RESUMO
The heterogeneous family of tau proteins interacts with microtubules, actin filaments, and intermediate filaments. The tau isoforms have been shown to play a major role in neuronal polarity. However, tau-like proteins have been found in several other types of cells. Previous studies have also indicated the presence of a nuclear tau. The relationships between nuclear and cytoplasmic tau as well as the functional aspects of the nuclear tau are unknown. In this study, we demonstrate by reverse transcriptase polymerase chain reaction using specific primers that a transcript with features of neuronal tau is present in human fibroblast and Huh-7 hepatoma cell lines. Additionally, we present the first isolation and characterization of cytosolic and nuclear tau-like proteins from nonneuronal cells. Nonneuronal cytosolic tau components were isolated using the perchloric acid precipitation approach, while nuclear tau was isolated after selective extractions using high-ionic strength buffers. The cytoplasmic tau of nonneuronal cells is composed of at least three isoforms, whereas two main isoforms were detected in nuclear tau. Interestingly, the cytoplasmic and nuclear tau components exhibited the capacity to promote tubulin polymerization in vitro. Immunofluorescence studies using monoclonal anti-tau antibodies indicated a discrete distribution of tau protein in both the interphase and mitotic nucleus. In the latter, tau colocalized with the chromosomal scaffold. These studies, together with previous evidence on tau roles in modulating microtubule growth from centrosomes, and its role in the interaction patterns that stabilize the integrity of the cytoskeletal network, strongly support the idea that tau is a multifunctional protein involved in fundamental cellular processes.
Assuntos
Encéfalo/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Primers do DNA/genética , Fibroblastos/metabolismo , Humanos , Microscopia Eletrônica , Microtúbulos/química , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Células Tumorais Cultivadas , Proteínas tau/genéticaRESUMO
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 nucleation in vitro over 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.
Assuntos
Centrossomo/metabolismo , Proteínas tau/metabolismo , Células Cultivadas , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Interfase , Microtúbulos/metabolismo , Mitose , Frações Subcelulares/metabolismo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Actinas/química , Proteínas Associadas aos Microtúbulos/química , Proteínas de Neurofilamentos/química , Proteínas tau/química , Animais , Encéfalo/ultraestrutura , Células Cultivadas , Citocalasina D/farmacologia , Imunofluorescência , Nocodazol/farmacologia , RatosRESUMO
After the finding of the involvement of the C-terminal moieties of tubulin subunits in the interaction of MAPs, different studies have focused on the substructure of the binding domains for the different MAPs. Current biochemical evidence point to the role of a low-homology sequence between alpha and beta-subunits within the conserved region of the C-terminal domain of tubulin, in the binding of MAP-2 and tau. Another line of studies indicates that a site for interaction of the high molecular weight MAPs is located in the variable region defined by the glutamic-rich C-terminus of beta-tubulin. Here, we report the usefulness of idiotypic site-directed antibodies, produced by immunization with peptides from different beta-tubulin isoforms, to study both MAP-1 and MAP-2 binding sites on tubulin. On the basis of these results with site-specific antibodies along with previous structural information (Cross et al., 1991, Biochemistry 30: 4362-4366), we propose the role of consensus sequences, from the invariant beta-tubulin C-terminal domain in the binding of MAP-2 and from the variable domain in the interactions of MAP-1 and MAP-2.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Tubulina (Proteína)/genética , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Bovinos , Microtúbulos/metabolismo , Dados de Sequência Molecular , Peso Molecular , Tubulina (Proteína)/imunologiaRESUMO
The cytoskeletal integrity of human and rodent cell lines was analyzed using site-directed monoclonal antibodies prepared from hybridomas. Secreting hybridomas were produced by immunizing mice with synthetic peptides from the C-terminal domain of the beta II-tubulin isotype, beta II(422-434), YQQYQDATADEQG, and the first imperfect repeat from brain tau, Tau-I(187-204), VRSKIGSTENLKHQPGGG. Two hybridomas were selected for this work: MTB6.22, an anti-idiotypic monoclonal antibody, which was obtained from a mouse immunized with the beta II-peptide and recognizes specific tubulin-binding domains on MAP-2 and tau; and Tau-I/1, which recognizes the repetitive binding sequences on tau and MAP-2. Immunoblots of cytoskeletal protein preparations from the five different tumor cell lines studied, showed the interaction of the site-directed antibodies MTB6.22 and Tau-I/1 with a group of proteins that co-migrate with brain tau. Immunoreactive tau components were also identified using an anti-tau monoclonal antibody (clone Tau-2), and several polyclonal anti-tau antibodies that interact with tau epitopes, other than those of the tubulin-binding domains. These tau-like proteins bound to a calmodulin-Sepharose affinity column and were eluted using 2 mM EGTA. Interestingly, washing the extracted cytoskeleton pellet with 5 x 10(-3) M Ca2+ for short periods of time selectively released the tau-like protein components, whilst most of the other cytoskeletal proteins remained in the pellet. On the other hand, immunofluorescence microscopy of detergent-extracted cells showed immunostaining of MAP components that appear to be co-localized in a discrete dot-like distribution along the stress fibers, which were revealed using rhodamine-phallacidin. Further support for the specificity of tau interaction with sites on tubulin and actin polymers was obtained with double-immunofluorescence, using the MAP-reactive monoclonal antibody MTB6.22 and a polyclonal antibody to a tubulin peptide containing part of the tau-binding domain on tubulin. Considering the anti-idiotypic nature of the MTB6.22 monoclonal antibody, our studies indicate that, in all the cell lines analyzed, a tau-like protein component is involved in mediating the interaction of both actin and tubulin polymers.
Assuntos
Actinas/metabolismo , Microtúbulos/metabolismo , Proteínas tau/metabolismo , Actinas/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Epitopos/análise , Humanos , Hibridomas , Imuno-Histoquímica , Mapas como Assunto , Dados de Sequência Molecular , Ratos , Proteínas tau/análiseRESUMO
The interaction of microtubule-associated proteins MAP-1 and MAP-2 with different peptides containing sequences covering the C-terminal region of beta-tubulin isoforms has been analyzed. Our results indicate that MAP-1 and MAP-2 bind to a common sequence within the variable C-terminal region of the different beta-tubulin isoforms, while MAP-2 also interacts with the subdomain beta (422-434) of the constant region, in agreement with previous results (Maccioni, R.B., Rivas, C., & Vera, J.C. (1988) EMBO J. 7, 1957-1963). The productive interaction of MAP-2 with the latter domain appears to be involved in the assembly of microtubules.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Animais , Autorradiografia , Sítios de Ligação , Bovinos , Microscopia Eletrônica , Dados de Sequência Molecular , Tubulina (Proteína)/genética , Tubulina (Proteína)/ultraestruturaRESUMO
The expression of the rat angiotensin-II receptor has been studied in Xenopus oocytes. Poly(A)+ RNA isolated from the brain and adrenal gland was injected into oocytes, and the expression of the receptor in the oocyte plasma membrane was assayed by measuring the change in membrane potential in the presence of angiotensin-II. Expression of the angiotensin-II receptor was detected 1.0-1.5 days after messenger RNA (mRNA) injection, and the degree of membrane depolarization was proportional to the amount of mRNA injected. Ca+2 channel blockers inhibited the angiotensin-II-induced depolarization. The total mRNA was fractionated by preparative agarose gel electrophoresis and each fraction was assayed for its ability to induce angiotensin-II depolarization. The mRNA encoding the angiotensin-II receptor was found in a single fraction of 4.4 kilobases.