Tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies

The intracellular polymerization of cytoskeletal proteins into their supramolecular assemblies raises many questions regarding the regulatory patterns that control this process. Binding experiments using the ELISA solid phase system, together with protein assembly assays and electron microscopical studies provided clues on the protein-protein associations in the polymerization of tubulin and actin networks. In vitro reconstitution experiments of these cytoskeletal filaments using purified tau, tubulin, and actin proteins were carried out. Tau protein association with tubulin immobilized in a solid phase support system was inhibited by actin monomer, and a higher inhibition was attained in the presence of preassembled actin filaments. Conversely, tubulin and assembled microtubules strongly inhibited tau interaction with actin in the solid phase system. Actin filaments decreased the extent of in vitro tau-induced tubulin assembly. Studies on the morphological aspects of microtubules and actin filaments coexisting in vitro, revealed the association between both cytoskeletal filaments, and in some cases, the presence of fine filamentous structures bridging these polymers. Immunogold studies showed the association of tau along polymerized microtubules and actin filaments, even though a preferential localization of labeled tau with microtubules was revealed. The studies provide further evidence for the involvement of tau protein in modulating the interactions of microtubules and actin polymers in the organization of the cytsokeletal network.     

The Association of Tau-Like Proteins with Vimentin Filaments in Cultured Cells

There is increasing evidence that the different polymers that constitute the cytoskeleton are interconnected to form a three-dimensional network. The macromolecular interaction patterns that stabilize this network and its intrinsic dynamics are the basis for numerous cellular processes. Within this context,in vitrostudies have pointed to the existence of specific associations between microtubules, microfilaments, and intermediate filaments. It has also been postulated that microtubule-associated proteins (MAPs) are directly involved in mediating these interactions. The interactions of tau with vimentin filaments, and its relationships with other filaments of the cytoskeletal network, were analyzed in SW-13 adenocarcinoma cells, through an integrated approach that included biochemical and immunological studies. This cell line has the advantage of presenting a wild-type clone (vim+) and a mutant clone (vim−) which is deficient in vimentin expression. We analyzed the cellular roles of tau, focusing on its interactions with vimentin filaments, within the context of its functional aspects in the organization of the cytoskeletal network. Cosedimentation experiments of microtubular protein with vimentin in cell extracts enriched in intermediate filaments, combined with studies on the direct interaction of tau with nitrocellulose-bound vimentin and analysis of tau binding to vimentin immobilized in single-strand DNA affinity columns, indicate that tau interacts with the vimentin network. These studies were confirmed by a quantitative analysis of the immunofluorescence patterns of cytoskeleton-associated tubulin, tau, and vimentin using flow cytometry. In this regard, a decrease in the levels of tau associated to the cytoskeletal network in the vim− cell mutant compared with the wild-type clones was observed. However, immunofluorescence data on SW-13 cells suggest that the absence of a structured network of vimentin in the mutant vim− cells does not affect the cytoplasmic organization formed by microtubules and actin filaments, when compared with the wild-type vim+ cells. These studies suggest that tau associates with vimentin filaments and that these interactions may play a structural role in cells containing these filaments.     

Cytoskeleton integrity influences XRCC1 and PCNA dynamics at DNA damage

On induction of DNA damage with 405-nm laser light, proteins involved in base excision repair (BER) are recruited to DNA lesions. We find that the dynamics of factors typical of either short-patch (XRCC1) or long-patch (PCNA) BER are altered by chemicals that perturb actin or tubulin polymerization in human cells. Whereas the destabilization of actin filaments by latrunculin B, cytochalasin B, or Jasplakinolide decreases BER factor accumulation at laser-induced damage, inhibition of tubulin polymerization by nocodazole increases it. We detect no recruitment of actin to sites of laser-induced DNA damage, yet the depolymerization of cytoplasmic actin filaments elevates both actin and tubulin signals in the nucleus. While published evidence suggested a positive role for F-actin in double-strand break repair in mammals, the enrichment of actin in budding yeast nuclei interferes with BER, augmenting sensitivity to Zeocin. Our quantitative imaging results suggest that the depolymerization of cytoplasmic actin may compromise BER efficiency in mammals not only due to elevated levels of nuclear actin but also of tubulin, linking cytoskeletal integrity to BER.     

The interaction of actin filaments with microtubules and microtubule-associated proteins

Purified actin and microtubule proteins polymerized together form a gel, while mixtures of actin with tubulin polymers lacking microtubule-associated proteins (MAPs) have low viscosities close to the sum of the viscosities of the constituents. Mixtures of actin and MAPs also have high viscosities. Our interpretation of these observations was that there is interaction of actin filaments and microtubules which is mediated by MAPs (Griffith, L. M., and Pollard, T. D. (1978) J. Cell Biol. 78, 958-965). We report here further evidence for this interaction. 1) Actin filaments and microtubules can form gels at physiological ionic strength providing the anion is glutamate rather than chloride. Both glutamate and chloride inhibit actin-MAPs interaction, but this is compensated for in glutamate where the microtubules are longer than in chloride. 2) The low shear viscosity of mixtures of isolated MAPs and actin filaments is enhanced by acidic pH and inhibited by high ionic strength. 3) MAPs can be fractionated to yield four different fractions with actin cross-linking activity: a subset of high molecular weight MAPs, purified "MAP-2" and two different fractions of tau polypeptides. 4) We have reconstituted a gel from actin, purified tubulin, and whole MAPs, but have not yet been successful with actin, purified tubulin, and any single purified MAP.     

Evidence for actin filament-microtubule interaction mediated by microtubule-associated proteins

We have used low shear viscometry and electron microscopy to study the interaction between pure actin filaments and microtubules. Mixtures of microtubules having microtubule-associated proteins (MAPs) with actin filament have very high viscosities compared with the viscosities of the separate components. MAPs themselves also cause a large increase in the viscosity of actin filaments. In contrast, mixtures of actin filaments with tubulin polymers lacking MAPs have low viscosities, close to the sum of the viscosities of the separate components. Our interpretation of these observations is that there is an interaction between actin filaments and microtubules which requires MAPs. This interaction is inhibited by ATP and some related compounds. Electron micrographs of thin sections through mixtures of actin and microtubules show numerous close associations between the two polymers which may be responsible for their high viscosity.     

Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization

To study the effects of microtubule-associated proteins (MAPs) on in vivo microtubule assembly, cDNAs containing the complete coding sequences of a Drosophila 205-kD heat stable MAP, human MAP 4, and human tau were stably transfected into CHO cells. Constitutive expression of the transfected genes was low in most cases and had no obvious effects on the viability of the transfected cell lines. High levels of expression, as judged by Western blots, immunofluorescence, and Northern blots, could be induced by treating cells with sodium butyrate. High levels of MAPs were maintained for at least 24-48 h after removal of the sodium butyrate. Immunofluorescence analysis indicated that all three MAPs bound to cellular microtubules, but only the transfected tau caused a rearrangement of microtubules into bundles. Despite high levels of expression of these exogenous MAPs and the bundling of microtubules in cells expressing tau, transfected cells had normal levels of assembled and unassembled tubulin. With the exception of the tau-induced bundles, microtubules in transfected cells showed the same sensitivity as control cells to microtubule depolymerization by Colcemid. Further, all three MAPs were ineffective in reversing the taxol-dependent phenotype of a CHO mutant cell line. The absence of a quantitative effect of any of these heterologous proteins on the assembly of tubulin suggests that these MAPs may have different roles in vivo from those inferred previously from in vitro experiments.     

Action of cytochalasin D on cytoskeletal networks

Extraction of SC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body; 10-nm filaments, actin microfilaments, microtubules, and 2- to 3-nm filaments. Actin filaments and 2- to 3-nm filaments form numerous end- to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the three-dimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased. Supernates of preparations treated in this way contain both short actin filaments and network fragments (i.e., actin filaments in end-to-side contact with other actin filaments). It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response. The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filament-filament interactions (i.e., disruption of end-to-side contacts). The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci.     

Microtubule-associated Protein 2c Reorganizes Both Microtubules and Microfilaments into Distinct Cytological Structures in an Actin-binding Protein-280–deficient Melanoma Cell Line

The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments.     

Plant microtubule cytoskeleton complexity: microtubule arrays as fractals

Biological systems are by nature complex and this complexity has been shown to be important in maintaining homeostasis. The plant microtubule cytoskeleton is a highly complex system, with contributing factors through interactions with microtubule-associated proteins (MAPs), expression of multiple tubulin isoforms, and post-translational modification of tubulin and MAPs. Some of this complexity is specific to microtubules, such as a redundancy in factors that regulate microtubule depolymerization. Plant microtubules form partial helical fractals that play a key role in development. It is suggested that, under certain cellular conditions, other categories of microtubule fractals may form including isotropic fractals, triangular fractals, and branched fractals. Helical fractal proteins including coiled-coil and armadillo/beta-catenin repeat proteins and the actin cytoskeleton are important here too. Either alone, or in combination, these fractals may drive much of plant development.     

The hyaluronate receptor is associated with actin filaments

The cell-surface receptor for hyaluronate is an integral membrane glycoprotein of Mr 85,000 (Underhill, C. B., A. L. Thurn, and B. E. Lacy, 1985, J. Biol. Chem., 260:8128-8133) that is thought to mediate many of the effects that hyaluronate has on cell behavior, such as migration, angiogenesis, and phagocytosis. To determine if the receptor is associated with the underlying cytoskeleton, Swiss 3T3 cells were extracted with a solution of Triton X-100, which solubilized most of the cellular components, but which left behind an insoluble residue containing the cytoskeleton. This detergent-insoluble residue was found to contain the bulk of the hyaluronate-binding activity, suggesting that the receptor might indeed be associated with the cytoskeleton. To further define the cytoskeletal element with which the receptor interacts, 3T3 cells were extracted with Triton X-100 under a variety of different ionic conditions. In each case, the amount of hyaluronate-binding activity in the detergent-insoluble residue was related to the amount of actin present, but not to either tubulin or vimentin. In addition, the recovery of hyaluronate-binding activity was dramatically enhanced (to 100% in most cases) if the cells were extracted in the presence of phalloidin, a drug that stabilizes actin filaments. However, the recovery of binding activity was dramatically decreased when whole cells were treated with cytochalasin B before extraction, and when extracted cells were treated with DNase I, which promotes the depolymerization of actin filaments. In addition, preincubating an extract of SV-40-transformed Swiss 3T3 cell membranes with DNase I caused a change in the elution profile of the receptor as judged by molecular-sieve chromatography. Presumably this decrease in the size of the receptor is due to the loss of associated actin filaments. The results of these experiments strongly suggest that the receptor for hyaluronate is associated either directly or indirectly with cytosolic actin filaments.     

Actin and microtubules in neurite initiation: are MAPs the missing link?

During neurite initiation microtubules align to form a tight bundle and actin filaments reorganize to produce a growth cone. The mechanisms that underlie these highly coordinated cytoskeletal rearrangements are not yet fully understood. Recently, various levels of coordination between the actin- and microtubule-based cytoskeletons have been observed during cellular migration and morphogenesis, processes that share some similarities to neurite initiation. Direct, physical association between both cytoskeletons has been suggested, because microtubules often preferentially grow along actin bundles and transiently target actin-rich adhesion complexes. We propose that such physical association might be involved in force-based interactions and spatial organization of the two networks during neurite initiation as well. In addition, many signaling cascades that affect actin filaments are also involved in the regulation of microtubule dynamics, and vice versa. Although several candidates for mediating these effects have been identified in non-neuronal cells, the general mechanism is still poorly understood. In neurons certain plakins and neuron-specific microtubule associated proteins (MAPs), like MAP1B and MAP2, which can bind to both microtubules and F-actin, are promising candidates to play key roles in the specific cytoskeletal rearrangements controlling the transition from an undifferentiated state to neurite-bearing morphology. Here we review the effects of MAPs on microtubules and actin, as well as the coordination of both cytoskeletons during neurite initiation.     

Tau-like Proteins Associated with Centrosomes in Cultured Cells

The subcellular association of tau-like proteins with centrosomes in cultured cell lines and its effects in nucleating microtubule assembly were analyzed using biochemical and immunocytochemical approaches. Tau proteins, major components of microtubules, appear to be tightly associated with actin filaments in a variety of cell lines, while in pathological conditions of neurons, they are part of paired helical filaments found in Alzheimer's disease. Different studies suggest that, in addition to tau interactions with the components of the cytoskeletal network, tau polypeptides appear to be associated with highly structured cellular elements, in both interphase and mitotic cells. An in-depth analysis of tau subcellular distribution us- ing different polyclonal and monoclonal antibodies showed colocalization of tau-like components with centrosomes in interphase cells of the human Huh-7 hepatoma, in SW-13 adenocarcinoma, and in normal human fibroblasts. Tau associated with centrosomes in mitotic Huh-7 cells was also identified. However, antibodies against the tau binding repeats did not stain centrosomes. A set of different tau isoforms was also identified by Western blot analysis on isolated centrosomal preparations from Huh-7 cells, obtained by differential centrifugation through sucrose gradients. Microtubule nucleationin vitroover isolated centrosomes was inhibited by both the polyclonal antibody against native tau and an antibody to the N-terminal tau sequence, as revealed by immunofluorescence analysis and assembly kinetics experiments. The antibody TRS1.2 against the fragment containing the first binding repeat on tau did not affect nucleation. These studies allowed us to characterize tau association with the isolated centrosomal preparation and its involvement in microtubule assembly nucleated over centrosomes, thus suggesting possible structural and functional roles for these interactions.     

Endoplasmic reticulum calcium release is modulated by actin polymerization

Intracellular calcium ions regulate the structure and functions of cytoskeletal proteins. On the other hand, recent studies have shown that the cytoskeleton, and actin filaments in particular, can modulate calcium influx through plasma membrane ligand- and voltage-gated channels. We now report that calcium release from inositol trisphosphate (IP3) and ryanodine-sensitive endoplasmic reticulum (ER) stores is modulated by polymerization and depolymerization of actin filaments in cultured hippocampal neurons. Depolymerization of actin filaments with cytochalasin D attenuates calcium release induced by carbamylcholine (CCh; a muscarinic agonist for IP3 pathway), caffeine (a ryanodine receptor agonist) and thapsigargin (an inhibitor of the ER calcium- ATPase) in both the presence and absence of extracellular calcium. Conversely, the actin polymerizing agent jasplakinolide potentiates calcium release induced by CCh, caffeine and thapsigargin. Cytochalasin D attenuated, while jasplakinolide augmented, thapsigargin-induced JNK activation and neuronal cell death. Our data show that the actin cytoskeleton regulates ER calcium release, suggesting roles for actin in the various physiological and pathological processes that involve calcium release.     

Transformation of actin-encapsulating liposomes induced by cytochalasin D.

Liposomes encapsulating actin filaments were prepared by swelling at 0 degrees C lipid film consisting of a mixture of dimyristoyl phosphatidylcholine and cardiolipin (equal amounts by weight) in 100 microM rabbit skeletal muscle actin and 0.5 mM CaCl2 followed by polymerization of actin at 30 degrees C. Liposomes initially assumed either disk or dumbbell shape, but when cytochalasin D was added to the medium surrounding the liposomes, they were found to become spindle shaped. Liposomes containing bovine serum albumin that were given cytochalasin D and actin-containing liposomes that were given dimethylformamide, the solvent for cytochalasin D, did not transform. These results indicated actin-cytochalasin interaction is involved in the transformation process. Falling-ball viscometry and sedimentation analysis of actin solution indicated that cytochalasin cleaved actin filaments and caused depolymerization. The observation of polarized fluorescence of encapsulated actin labeled with acrylodan indicated that the actin filaments in the transformed liposomes aligned along the long axis of the liposomes. Because the actin filaments in the disk- or dumbbell-shaped liposomes formed bundles running along the liposome contour, the transformation was likely to be accompanied by the change in the actin filament arrangement in the liposomes, which was induced by actin-cytochalasin interaction.     

How actin filaments and microtubules steer growth cones to their targets

Guidance molecules steer growth cones to their targets by attracting or repelling them. Turning in a new direction requires remodeling of the growth cone and bending of the axon. This depends upon reorganization of actin filaments and microtubules, which are the primary cytoskeletal components of growth cones. This article discusses how these cytoskeletal components induce turning. The importance of each component as well as how interactions between them result in axon guidance is discussed. Current evidence shows that microtubules are influenced by both the organization and dynamics of actin filaments in the peripheral domain of growth cones. Cytoskeletal models for repulsive and attractive turning are presented. Molecular candidates that may link actin filaments with microtubules are suggested and potential signal transduction pathways that allow these cytoskeletal components to affect each other are discussed.     

Tropomyosins Regulate the Severing Activity of Gelsolin in Isoform-Dependent and Independent Manners

The actin cytoskeleton fulfills numerous key cellular functions, which are tightly regulated in activity, localization, and temporal patterning by actin binding proteins. Tropomyosins and gelsolin are two such filament-regulating proteins. Here, we investigate how the effects of tropomyosins are coupled to the binding and activity of gelsolin. We show that the three investigated tropomyosin isoforms (Tpm1.1, Tpm1.12, and Tpm3.1) bind to gelsolin with micromolar or submicromolar affinities. Tropomyosin binding enhances the activity of gelsolin in actin polymerization and depolymerization assays. However, the effects of the three tropomyosin isoforms varied. The tropomyosin isoforms studied also differed in their ability to protect pre-existing actin filaments from severing by gelsolin. Based on the observed specificity of the interactions between tropomyosins, actin filaments, and gelsolin, we propose that tropomyosin isoforms specify which populations of actin filaments should be targeted by, or protected from, gelsolin-mediated depolymerization in living cells.     

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.     

Differential Association of Tau With Subsets of Microtubules Containing Posttranslationally-Modified Tubulin Variants in Neuroblastoma Cells

Neuronal cells display different subsets of dynamic microtubules. In axons and extending neurites, this intrinsic dynamics is modulated by the microtubule-associated protein tau. Moreover, posttranslational modifications of tubulin, namely acetylation, tyrosination or glutamylation are directly involved in determining the stability of neuronal microtubules. Studies were carried out to analyze the interaction patterns of tau with subsets of microtubules in N2A neuroblastoma cells, which can differentiate in the presence of dibutyryl cAMP. Double labeling studies showed a differential pattern of tau association with microtubules containing acetylated and tyrosinated tubulin. Furthermore, studies using depolymerizing drugs revealed a selectivity in the association of tau with microtubular polymers and microfilaments, within the organization of the neuronal cytoskeleton. In order to study the association of specific tau isoforms with microtubules containing modified tubulin variants, immunoprecipitation studies were carried out. The coimmunoprecipitation data indicated a selective binding of specific tau isoforms to either modified tubulin variant. To assess the hypothesis on the roles of tau isoforms in the stabilization of microtubules containing modified tubulins, the association of those variants with tau isoforms was analyzed in overlay experiments. A preferential binding of acetylated tubulin from undifferentiated N2A cell extracts, to at least one slow-migrating tau isoform was revealed. However, acetylated tubulin from N2A cells containing long neurites displayed a preferential association with two isoforms of tau. On the other hand, tyrosinated tubulin from N2A extracts bound to the entire set of neuronal tau isoforms. These studies, along with the tau association with microtubules with different stability, indicate that tau segregates into subsets of microtubules in the axonal processes. The studies also suggest that these interactions may respond to a functional versatility of these polymers in differentiating neurons.