RESUMO
The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of ß-tubulin using human prefoldin and TRiC. We find unstructured ß-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded ß-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin's core and middle domains and GTP-binding. Thus, TRiC's chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.
Assuntos
Chaperonina com TCP-1 , Tubulina (Proteína) , Humanos , Chaperonina com TCP-1/química , Tubulina (Proteína)/metabolismo , Dobramento de Proteína , Proteostase , Trifosfato de Adenosina/metabolismoRESUMO
Microtubules are essential dynamic polymers composed of α/ß-tubulin heterodimers. They support intracellular trafficking, cell division, cellular motility, and other essential cellular processes. In many species, both α-tubulin and ß-tubulin are encoded by multiple genes with distinct expression profiles and functionality. Microtubules are further diversified through abundant posttranslational modifications, which are added and removed by a suite of enzymes to form complex, stereotyped cellular arrays. The genetic and chemical diversity of tubulin constitute a tubulin code that regulates intrinsic microtubule properties and is read by cellular effectors, such as molecular motors and microtubule-associated proteins, to provide spatial and temporal specificity to microtubules in cells. In this review, we synthesize the rapidly expanding tubulin code literature and highlight limitations and opportunities for the field. As complex microtubule arrays underlie essential physiological processes, a better understanding of how cells employ the tubulin code has important implications for human disease ranging from cancer to neurological disorders.
Assuntos
Microtúbulos , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/genética , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional/genética , Movimento CelularRESUMO
The microtubule (MT) cytoskeleton provides the architecture that governs intracellular organization and the regulated motion of macromolecules through the crowded cytoplasm. The key to establishing a functioning cytoskeletal architecture is regulating when and where new MTs are nucleated. Within the spindle, the vast majority of MTs are generated through a pathway known as branching MT nucleation, which exponentially amplifies MT number in a polar manner. Whereas other MT nucleation pathways generally require a complex organelle such as the centrosome or Golgi apparatus to localize nucleation factors, the branching site is based solely on a simple, preformed MT, making it an ideal system to study MT nucleation. In this review, we address recent developments in characterizing branching factors, the branching reaction, and its regulation, as well as branching MT nucleation in systems beyond the spindle and within human disease.
Assuntos
Centro Organizador dos Microtúbulos , Fuso Acromático , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
The γ-tubulin ring complex (γ-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation, yet its structure is not known. Here, we present a cryo-EM reconstruction of the native human γ-TuRC at â¼3.8 Å resolution, revealing an asymmetric, cone-shaped structure. Pseudo-atomic models indicate that GCP4, GCP5, and GCP6 form distinct Y-shaped assemblies that structurally mimic GCP2/GCP3 subcomplexes distal to the γ-TuRC "seam." We also identify an unanticipated structural bridge that includes an actin-like protein and spans the γ-TuRC lumen. Despite its asymmetric architecture, the γ-TuRC arranges γ-tubulins into a helical geometry poised to nucleate microtubules. Diversity in the γ-TuRC subunits introduces large (>100,000 Å2) surfaces in the complex that allow for interactions with different regulatory factors. The observed compositional complexity of the γ-TuRC could self-regulate its assembly into a cone-shaped structure to control microtubule formation across diverse contexts, e.g., within biological condensates or alongside existing filaments.
Assuntos
Centro Organizador dos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/ultraestrutura , Tubulina (Proteína)/ultraestrutura , Actinas/metabolismo , Microscopia Crioeletrônica/métodos , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells. In humans, impaired axoneme function causes a range of ciliopathies. Axoneme assembly, structure, and motility require a radially arranged set of doublet microtubules, each decorated in repeating patterns with non-tubulin components. We use single-particle cryo-electron microscopy to visualize and build an atomic model of the repeating structure of a native axonemal doublet microtubule, which reveals the identities, positions, repeat lengths, and interactions of 38 associated proteins, including 33 microtubule inner proteins (MIPs). The structure demonstrates how these proteins establish the unique architecture of doublet microtubules, maintain coherent periodicities along the axoneme, and stabilize the microtubules against the repeated mechanical stress induced by ciliary motility. Our work elucidates the architectural principles that underpin the assembly of this large, repetitive eukaryotic structure and provides a molecular basis for understanding the etiology of human ciliopathies.
Assuntos
Axonema/ultraestrutura , Cílios/ultraestrutura , Ciliopatias/patologia , Microtúbulos/ultraestrutura , Axonema/química , Axonema/genética , Movimento Celular/genética , Cílios/química , Cílios/genética , Ciliopatias/genética , Ciliopatias/metabolismo , Microscopia Crioeletrônica , Humanos , Proteínas dos Microtúbulos/química , Proteínas dos Microtúbulos/ultraestrutura , Microtúbulos/química , Microtúbulos/genética , Estresse MecânicoRESUMO
Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and â¼200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes.
Assuntos
Centrossomo/metabolismo , Cinetocoros/metabolismo , Metáfase , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Centrossomo/ultraestrutura , Segregação de Cromossomos , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Cinetocoros/ultraestrutura , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Transdução de Sinais , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Zigoto/citologia , Zigoto/metabolismoRESUMO
The organization of microtubule networks is crucial for controlling chromosome segregation during cell division, for positioning and transport of different organelles, and for cell polarity and morphogenesis. The geometry of microtubule arrays strongly depends on the localization and activity of the sites where microtubules are nucleated and where their minus ends are anchored. Such sites are often clustered into structures known as microtubule-organizing centers, which include the centrosomes in animals and spindle pole bodies in fungi. In addition, other microtubules, as well as membrane compartments such as the cell nucleus, the Golgi apparatus, and the cell cortex, can nucleate, stabilize, and tether microtubule minus ends. These activities depend on microtubule-nucleating factors, such as γ-tubulin-containing complexes and their activators and receptors, and microtubule minus end-stabilizing proteins with their binding partners. Here, we provide an overview of the current knowledge on how such factors work together to control microtubule organization in different systems.
Assuntos
Centro Organizador dos Microtúbulos/metabolismo , Animais , Divisão Celular , Centrossomo/metabolismo , Complexo de Golgi/metabolismo , Humanos , Modelos Biológicos , Membrana Nuclear/metabolismoRESUMO
Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates the decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER, in turn, engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.
Assuntos
Ribossomos , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Microtúbulos/metabolismo , Homeostase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estabilidade de RNA , Proteínas de Transporte/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Small molecules that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biology research and as clinical anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, we introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biology, and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.
Assuntos
Antimitóticos/química , Morte Celular , Microtúbulos/efeitos dos fármacos , Mitose , Estilbenos/química , Animais , Antimitóticos/toxicidade , Linhagem Celular Tumoral , Citoesqueleto/química , Humanos , Luz , Camundongos , Polimerização , Estilbenos/toxicidadeRESUMO
Although it is known that the centrioles play instructive roles in pericentriolar material (PCM) assembly and that the PCM is essential for proper centriole formation, the mechanism that governs centriole-PCM interaction is poorly understood. Here, we show that ATF5 forms a characteristic 9-fold symmetrical ring structure in the inner layer of the PCM outfitting the proximal end of the mother centriole. ATF5 controls the centriole-PCM interaction in a cell-cycle- and centriole-age-dependent manner. Interaction of ATF5 with polyglutamylated tubulin (PGT) on the mother centriole and with PCNT in the PCM renders ATF5 as a required molecule in mother centriole-directed PCM accumulation and in PCM-dependent centriole formation. ATF5 depletion blocks PCM accumulation at the centrosome and causes fragmentation of centrioles, leading to the formation of multi-polar mitotic spindles and genomic instability. These data show that ATF5 is an essential structural protein that is required for the interaction between the mother centriole and the PCM.
Assuntos
Fatores Ativadores da Transcrição/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Citoesqueleto/metabolismo , Regulação para Baixo , Instabilidade Genômica , Células HeLa , Humanos , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/ß-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.
Assuntos
Proteínas Associadas aos Microtúbulos , Microtúbulos , Tubulina (Proteína) , Xenopus laevis , Proteína ran de Ligação ao GTP , Animais , Microscopia Crioeletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Proteína ran de Ligação ao GTP/genética , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genéticaRESUMO
Rigosertib is a styryl benzyl sulfone that inhibits growth of tumor cells and acts as a RAS mimetic by binding to Ras binding domains of RAS effectors. A recent study attributed rigosertib's mechanism of action to microtubule binding. In that study, rigosertib was obtained from a commercial vendor. We compared the purity of clinical-grade and commercially sourced rigosertib and found that commercially sourced rigosertib contains approximately 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical-grade rigosertib, which is free of this impurity, does not exhibit tubulin-binding activity. Cell lines expressing mutant ß-tubulin have also been reported to be resistant to rigosertib. However, our study showed that these cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Rigosertib induced a senescence-like phenotype in the small percentage of surviving cells, which could be incorrectly scored as resistant using short-term cultures.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células , Glicina/análogos & derivados , Neoplasias Pulmonares/patologia , Sulfonas/farmacologia , Tubulina (Proteína)/metabolismo , Contaminação de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Glicina/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Mutação , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Células Tumorais CultivadasRESUMO
The cell orchestrates the dance of chromosome segregation with remarkable speed and fidelity. The mitotic spindle is built from scratch after interphase through microtubule (MT) nucleation, which is dependent on the γ-tubulin ring complex (γ-TuRC), the universal MT template. Although several MT nucleation pathways build the spindle framework, the question of when and how γ-TuRC is targeted to these nucleation sites in the spindle and subsequently activated remains an active area of investigation. Recent advances facilitated the discovery of new MT nucleation effectors and their mechanisms of action. In this review, we illuminate each spindle assembly pathway and subsequently consider how the pathways are merged to build a spindle.
Assuntos
Proteínas Associadas aos Microtúbulos , Tubulina (Proteína) , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismo , Centro Organizador dos Microtúbulos/metabolismoRESUMO
Intracellular organelle organization is conserved in eukaryotic cells and is primarily achieved through active transport by motor proteins along the microtubule cytoskeleton. Microtubule post-translational modifications (PTMs) can contribute to microtubule diversity and differentially regulate motor-mediated transport. Here, we show that centrosome amplification, commonly observed in cancer and shown to promote aneuploidy and invasion, induces a global change in organelle positioning towards the cell periphery and facilitates nuclear migration through confined spaces. This reorganization requires kinesin-1 and is analogous to the loss of dynein. Cells with amplified centrosomes display increased levels of acetylated tubulin, a PTM that could enhance kinesin-1-mediated transport. Depletion of α-tubulin acetyltransferase 1 (αTAT1) to block tubulin acetylation rescues the displacement of centrosomes, mitochondria, and vimentin but not Golgi or endosomes. Analyses of the distribution of total and acetylated microtubules indicate that the polarized distribution of modified microtubules, rather than levels alone, plays an important role in the positioning of specific organelles, such as the centrosome. We propose that increased tubulin acetylation differentially impacts kinesin-1-mediated organelle displacement to regulate intracellular organization.
Assuntos
Cinesinas , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Acetilação , Microtúbulos/metabolismo , Centrossomo/metabolismo , Dineínas/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
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.
Assuntos
Tubulina (Proteína) , Animais , Camundongos , Citoesqueleto/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Peptídeo Sintases , Proteínas Associadas aos MicrotúbulosRESUMO
Cilia are microtubule (MT)-based organelles present on the surface of nearly all vertebrate cells. MTs are polymers of α- and ß-tubulins that are each encoded by multiple, individual isotype genes. Tubulin isotype composition is thought to influence MT behaviors. Ciliary MTs differ from other MTs in the cell in terms of organization, stability and post-translational modifications. However, little is known about the tubulin isotypes that build ciliary MTs and the functional requirements for tubulin isotypes in cilia have not been examined in vertebrates. Here, we have tested the role of the ß-tubulin isotype genes in the mouse that harbor a conserved amino acid motif associated with ciliated organisms. We found that Tubb4b localizes to cilia in multi-ciliated cells (MCCs) specifically. In respiratory and oviduct MCCs, Tubb4b is asymmetrically localized within multi-cilia, indicating that the tubulin isotype composition changes along the length of the ciliary axonemal MTs. Deletion of Tubb4b resulted in striking structural defects within the axonemes of multi-cilia, without affecting primary cilia. These studies show that Tubb4b is essential for the formation of a specific MT-based subcellular organelle and sheds light on the requirements of tubulin isotypes in cilia.
Assuntos
Cílios , Tubulina (Proteína) , Animais , Camundongos , Axonema/metabolismo , Cílios/metabolismo , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Nanomaterials acquire a biomolecular corona upon introduction to biological media, leading to biological transformations such as changes in protein function, unmasking of epitopes, and protein fibrilization. Ex vivo studies to investigate the effect of nanoparticles on protein-protein interactions are typically performed in buffer and are rarely measured quantitatively in live cells. Here, we measure the differential effect of silica nanoparticles on protein association in vitro vs. in mammalian cells. BtubA and BtubB are a pair of bacterial tubulin proteins identified in Prosthecobacter strains that self-assemble like eukaryotic tubulin, first into dimers and then into microtubules in vitro or in vivo. Förster resonance energy transfer labeling of each of the Btub monomers with a donor (mEGFP) and acceptor (mRuby3) fluorescent protein provides a quantitative tool to measure their binding interactions in the presence of unfunctionalized silica nanoparticles in buffer and in cells using fluorescence spectroscopy and microscopy. We show that silica nanoparticles enhance BtubAB dimerization in buffer due to protein corona formation. However, these nanoparticles have little effect on bacterial tubulin self-assembly in the complex mammalian cellular environment. Thus, the effect of nanomaterials on protein-protein interactions may not be readily translated from the test tube to the cell in the absence of particle surface functionalization that can enable targeted protein-nanoparticle interactions to withstand competitive binding in the nanoparticle corona from other biomolecules.
Assuntos
Proteínas de Bactérias , Nanopartículas , Dióxido de Silício , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Nanopartículas/química , Dióxido de Silício/química , Dióxido de Silício/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Transferência Ressonante de Energia de Fluorescência , Humanos , Microtúbulos/metabolismo , Multimerização Proteica , Ligação ProteicaRESUMO
Mutations in Cytosolic Carboxypeptidase-like Protein 5 (CCP5) are associated with vision loss in humans. To decipher the mechanisms behind CCP5-associated blindness, we generated a novel mouse model lacking CCP5. In this model, we found that increased tubulin glutamylation led to progressive cone-rod dystrophy, with cones showing a more pronounced and earlier functional loss than rod photoreceptors. The observed functional reduction was not due to cell death, levels, or the mislocalization of major phototransduction proteins. Instead, the increased tubulin glutamylation caused shortened photoreceptor axonemes and the formation of numerous abnormal membranous whorls that disrupted the integrity of photoreceptor outer segments (OS). Ultimately, excessive tubulin glutamylation led to the progressive loss of photoreceptors, affecting cones more severely than rods. Our results highlight the importance of maintaining tubulin glutamylation for normal photoreceptor function. Furthermore, we demonstrate that murine cone photoreceptors are more sensitive to disrupted tubulin glutamylation levels than rods, suggesting an essential role for axoneme in the structural integrity of the cone outer segment. This study provides valuable insights into the mechanisms of photoreceptor diseases linked to excessive tubulin glutamylation.
Assuntos
Distrofias de Cones e Bastonetes , Tubulina (Proteína) , Humanos , Camundongos , Animais , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Distrofias de Cones e Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , MutaçãoRESUMO
During development, neurons achieve a stereotyped neuron type-specific morphology, which relies on dynamic support by microtubules (MTs). An important player is the augmin complex (hereafter augmin), which binds to existing MT filaments and recruits the γ-tubulin ring complex (γ-TuRC), to form branched MTs. In cultured neurons, augmin is important for neurite formation. However, little is known about the role of augmin during neurite formation in vivo. Here, we have revisited the role of mammalian augmin in culture and then turned towards the class four Drosophila dendritic arborization (c4da) neurons. We show that MT density is maintained through augmin in cooperation with the γ-TuRC in vivo. Mutant c4da neurons show a reduction of newly emerging higher-order dendritic branches and in turn also a reduced number of their characteristic space-filling higher-order branchlets. Taken together, our data reveal a cooperative function for augmin with the γ-TuRC in forming enough MTs needed for the appropriate differentiation of morphologically complex dendrites in vivo.
Assuntos
Dendritos , Proteínas de Drosophila , Proteínas Associadas aos Microtúbulos , Microtúbulos , Animais , Microtúbulos/metabolismo , Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Drosophila melanogaster/metabolismo , Tubulina (Proteína)/metabolismo , Drosophila/metabolismo , Humanos , Neurônios/metabolismo , Neurônios/citologiaRESUMO
In neurons, the microtubule (MT) cytoskeleton forms the basis for long-distance protein transport from the cell body into and out of dendrites and axons. To maintain neuronal polarity, the axon initial segment (AIS) serves as a physical barrier, separating the axon from the somatodendritic compartment and acting as a filter for axonal cargo. Selective trafficking is further instructed by axonal enrichment of MT post-translational modifications, which affect MT dynamics and the activity of motor proteins. Here, we compared two knockout mouse lines lacking the respective enzymes for MT tyrosination and detyrosination, and found that both knockouts led to a shortening of the AIS. Neurons from both lines also showed an increased immobile fraction of endolysosomes present in the axon, whereas mobile organelles displayed shortened run distances in the retrograde direction. Overall, our results highlight the importance of maintaining the balance of tyrosinated and detyrosinated MTs for proper AIS length and axonal transport processes.