The tubulin-binding sequence of brain microtubule-associated proteins, tau and MAP-2, is also involved in actin binding.
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ABSTRACT:
SUBMITTER: Correas I
PROVIDER: S-EPMC1131531 | biostudies-other | 1990 Jul
REPOSITORIES: biostudies-other
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Project description:Abnormal mitochondrial function is a widely reported contributor to neurodegenerative disease including Alzheimer's disease (AD), however, a mechanistic link between mitochondrial dysfunction and the initiation of neuropathology remains elusive. In AD, one of the earliest hallmark pathologies is neuropil threads comprising accumulated hyperphosphorylated microtubule-associated protein (MAP) tau in neurites. Rod-like aggregates of actin and its associated protein cofilin (AC rods) also occur in AD. Using a series of antibodies--AT270, AT8, AT100, S214, AT180, 12E8, S396, S404 and S422--raised against different phosphoepitopes on tau, we characterize the pattern of expression and re-distribution in neurites of these phosphoepitope labels during mitochondrial inhibition. Employing chick primary neuron cultures, we demonstrate that epitopes recognized by the monoclonal antibody 12E8, are the only species rapidly recruited into AC rods. These results were recapitulated with the actin depolymerizing drug Latrunculin B, which induces AC rods and a concomitant increase in the 12E8 signal measured on Western blot. This suggests that AC rods may be one way in which MAP redistribution and phosphorylation is influenced in neurons during mitochondrial stress and potentially in the early pathogenesis of AD.
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Project description:Tau is an intrinsically disordered protein with a central role in the pathology of a number of neurodegenerative diseases. Tau normally functions to stabilize neuronal microtubules, although the mechanism underlying this function is not well understood. Of note is that the interaction between tau and soluble tubulin, which has implications both in understanding tau function as well as its role in disease, is underexplored. Here we investigate the relationship between heterogeneity in tau-tubulin complexes and tau function. Specifically, we created a series of truncated and scrambled tau constructs and characterized the size and heterogeneity of the tau-tubulin complexes formed under nonpolymerizing conditions. Function of the constructs was verified by tubulin polymerization assays. We find that, surprisingly, the pseudo-repeat region of tau, which flanks the core microtubule-binding domain of tau, contributes largely to the formation of large, heterogeneous tau tubulin complexes; additional independent tubulin binding sites exist in repeats two and three of the microtubule binding domain. Of particular interest is that we find positive correlation between the size and heterogeneity of the complexes and rate of tau-promoted microtubule polymerization. We propose that tau-tubulin can be described as a "fuzzy" complex, and our results demonstrate the importance of heterogeneous complex formation in tau function. This work provides fundamental insights into the functional mechanism of tau, and more broadly underscores the relevance of heterogeneous and dynamic complexes in the functions of intrinsically disordered proteins.
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Project description:Primary cilia are organelles necessary for proper implementation of developmental and homeostasis processes. To initiate their assembly, coordinated actions of multiple proteins are needed. Tau tubulin kinase 2 (TTBK2) is a key player in the cilium assembly pathway, controlling the final step of cilia initiation. The function of TTBK2 in ciliogenesis is critically dependent on its kinase activity; however, the precise mechanism of TTBK2 action has so far not been fully understood due to the very limited information about its relevant substrates. In this study, we demonstrate that CEP83, CEP89, CCDC92, Rabin8, and DVL3 are substrates of TTBK2 kinase activity. Further, we characterize a set of phosphosites of those substrates and CEP164 induced by TTBK2 in vitro and in vivo. Intriguingly, we further show that identified TTBK2 phosphosites and consensus sequence delineated from those are distinct from motifs previously assigned to TTBK2. Finally, we show that TTBK2 is also required for efficient phosphorylation of many S/T sites in CEP164 and provide evidence that TTBK2-induced phosphorylations of CEP164 modulate its function, which in turn seems relevant for the process of cilia formation. In summary, our work provides important insight into the substrates-TTBK2 kinase relationship and suggests that phosphorylation of substrates on multiple sites by TTBK2 is probably involved in the control of ciliogenesis in human cells.
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Project description:The centrosome-associated C1orf96/Centriole, Cilia and Spindle-Associated Protein (CSAP) targets polyglutamylated tubulin in mitotic microtubules (MTs). Loss of CSAP causes critical defects in brain development; however, it is unclear how CSAP association with MTs affects mitosis progression. In this study, we explored the molecular mechanisms of the interaction of CSAP with mitotic spindles. Loss of CSAP caused MT instability in mitotic spindles and resulted in mislocalization of Nuclear protein that associates with the Mitotic Apparatus (NuMA), with defective MT dynamics. Thus, CSAP overload in the spindles caused extensive MT stabilization and recruitment of NuMA. Moreover, MT stabilization by CSAP led to high levels of polyglutamylation on MTs. MT depolymerization by cold or nocodazole treatment was inhibited by CSAP binding. Live-cell imaging analysis suggested that CSAP-dependent MT-stabilization led to centrosome-free MT aster formation immediately upon nuclear envelope breakdown without γ-tubulin. We therefore propose that CSAP associates with MTs around centrosomes to stabilize MTs during mitosis, ensuring proper bipolar spindle formation and maintenance.
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Project description:The crosstalk between microtubules and actin is essential for cellular functions. However, mechanisms underlying the microtubule-actin organization by cross-linkers remain largely unexplored. Here, we report that tau, a neuronal microtubule-associated protein, binds to microtubules and actin simultaneously, promoting in vitro co-organization and coupled growth of both networks. By developing an original assay to visualize concomitant microtubule and actin assembly, we show that tau can induce guided polymerization of actin filaments along microtubule tracks and growth of single microtubules along actin filament bundles. Importantly, tau mediates microtubule-actin co-alignment without changing polymer growth properties. Mutagenesis studies further reveal that at least two of the four tau repeated motifs, primarily identified as tubulin-binding sites, are required to connect microtubules and actin. Tau thus represents a molecular linker between microtubule and actin networks, enabling a coordination of the two cytoskeletons that might be essential in various neuronal contexts.
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Project description:Tubulin posttranslational modifications have been predicted to control cytoskeletal functions by coordinating the molecular interactions between microtubules and their associating proteins. A prominent tubulin modification in neurons is polyglutamylation, the deregulation of which causes neurodegeneration. Yet, the underlying molecular mechanisms have remained elusive. Here, using in-vitro reconstitution, we determine how polyglutamylation generated by the two predominant neuronal polyglutamylases, TTLL1 and TTLL7, specifically modulates the activities of three major microtubule interactors: the microtubule-associated protein Tau, the microtubule-severing enzyme katanin and the molecular motor kinesin-1. We demonstrate that the unique modification patterns generated by TTLL1 and TTLL7 differentially impact those three effector proteins, thus allowing for their selective regulation. Given that our experiments were performed with brain tubulin from mouse models in which physiological levels and patterns of polyglutamylation were altered by the genetic knockout of the main modifying enzymes, our quantitative measurements provide direct mechanistic insight into how polyglutamylation could selectively control microtubule interactions in neurons.
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Project description:Actin binding proteins expression levels when modifying the actin sequence
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Project description:Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on ?-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo-electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, ?TAT1, with microtubules and find that ?TAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of ?TAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.
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Project description:BackgroundThe microtubule associated protein Tau (MAPT) promotes assembly and interaction of microtubules with the cytoskeleton, impinging on axonal transport and synaptic plasticity. Its neuronal expression and intrinsic disorder implicate it in some 30 tauopathies such as Alzheimer's disease and frontotemporal dementia. These pathophysiological studies have yet to be complemented by computational analyses of its molecular evolution and structural models of all its functional domains to explain the molecular basis for its conservation profile, its site-specific interactions and the propensity to conformational disorder and aggregate formation.ResultsWe systematically annotated public sequence data to reconstruct unspliced MAPT, MAP2 and MAP4 transcripts spanning all represented genomes. Bayesian and maximum likelihood phylogenetic analyses, genetic linkage maps and domain architectures distinguished a nonvertebrate outgroup from the emergence of MAP4 and its subsequent ancestral duplication to MAP2 and MAPT. These events were coupled to other linked genes such as KANSL1L and KANSL and may thus be consequent to large-scale chromosomal duplications originating in the extant vertebrate genomes of hagfish and lamprey. Profile hidden Markov models (pHMMs), clustered subalignments and 3D structural predictions defined potential interaction motifs and specificity determining sites to reveal distinct signatures between the four homologous microtubule binding domains and independent divergence of the amino terminus.ConclusionThese analyses clarified ambiguities of MAPT nomenclature, defined the order, timing and pattern of its molecular evolution and identified key residues and motifs relevant to its protein interaction properties and pathogenic role. Additional unexpected findings included the expansion of cysteine-containing, microtubule binding domains of MAPT in cold adapted Antarctic icefish and the emergence of a novel multiexonic saitohin (STH) gene from repetitive elements in MAPT intron 11 of certain primate genomes.
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