Control of tubulin and actin gene expression in Tetrahymena pyriformis during the cell cycle

Poly(A)-containing mRNA was isolated from division synchronized populations of the ciliated protozoan, Tetrahymena pyriformis. The level of tubulin and actin mRNA at specific cell cycle stages was analyzed by hybridization to tubulin and actin cDNA probes and by gel analysis of their in vitro translation products. The pattern of fluctuation of tubulin mRNA levels was similar to that observed for the in vivo tubulin synthesis previously reported [1]. This suggests that as the cells progress through the cell cycle, tubulin synthesis is controlled at the mRNA level. There was little fluctuation of actin synthesis or actin mRNA levels during the cell cycle, which may be indicative of a different regulatory mechanism for actin than for tubulin.     

Antibodies to tubulin and actin bind to the surface of a human monocytic cell line, U937

Intermittent reports of cytoskeleton proteins (actin and tubulin) on the cell surface have appeared over the last 13 years. Whereas most have concentrated on lymphocytes, this study provides evidence for the presence of these proteins on the surface of a human cultured monocyte-like cell line, U937. Both actin and tubulin were detected on the surface of U937 cells by flow cytometry, using an indirect staining procedure based on biotin-streptavidin-phycoerythrin, chosen for greater sensitivity. By use of this procedure, the majority of viable unstimulated U937 cells stained positively for actin and tubulin, although the level of fluorescence intensity was low. With an antibody specific for tyrosine-tubulin, most of the surface tubulin was also found to be tyrosinylated. For vimentin, an intermediate filament protein abundantly present in the cytoplasm of U937 cells, no staining could be detected. Confirmation of the flow cytometry data for surface actin and tubulin on unstimulated U937 cells was achieved by direct vesualization using a confocal laser scanning microscope. When U937 cells were activated with PMA and LPS, a marked reduction in the level of cell surface actin and tubulin occurred. The role of cell surface actin and tubulin on unstimulated U937 cells, in terms of monocyte function, remains to be elucidated.     

Interaction of rat brain microtubule proteins and 6S tubulin with rabbit skeletal muscle actomysin.

The interaction between actomyosin from rabbit skeletal muscle and microtubule proteins or 6S tubulin from rat brain was investigated with respect to the change in ATPase activity and physicochemical properties. Myosin bound to both microtubule proteins and 6S tubulin at low ionic strength. In the aggregates the molar ratio of microtubule proteins or 6S tubulin to myosin was 0.5–1.5 or 1.5–2.5. The superprecipitation of actomyosin was inhibited by 6S tubulin. The degree of superprecipitation inhibition was dependent on the mixing order of myosin, actin, 6S tubulin, and ATP. When myosin was preincubated first with 6S tubulin, the inhibition was most marked. The actin activation of myosin Mg-ATPase was inhibited by both microtubule proteins and 6S tubulin with stronger effects by the latter. The preincubation of myosin with 6S tubulin prior to the addition of actin induced not only greater inhibition of ATPase but also the binding of a larger quantity of 6S tubulin to myosin than the preincubation of myosin with actin. The similar results were obtained with microtubule proteins.     

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.     

Localization of actin and tubulin in developing and adult mammalian photoreceptors

Summary In order to define cytoskeletal domains of the mammalian photoreceptor, actin and tubulin were localized in adult retinae of mouse and human. For light-microscopic localization, actin was labeled using fluorescent phalloidin or monoclonal antibodies against actin, and tubulin was labeled using monoclonal antibodies against alpha- and beta-tubulin in an immunocytochemical method. Actin and tubulin were also localized by ultrastructural immunocytochemistry in the mouse. Filamentous actin was present in the retina at the outer limiting membrane and in synaptic terminals, especially of the cones, while globular actin was observed additionally in the inner segments. Müller cell cytoplasm and apical microvilli at the outer limiting membrane were also labeled for filamentous actin. Alpha- and beta-tubulin were evident throughout the photoreceptors, including the inner segments, but not in the synaptic terminals or at the outer limiting membrane. In the early postnatal retina of mouse, actin and tubulin were present at the ventricular surface. This pattern changed as photoreceptors fully elongated and as synaptogenesis occurred in the outer plexiform layer.     

Interactions of actin and tubulin with human deoxyhemoglobin. Their possible occurrence within erythrocytes

Short actin filaments are an essential component of the red-cell membrane skeleton, and microtubules are also present in nucleated erythrocytes as a marginal band. Actin and tubulin share the property of possessing a very anionic terminal peptide. Since deoxyhemoglobin (Hb) is known to be a strong polyanion-binding protein, we have considered how it may interact with actin and tubulin within the intact cell. Here we demonstrate that actin and tubulin form in vitro a high-affinity complex with Hb. This is shown by measuring, by stopped-flow experiments, the decrease of the binding rate constant of CO to Hb in the presence of increasing amounts of actin and tubulin. One tetramer of Hb is bound by an actin monomer, and about two tetramers by an alpha, beta-tubulin heterodimer. Binding assays in batch experiments with immobilized tubulin give the same stoichiometry. Formation of the complexes involves the 2,3-bisphosphoglycerate-binding site of Hb and a negatively charged domain, most likely the highly acidic N and C-terminal peptides of actin and tubulin. In addition to providing new opportunities to study the structural and functional properties of actin and tubulin, these results support the idea that in the case of partial metabolic depletion of bisphosphoglycerate and ATP in erythrocytes, Hb may interact with oligomeric actin and tubulin present in the cytoskeleton.     

Are histones, tubulin, and actin derived from a common ancestral protein?

Histones and the cytoskeletal components tubulin and actin all act as thermal ratchets, using the energy present in Brownian motion to do work. All three also bind to nucleotides. Here we suggest that histones, tubulin, and actin derive from a common ancestral protein. There is some sequence similarity between histone 2A and the bacterial tubulin homologue FtsZ. Histones and actin also share some sequence similarity in the nucleotides and at phosphate-binding sites. Thus, actin and tubulin may also be related, although this is not obvious from sequence analysis. Indeed, actin and tubulin are closely functionally related and cooperate in many cellular processes. Interestingly, recent advances in nanotechnology suggest that thermal ratchets may be able to impart lifelike properties; thus, the evolution of the ancestral histone, tubulin, and actin thermal ratchet may have been crucial in the development of complexity in living organisms.     

Localization of cytoskeletal proteins in Pneumocystis carinii by immuno-electron microscopy

Pneumocystis carinii causes serious pulmonary infection in immunosuppressed patients. This study was undertaken to observe the cytoskeletal proteins of P. carinii by immuno-electron microscopy. P. carinii infection was experimentally induced by immunosuppression of Sprague-Dawley rats for seven weeks, and their lungs were used for the observations of this study. The gold particles localized actin, tropomyosin, and tubulin. The actin was irregularly scattered in the cytoplasm of the trophic forms but was much more concentrated in the inner space of the cell wall of the cystic forms called the inner electron-lucent layer. No significant amount of tropomyosin was observed in either trophic forms or cystic forms. The tubulin was distributed along the peripheral cytoplasm and filopodia of both the trophic and cystic forms rather than in the inner side of the cytoplasm. Particularly, in the cystic forms, the amount of tubulin was increased and located mainly in the inner electron-lucent layer of the cell wall where the actin was concentrated as well. The results of this study showed that the cell wall of P. carinii cystic forms is a structure whose inner side is rich in actin and tubulin. The location of the actin and tubulin in P. carinii suggests that the main role of these proteins is an involvement in the protection of cystic forms from the outside environment by maintaining rigidity of the cystic forms.     

Interaction of the betaIV-tubulin isotype with actin stress fibers in cultured rat kidney mesangial cells.

Microtubules and actin filaments are two of the major components of the cytoskeleton. There is accumulating evidence for interaction between the two networks. Both the alpha- and beta-subunits of tubulin exist as numerous isotypes, some of which have been highly conserved in evolution. In an effort to better understand the functional significance of tubulin isotypes, we used a double immunofluorescence labeling technique to investigate the interactions between the tubulin beta-isotypes and the actin stress fiber network in cultured rat kidney mesangial cells, smooth-muscle-like cells from the renal glomerulus. Removal of the soluble cytoplasmic and nucleoplasmic proteins by detergent extraction caused the microtubule network to disappear while the stress fiber network was still present. In these extracted cells, the betaI- and betaII-tubulin isotypes were no longer present in the cytoplasm while the betaIV-isotype co-localized with actin stress fibers. Co-localization between betaIV-tubulin and actin stress fibers was also observed when the microtubule network was disrupted by the anti-tubulin drug colchicine and also by microinjection of the betaIV-tubulin antibody. Our results suggest that the betaIV isotype of tubulin may be involved in interactions between microtubules and actin.     

Analysis of the aplyronine A-induced protein–protein interaction between actin and tubulin by surface plasmon resonance

The antitumor macrolide aplyronine A induces protein–protein interaction (PPI) between actin and tubulin to exert highly potent biological activities. The interactions and binding kinetics of these molecules were analyzed by the surface plasmon resonance with biotinylated aplyronines or tubulin as ligands. Strong binding was observed for tubulin and actin with immobilized aplyronine A. These PPIs were almost completely inhibited by one equivalent of either aplyronine A or C, or mycalolide B. In contrast, a non-competitive actin-depolymerizing agent, latrunculin A, highly accelerated their association. Significant binding was also observed for immobilized tubulin with an actin–aplyronine A complex, and the dissociation constant K_D was 1.84 μM. Our method could be used for the quantitative analysis of the PPIs between two polymerizing proteins stabilized with small agents.     

The differential expression of the actins and tubulins during spermatogenesis in the mouse

Following intratesticular injection of [35S]methionine, the multiple isoforms of actin and tubulin from highly purified mouse testicular meiotic and post-meiotic cells have been analysed by high resolution two-dimensional gel electrophoresis. In pachytene spermatocytes both beta and gamma actin are synthesized, gamma actin being made in a significantly greater amount. The relative proportion of synthesis of beta and gamma actin changes during spermiogenesis, beta actin increasing and gamma actin decreasing in round spermatids, elongating spermatids, and residual bodies. Both alpha and beta tubulin are synthesized in approximately equal proportion in pachytene spermatocytes. In addition to the tubulin isoforms synthesized during meiosis, at least one new form of both alpha and beta tubulin first appears in post-meiotic (haploid) cells. In elongating spermatids and residual bodies, the synthesis of alpha tubulin is drastically reduced.     

Quantitative analysis of cytoskeletal proteins throughout the cell cycle of the MRC-5 fibroblastic cell line

Fluorescent dyes were used to stain actin, vimentin, tubulin and DNA in the same MRC-5 fibroblastic cells. Cytofluorometry and image analysis were then used to quantitatively evaluate the F actin, vimentin and tubulin content throughout the cell cycle. The results showed that different cells can have the same DNA content while their cytoskeletal protein content is variable. The data also showed that cytoskeletal protein content variations exist throughout the cell cycle of the fibroblastic cell line. The F actin content increased during the cell cycle from G1 to G2 phases and decreased in M phase. The amount of tubulin in the G2 was about twice as much as that in the G1 phase, before decreasing in the M phase; there was a threshold of tubulin content for G2 cells entering S phase.     

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.     

Structural Variations in Protein Superfamilies: Actin and Tubulin

Structures of homologous proteins are usually conserved during evolution, as are critical active site residues. This is the case for actin and tubulin, the two most important cytoskeleton proteins in eukaryotes. Actins and their related proteins (Arps) constitute a large superfamily whereas the tubulin family has fewer members. Unaligned sequences of these two protein families were analysed by searching for short groups of family-specific amino acid residues, that we call motifs, and by counting the number of residues from one motif to the next. For each sequence, the set of motif-to-motif residue counts forms a subfamily-specific pattern (landmark pattern) allowing actin and tubulin superfamily members to be identified and sorted into subfamilies. The differences between patterns of individual subfamilies are due to inserts and deletions (indels). Inserts appear to have arisen at an early stage in eukaryote evolution as suggested by the small but consistent kingdom-dependent differences found within many Arp subfamilies and in γ-tubulins. Inserts tend to be in surface loops where they can influence subfamily-specific function without disturbing the core structure of the protein. The relatively few indels found for tubulins have similar positions to established results, whereas we find many previously unreported indel positions and lengths for the metazoan Arps.     

Decreased actin and tubulin synthesis in 3T3 cells after transformation by SV40 virus.

Actin and tubulin are major protein constituents of 3T3 and SV40 virus-transformed 3T3 cells. We have fractionated growing, confluent and SV403T3 cells into particulate and soluble fractions using conditions designed to sediment microtubules, actin filaments or membrane associated actin or tubulin. The ratio of particulate to soluble actin synthesized in growing or confluent 3T3 cells is 2 to 1, while the ratio is reversed in transformed cells. There is also a 60% decrease in particulate tubulin synthesis in SV403T3 cells when compared with that in normal cells. Similar results are obtained when total actin and tubulin amounts are determined. The half-lives of actin, tubulin and total protein are over 3 days in growing 3T3 and SV40 cells and decrease over two-fold in confluent 3T3 cells. The significance of these results with respect to loss of contact inhibition and development of malignancy by these cells after transformation is discussed.     

Actin and tubulin binding domains of synapsins Ia and Ib

Synapsins Ia and Ib are neuronal phosphoproteins involved with the regulated clustering of small synaptic vesicles at the presynaptic terminus. In vitro they bind and bundle filaments of both actin and tubulin. Previously, we identified an actin binding domain in the NH2-terminal 25-kDa fragment (N25) generated by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage of synapsin I and found that a complementary COOH-terminal 52-kDa portion of the molecule (N52) contained either a second actin binding site or a site of self-association [Petrucci, T. P., & Morrow, J. S. (1987) J. Cell. Biol. 105, 1355]. Using direct binding assays between actin, tubulin, and specific synapsin NTCB-derived peptides, we confirm the ability of purified N25 to bind but not bundle actin and demonstrate that the complementary N52 (or N50) fragments from synapsins Ia and Ib and a 14-kDa fragment derived from the middle of the molecule also associate directly with actin. An antibody specific for N25 inhibits the actin binding activity of N25 and the actin bundling but not the actin binding activity of intact synapsin I. Similar studies conducted with purified tubulin and tubulin immobilized on Sepharose demonstrate that both tubulin and actin bind at approximately the same sites in the NH2-terminal half of synapsin I. Although the fragments derived from the COOH terminus of both synapsin Ia and synapsin Ib (N40b/N34) were devoid of measurable actin binding activity after NTCB cleavage, they were specifically labeled in the intact molecule by a photoactivated cross-linker bound to F-actin. Collectively, these results indicate that synapsins Ia and Ib possess two actin and tubulin binding domains located in the NH2-terminal half of the molecule and suggest that a third actin binding domain is located in the COOH-terminal region. The NH2-terminal sites are found in NTCB peptides N25 and N14, while the third site, apparently of lower affinity, resides in N40b/N34. It is hypothesized that, in the intact molecule, the two NH2-terminal domains contribute to a single high-affinity actin and/or tubulin binding site in the "globular" head region of synapsin I, while the third actin binding domain constitutes the topographically distinct site required for the actin bundling activity of the native molecule. The 45-residue COOH extension that distinguishes synapsin Ia from synapsin Ib appears not to be involved with actin binding, since no differences were found in the ability of N40b and N34 to be photo-cross-linked to actin.(ABSTRACT TRUNCATED AT 400 WORDS)     

The cytosolic class II chaperonin CCT recognizes delineated hydrophobic sequences in its target proteins

The nonhomologous proteins actin and alpha- and beta-tubulin need the assistance of the cytosolic chaperonin containing TCP-1 (CCT) to reach their correct native state, and their folding requires a transient binary complex formation with CCT. We show that separate or combined deletion of three delineated hydrophobic sequences in actin disturbs the interaction with CCT. These sites are situated between residues 125-179, 244-285, and 340-375. Also, alpha- and beta-tubulin contain at least one recognition region, and intriguingly, it has a similar distribution of hydrophobic residues as region 244-285 in actin. Internal deletion of the sites in actin favor a model for cooperative binding of target proteins to CCT. Peptide mimetics, representing the binding regions, inhibit target polypeptide binding to CCT, suggesting that actin and tubulin contact similar CCT subunits. In addition, we show that actin recognition by class II chaperonins is different from that by class I.     

Tubulin and Neurofilament Proteins Are Transported Differently in Axons of Chicken Motoneurons

SUMMARY 1. We previously showed that actin is transported in an unassembled form with its associated proteins actin depolymerizing factor, cofilin, and profilin. Here we examine the specific activities of radioactively labeled tubulin and neurofilament proteins in subcellular fractions of the chicken sciatic nerve following injection of L-[³⁵S]methionine into the lumbar spinal cord.2. At intervals of 12 and 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Analysis of the fractions by high-resolution two-dimensional gel electrophoresis, immunoblotting, fluorography, and computer densitometry showed that tubulin was transported as a unimodal wave at a slower average rate (2–2.5 mm/day) than actin (4–5 mm/day). Moreover, the specific activity of soluble tubulin was five times that of its particulate form, indicating that tubulin is transported in a dimeric or small oligomeric form and is assembled into stationary microtubules.3. Neurofilament triplet proteins were detected only in the particulate fractions and transported at a slower average rate (1 mm/day) than either actin or tubulin.4. Our results indicate that the tubulin was transported in an unpolymerized form and that the neurofilament proteins were transported in an insoluble, presumably polymerized form.     

Cytoskeleton disruption in chondrocytes from a rat osteoarthrosic (OA) -induced model: its potential role in OA pathogenesis

Morphological and functional changes of chondrocytes are typical in OA cartilage. In this work, we have described noteworthy changes in intermediate filaments cytoskeleton evidenced by transmission electron microscopy. Alterations in the distribution as well as in the content of vimentin, actin, and tubulin have been described by specific fluorescence labelling of each cytoskeletal component and confocal analysis. Normal vs OA cartilages showed a reduction in the percentage of labelled chondrocytes of 37.1% for vimentin, 4.7% for actin, and 20.1% for tubulin. Statistical analysis of fluorescence intensities (mean % +/- SEM) between normal and OA rat cartilage revealed a highly significant difference in vimentin, a significant difference in tubulin, and a non-significant difference in actin. Moreover, by western blot, altered electrophoretic patterns were observed mainly for vimentin and tubulin in OA cartilage in comparison with normal cartilage. These results allow us to suggest that substantial changes in vimentin and tubulin cytoskeleton of chondrocytes might be involved in OA pathogenesis.