This protein is rod shaped, approximately 40 nm long in the electron microscope, contains two subunits measuring 95,000 daltons each, and cross-links actin filaments. Cross-linking activity was demonstrated by using falling-ball viscometry, Ostwald viscometry, and electron microscopy. Cross-linking activity is optimal at 0.1 microM Ca++ and pH 6.8, but is progressively inhibited at higher Ca++ and pH levels over a physiological range. Half-maximal inhibition occurs at 1.6 microM free Ca++ and pH 7.3, respectively. Sedimentation experiments demonstrate that elevated Ca++ and pH inhibit the binding of 95K to F-actin which explains the loss of cross-linking activity. Electron microscopy demonstrates that under optimal conditions for cross-linking, 95K protein bundles actin filaments and that this bundling is inhibited by microM Ca++. Severing of actin filaments by 95K was not observed in any of the various assays under any of the solution conditions used. Hence, 95K protein is a rod-shaped, dimeric, Ca++- and pH-regulated actin binding protein that cross-links but does not sever actin filaments.     

Cytoskeletal proteins at the cholinergic synapse: distribution of desmin, actin, fodrin, neurofilaments, and tubulin in Torpedo electric organ

Treatment of the electric organ of Torpedo marmorata with Triton X-100 in the presence of 2 mM MgCl2 generated a cytoskeletal fraction in which a 54 kDa polypeptide is a major constituent. This 54 kDa polypeptide accounted for about 8% of the cellular protein when total electric organ tissue was analyzed by two-dimensional gel electrophoresis. Immunoblotting experiments showed that this protein reacts with monoclonal antibodies to desmin, the major intermediate filament protein of avian and mammalian muscle tissue. Negative stain analysis revealed that filaments of about 10 nm diameter are the major structural elements of the electric organ cytoskeleton. In the presence of Ca2+ there was a rapid degradation of the desmin-like protein and intermediate filaments due to a Ca2+-activated protease. Some of the resulting fragments retained antigenic activity against the desmin antibodies. Immunoblotting of membrane fractions enriched in acetylcholine receptor revealed desmin in addition to some actin. A further cytoskeletal component was identified from biochemical and immunological properties as a homologue of the mammalian neurofilament L-polypeptide. Thus Torpedo expresses proteins homologous to the mammalian desmin and neurofilament L-protein which can be detected using immunological approaches. Immunofluorescence microscopy was used to map the location of various cytoskeletal proteins of the cholinergic synapse on paraffin sections and on en face preparations of membranes. Desmin staining was restricted to electrocytes and in en face preparations was seen associated with both the ventral receptor-containing membrane and with the non-innervated dorsal membrane. Antibodies to neurofilament L-protein stained only the axons and not the electrocytes. Staining for fodrin, a non-erythrocyte spectrin, resulted in submembraneous decoration of both the axons and the electrocytes. Axonal staining for neurofilaments and microtubules did not extend into the ends of the nerve terminal arborizations.     

Antibodies to the ciliary membrane of Paramecium tetraurelia alter membrane excitability

Immobilization of Paramecium followed the binding of antibodies to the major proteins of the ciliary membrane (the immobilization antigens, i- antigens, approximately 250,000 mol wt). Immunoelectron microscopy showed this binding to be serotype-specific and to occur over the entire cell surface. Antibody binding also reduced the current through the Ca-channel of the excitable ciliary membrane as monitored using a voltage-clamp. The residual Ca-current appeared normal in its voltage sensitivity and kinetics. As a secondary consequence of antibody binding, the Ca-induced K-current was also reduced. The resting membrane characteristics and other activatable currents, however, were not significantly altered by the antibody treatment. Since monovalent fragments of the antibodies also reduced the current but did not immobilize the cell, the electrophysiological effects were not the secondary consequences of immobilization. Antibodies against the second most abundant family of proteins (42,000-45,000 mol wt) had similar electrophysiological effects as revealed by experiments in which the Paramecia and the serum were heterologous with respect to the i-antigen but homologous with respect to the 42,000-45,000-mol-wt proteins. Protease treatment, shown to remove the surface antigen, also caused a reduction of the Ca-inward current. The loss of the inward Ca-current does not seem to be due to a drop in the driving force for Ca++ entry since increasing the external Ca++ or reducing the internal Ca++ (through EGTA injection) did not restore the current. Here we discuss the possibilities that (a) the major proteins define the functional environment of the Ca-channel and that (b) the Ca-channel is more susceptible to certain general changes in the membrane.     

F actin assembly modulated by villin: Ca++-dependent nucleation and capping of the barbed end

We have studied the mechanism of Ca++-dependent restriction of actin filament length by villin, one of the major actin-associated proteins of intestinal microvilli microfilament bundles. Villin acts, even at a ratio of 1 to 1000 with respect to actin, very efficiently as a Ca++-dependent nucleation factor on actin assembly. This gives rise to unidirectional assembly, with the morphologically defined "barbed" end of the resulting filament being capped. Consequently, at steady state treadmilling of actin monomers through the filament is inhibited. Increase of the villin-to-actin ratio enhances the number of nucleated filaments necessarily shorter in length. This results finally in nonsedimentable F actin and a low molecular weight complex of one villin and three monomeric actins, which itself is a potent nucleator. Thus restriction of actin assembly by villin is not due to a direct inhibition of assembly but arises as the consequence of strongly enhanced nucleation followed by unidirectional elongation at the pointed end of the nucleated filaments. In addition, in the presence of Ca++-villin, but not the villin-actin complex, seems able to "break" or "sever" preformed F actin filaments. Thus a variety of cellular phenomena-nucleation, unidirectional assembly, filament end capping, nonpolymerizable actin and F actin bundles-can be observed in vitro in a two-protein component system modulated by the concentration of free Ca++.     

Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells

Although the actin cytoskeleton has been implicated in vesicle trafficking, docking and fusion, its site of action and relation to the Ca(2+)-mediated activation of the docking and fusion machinery have not been elucidated. In this study, we examined the role of actin filaments in regulated exocytosis by introducing highly specific actin monomer- binding proteins, the beta-thymosins or a gelsolin fragment, into streptolysin O-permeabilized pancreatic acinar cells. These proteins had stimulatory and inhibitory effects. Low concentrations elicited rapid and robust exocytosis with a profile comparable to the initial phase of regulated exocytosis, but without raising [Ca2+], and even when [Ca2+] was clamped at low levels by EGTA. No additional cofactors were required. Direct visualization and quantitation of actin filaments showed that beta-thymosin, like agonists, induced actin depolymerization at the apical membrane where exocytosis occurs. Blocking actin depolymerization by phalloidin or neutralizing beta- thymosin by complexing with exogenous actin prevented exocytosis. These findings show that the cortical actin network acts as a dominant negative clamp which blocks constitutive exocytosis. In addition, actin filaments also have a positive role. High concentrations of the actin depolymerizing proteins inhibited all phases of exocytosis. The inhibition overrides stimulation by agonists and all downstream effectors tested, suggesting that exocytosis cannot occur without a minimal actin cytoskeletal structure.     

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.     

Preparation and purification of polymerized actin from sea urchin egg extracts

Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F- actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when 1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.     

New actin-binding proteins from Dictyostelium discoideum

Dictyostelium discoideum contains a soluble actin-binding protein that caps actin filaments at their fast growing ends. The purified protein consists of two subunits with 34 kd and 32 kd apparent mol. wts. Like similar proteins from Acanthamoeba and bovine brain the capping protein from D. discoideum acts in a Ca²⁺ -independent manner. It lacks severing activity as indicated by its inability to disrupt the stress fibers and the microfilament network in detergent-extracted cells. Two actin-binding proteins from a plasma membrane-enriched fraction were labeled with [¹²⁵I]actin using a gel overlay technique. One of these proteins, with an apparent mol. wt. of 17 kd in SDS-polyacrylamide gels, has been purified from high-salt extracts, the other protein with an apparent mol. wt. of 31 kd has been purified from Triton X-100 extracted membranes. Monoclonal antibodies were raised against D. discoideum severin, α-actinin, the larger subunit of the capping protein, and the 17-kd membrane-associated protein. Immunoblotting of proteins from whole cell lysates showed that all these actin-binding proteins were present in both growth phase and aggregation-competent cells.     

Isolation and characterization of glial filaments from human brain

Intermediate (8--9 nm) filaments of human central nervous system astrocytes were isolated from the gliosed white matter of cases of adrenoleukodystrophy (ALD). This hereditary lipidosis is characterized pathologically by demyelination, loss of axons, and replacement of the white matter of the caudal cerebrum by a glial scar. Glial filaments were composed largely of a single protein component with a mol wt of about 49,000 daltons. Smaller components (44,000--39,000 daltons) were detected in some samples, and appear to represent degradation products of the filament protein. Human neurofilaments were isolated from the normal frontal white matter of ALD cases by the standard myelin-free axon technique. Isolated glial and neurofilament proteins comigrated during acrylamide gel electrophoresis in SDS. Polypeptides resulting from cyanogen bromide cleavage of the two filament proteins were the same. Both proteins reacted with rabbit antisera raised against isolated bovine neurofilament protein and human glial fibrillary acidic protein.