RESUMO
Microtubule (MT) filaments, composed of α/ß-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They are nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, the molecular mechanism of nucleation remains elusive. Here we used cryo-electron tomography to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, γTuRC presents a ring of γ-tubulin subunits to seed nucleation of exclusively 13-protofilament MTs, adopting an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-electron tomography reconstruction revealed that a coiled-coil protein staples the first row of α/ß-tubulin of the MT to alternating positions along the γ-tubulin ring of γTuRC. This positioning of α/ß-tubulin onto γTuRC suggests a role for the coiled-coil protein in augmenting γTuRC-mediated MT nucleation. Based on our results, we describe a molecular model for budding yeast γTuRC activation and MT nucleation.
Assuntos
Microscopia Crioeletrônica , Microtúbulos , Modelos Moleculares , Saccharomyces cerevisiae , Fuso Acromático , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Microtúbulos/química , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Tomografia com Microscopia Eletrônica , Conformação Proteica , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/ultraestruturaRESUMO
Microtubules are composed of α-tubulin and ß-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale α/ß-tubulin tracks for intracellular transport that align with, rather than spiral along, the long axis of the filament. We report that the human ~2.3 MDa γ-tubulin ring complex (γ-TuRC), an essential regulator of microtubule formation that contains 14 γ-tubulins, selectively nucleates 13-protofilament microtubules. Cryogenic electron microscopy reconstructions of γ-TuRC-capped microtubule minus ends reveal the extensive intra-domain and inter-domain motions of γ-TuRC subunits that accommodate luminal bridge components and establish lateral and longitudinal interactions between γ-tubulins and α-tubulins. Our structures suggest that γ-TuRC, an inefficient nucleation template owing to its splayed conformation, can transform into a compacted cap at the microtubule minus end and set the lattice architecture of cellular microtubules.
Assuntos
Microscopia Crioeletrônica , Microtúbulos , Modelos Moleculares , Tubulina (Proteína) , Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/ultraestrutura , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestrutura , Humanos , Conformação Proteica , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/ultraestruturaRESUMO
The microtubule cytoskeleton forms complex macromolecular assemblies with a range of microtubule-associated proteins (MAPs) that have fundamental roles in cell architecture, division and motility. Determining how an individual MAP modulates microtubule behaviour is an important step in understanding the physiological roles of various microtubule assemblies. To characterize how MAPs control microtubule properties and functions, we developed an approach allowing for medium-throughput analyses of MAPs in cell-free conditions using lysates of mammalian cells. Our pipeline allows for quantitative as well as ultrastructural analyses of microtubule-MAP assemblies. Analysing 45 bona fide and potential mammalian MAPs, we uncovered previously unknown activities that lead to distinct and unique microtubule behaviours such as microtubule coiling or hook formation, or liquid-liquid phase separation along the microtubule lattice that initiates microtubule branching. We have thus established a powerful tool for a thorough characterization of a wide range of MAPs and MAP variants, thus opening avenues for the determination of mechanisms underlying their physiological roles and pathological implications.
Assuntos
Ensaios de Triagem em Larga Escala , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Imagem Individual de Molécula , Frações Subcelulares , Animais , Linhagem Celular Tumoral , Microscopia Crioeletrônica , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Microscopia de Vídeo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/genética , Microtúbulos/ultraestrutura , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/ultraestrutura , Transdução de Sinais , Fatores de Tempo , Imagem com Lapso de Tempo , Tubulina (Proteína)/metabolismoRESUMO
Microtubules (MTs) are polymers of αß-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.
Assuntos
Microscopia Crioeletrônica , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Humanos , Hidrólise , Cinesinas , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/genética , Proteínas Recombinantes , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestruturaRESUMO
Centriole biogenesis and maintenance are crucial for cells to generate cilia and assemble centrosomes that function as microtubule organizing centers (MTOCs). Centriole biogenesis and MTOC function both require the microtubule nucleator γ-tubulin ring complex (γTuRC). It is widely accepted that γTuRC nucleates microtubules from the pericentriolar material that is associated with the proximal part of centrioles. However, γTuRC also localizes more distally and in the centriole lumen, but the significance of these findings is unclear. Here we identify spatially and functionally distinct subpopulations of centrosomal γTuRC. Luminal localization is mediated by augmin, which is linked to the centriole inner scaffold through POC5. Disruption of luminal localization impairs centriole integrity and interferes with cilium assembly. Defective ciliogenesis is also observed in γTuRC mutant fibroblasts from a patient suffering from microcephaly with chorioretinopathy. These results identify a non-canonical role of augmin-γTuRC in the centriole lumen that is linked to human disease.
Assuntos
Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/ultraestrutura , Linhagem Celular , Centríolos/ultraestrutura , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Cílios , Feminino , Humanos , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/ultraestrutura , Centro Organizador dos Microtúbulos/ultraestrutura , Microtúbulos/metabolismo , NeurôniosRESUMO
The chromatin reader protein Spindlin1 plays an important role in epigenetic regulation, through which it has been linked to several types of malignant tumors. In the current work, we report on the development of novel analogs of the previously published lead inhibitor A366. In an effort to improve the activity and explore the structure-activity relationship (SAR), a series of 21 derivatives was synthesized, tested in vitro, and investigated by means of molecular modeling tools. Docking studies and molecular dynamics (MD) simulations were performed to analyze and rationalize the structural differences responsible for the Spindlin1 activity. The analysis of MD simulations shed light on the important interactions. Our study highlighted the main structural features that are required for Spindlin1 inhibitory activity, which include a positively charged pyrrolidine moiety embedded into the aromatic cage connected via a propyloxy linker to the 2-aminoindole core. Of the latter, the amidine group anchor the compounds into the pocket through salt bridge interactions with Asp184. Different protocols were tested to identify a fast in silico method that could help to discriminate between active and inactive compounds within the A366 series. Rescoring the docking poses with MM-GBSA calculations was successful in this regard. Because A366 is known to be a G9a inhibitor, the most active developed Spindlin1 inhibitors were also tested over G9a and GLP to verify the selectivity profile of the A366 analogs. This resulted in the discovery of diverse selective compounds, among which 1s and 1t showed Spindlin1 activity in the nanomolar range and selectivity over G9a and GLP. Finally, future design hypotheses were suggested based on our findings.
Assuntos
Fenômenos Biofísicos , Proteínas de Ciclo Celular/química , Epigênese Genética , Proteínas Associadas aos Microtúbulos/química , Fosfoproteínas/química , Conformação Proteica , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/ultraestrutura , Entropia , Humanos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/ultraestrutura , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/ultraestrutura , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Chromosomes pair and synapse with their homologous partners to segregate correctly at the first meiotic division. Association of telomeres with the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex composed of SUN1 and KASH5 enables telomere-led chromosome movements and telomere bouquet formation, facilitating precise pairwise alignment of homologs. Here, we identify a direct interaction between SUN1 and Speedy A (SPDYA) and determine the crystal structure of human SUN1-SPDYA-CDK2 ternary complex. Analysis of meiosis prophase I process in SPDYA-binding-deficient SUN1 mutant mice reveals that the SUN1-SPDYA interaction is required for the telomere-LINC complex connection and the assembly of a ring-shaped telomere supramolecular architecture at the nuclear envelope, which is critical for efficient homologous pairing and synapsis. Overall, our results provide structural insights into meiotic telomere structure that is essential for meiotic prophase I progression.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Prófase Meiótica I , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Telômero/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/ultraestrutura , Linhagem Celular Tumoral , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/isolamento & purificação , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 2 Dependente de Ciclina/ultraestrutura , Feminino , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/ultraestrutura , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Proteínas Associadas aos Microtúbulos/ultraestrutura , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
Microtubules are inherently dynamic cytoskeletal polymers whose length and organization can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle. It is comprised of a single layer of microtubules that are crosslinked to each other and to the overlying plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins at the array posterior, middle, and anterior. The array-associated protein PAVE1 stabilizes array microtubules at the cell posterior and is essential for maintaining its tapered shape. PAVE1 and the newly identified protein PAVE2 form a complex that binds directly to the microtubule lattice, demonstrating that they are a true kinetoplastid-specific MAP. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for maintaining the localization of array-associated proteins within their respective subdomains of the array. The arrangement of proteins within the array likely tunes the local properties of array microtubules and creates the asymmetric shape of the cell, which is essential for parasite viability.
Assuntos
Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/ultraestrutura , Trypanosoma brucei brucei/ultraestrutura , Tripanossomíase Africana/parasitologia , Ciclo Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/ultraestruturaRESUMO
The structural conservation across the AAA (ATPases associated with diverse cellular activities) protein family makes designing selective chemical inhibitors challenging. Here, we identify a triazolopyridine-based fragment that binds the AAA domain of human katanin, a microtubule-severing protein. We have developed a model for compound binding and designed ASPIR-1 (allele-specific, proximity-induced reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin does ASPIR-1 treatment increase the accumulation of CAMSAP2 at microtubule minus ends, confirming specific on-target cellular activity. Importantly, ASPIR-1 also selectively inhibits engineered cysteine mutants of human VPS4B and FIGL1-AAA proteins, involved in organelle dynamics and genome stability, respectively. Structural studies confirm our model for compound binding at the AAA ATPase site and the proximity-induced reactivity-based inhibition. Together, our findings suggest a chemical genetics approach to decipher AAA protein functions across essential cellular processes and to test hypotheses for developing therapeutics.
Assuntos
Proteínas AAA/genética , Katanina/genética , Proteínas Associadas aos Microtúbulos/genética , Piridinas/química , Proteínas AAA/antagonistas & inibidores , Proteínas AAA/ultraestrutura , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/ultraestrutura , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Humanos , Katanina/ultraestrutura , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/genética , Microtúbulos/ultraestrutura , Conformação Proteica/efeitos dos fármacos , Domínios Proteicos/genética , Piridinas/farmacologia , Triazóis/químicaRESUMO
Cortical dysplasia, complex, with other brain malformations 3 (CDCBM3) is a rare autosomal dominant syndrome caused by Kinesin family Member 2A (KIF2A) gene mutation. Patients with CDCBM3 exhibit posterior dominant agyria/pachygyria with severe motor dysfunction. Here, we report an 8-year-old boy with CDCBM3 showing a typical, but relatively mild, clinical presentation of CDCBM3 features. Whole-exome sequencing identified a heterozygous mutation of NM_001098511.2:c.1298C>A [p.(Ser433Tyr)]. To our knowledge, the mutation has never been reported previously. The variant was located distal to the nucleotide binding domain (NBD), in which previously-reported variants in CDCBM3 patients have been located. The computational structural analysis showed the p.433 forms the pocket with NBD. Variants in KIF2A have been reported in the NBD for CDCBM3, in the kinesin motor 3 domain, but not in the NBD in epilepsy, and outside of the kinesin motor domain in autism spectrum syndrome, respectively. Our patient has a variant, that is not in the NBD but at the pocket with the NBD, resulting in a clinical features of CDCBM3 with mild symptoms. The clinical findings of patients with KIF2A variants appear restricted to the central nervous system and facial anomalies. We can call this spectrum "KIF2A syndrome" with variable severity.
Assuntos
Epilepsia/genética , Cinesinas/genética , Malformações do Desenvolvimento Cortical/genética , Proteínas Associadas aos Microtúbulos/genética , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Criança , Epilepsia/diagnóstico , Epilepsia/diagnóstico por imagem , Epilepsia/patologia , Heterozigoto , Humanos , Cinesinas/ultraestrutura , Masculino , Malformações do Desenvolvimento Cortical/diagnóstico , Malformações do Desenvolvimento Cortical/diagnóstico por imagem , Malformações do Desenvolvimento Cortical/patologia , Proteínas Associadas aos Microtúbulos/ultraestrutura , Mutação de Sentido Incorreto/genética , Conformação Proteica , Tubulina (Proteína)/genética , Sequenciamento do ExomaRESUMO
Microtubules are essential cytoskeletal elements assembled from αß-tubulin dimers. In high eukaryotes, microtubule nucleation, the de novo assembly of a microtubule from its minus end, is initiated by the γ-tubulin ring complex (γ-TuRC). Despite many years of research, the structural and mechanistic principles of the microtubule nucleation machinery remained poorly understood. Only recently, cryoelectron microscopy studies uncovered the molecular organization and potential activation mechanisms of γ-TuRC. In vitro assays further deciphered the spatial and temporal cooperation between γ-TuRC and additional factors, for example, the augmin complex, the phase separation protein TPX2, and the microtubule polymerase XMAP215. These breakthroughs deepen our understanding of microtubule nucleation mechanisms and will link the assembly of individual microtubules to the organization of cellular microtubule networks.
Assuntos
Centro Organizador dos Microtúbulos/química , Microtúbulos/química , Tubulina (Proteína)/química , Animais , Microscopia Crioeletrônica , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/ultraestrutura , Centro Organizador dos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Polimerização , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestruturaRESUMO
OBJECTIVE: Most eukaryotic cells contain microtubule filaments, which play central roles in intra-cellular organization. However, microtubule networks have a wide variety of architectures from one cell type and organism to another. Nonetheless, the sequences of tubulins, of Microtubule Associated proteins (MAPs) and the structure of microtubules are usually well conserved throughout the evolution. MAPs being known to be responsible for regulating microtubule organization and dynamics, this raises the question of the conservation of their intrinsic properties. Indeed, knowing how the intrinsic properties of individual MAPs differ between organisms might enlighten our understanding of how distinct microtubule networks are built. End-Binding protein 1 (EB1), first described as a MAP in yeast, is conserved in plants and mammals. The intrinsic properties of the mammalian and the yeast EB1 proteins have been well described in the literature but, to our knowledge, the intrinsic properties of EB1 from plant and mammals have not been compared thus far. RESULTS: Here, using an in vitro assay, we discovered that plant and mammalian EB1 purified proteins have different intrinsic properties on microtubule dynamics. Indeed, the mammalian EB1 protein increases microtubules dynamic while the plant EB1 protein stabilizes them.
Assuntos
Proteínas de Arabidopsis , Proteínas Associadas aos Microtúbulos , Microtúbulos , Animais , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/ultraestrutura , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestruturaRESUMO
BACKGROUND: TgDCX is a doublecortin-domain protein associated with the conoid fibers, a set of strongly curved non-tubular tubulin-polymers in Toxoplasma. TgDCX deletion impairs conoid structure and parasite invasion. TgDCX contains two tubulin-binding domains: a partial P25α and the DCX/doublecortin domain. Orthologues are found in apicomplexans and their free-living relatives Chromera and Vitrella. RESULTS: We report that isolated TgDCX-containing conoid fibers retain their pronounced curvature, but loss of TgDCX destabilizes the fibers. We crystallized and determined the 3D-structure of the DCX-domain, which is similar to those of human doublecortin and well-conserved among TgDCX orthologues. However, the orthologues vary widely in targeting to the conoid in Toxoplasma and in modulating microtubule organization in Xenopus cells. Several orthologues bind to microtubules in Xenopus cells, but only TgDCX generates short, strongly curved microtubule arcs. EM analysis shows microtubules decorated with TgDCX bundled into rafts, often bordered on one edge by a "C"-shaped incomplete tube. A Chromera orthologue closely mimics TgDCX targeting in Toxoplasma and binds to microtubules in Xenopus cells, but does not generate arcs or "C"-shaped tubes, and fails to rescue the defects of the TgDCX-knockout parasite. CONCLUSIONS: These observations suggest that species-specific features of TgDCX enable it to generate strongly curved tubulin-polymers to support efficient host-cell invasion.
Assuntos
Proteínas Associadas aos Microtúbulos/química , Neuropeptídeos/química , Toxoplasma/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Técnicas de Inativação de Genes , Interações Hospedeiro-Parasita/genética , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Polímeros/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos/genética , Proteínas Recombinantes , Toxoplasma/química , Toxoplasma/efeitos dos fármacos , Toxoplasma/ultraestrutura , Tubulina (Proteína)/química , XenopusRESUMO
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
Microtubules are dynamic polymers of α- and ß-tubulin and have crucial roles in cell signalling, cell migration, intracellular transport and chromosome segregation1. They assemble de novo from αß-tubulin dimers in an essential process termed microtubule nucleation. Complexes that contain the protein γ-tubulin serve as structural templates for the microtubule nucleation reaction2. In vertebrates, microtubules are nucleated by the 2.2-megadalton γ-tubulin ring complex (γ-TuRC), which comprises γ-tubulin, five related γ-tubulin complex proteins (GCP2-GCP6) and additional factors3. GCP6 is unique among the GCP proteins because it carries an extended insertion domain of unknown function. Our understanding of microtubule formation in cells and tissues is limited by a lack of high-resolution structural information on the γ-TuRC. Here we present the cryo-electron microscopy structure of γ-TuRC from Xenopus laevis at 4.8 Å global resolution, and identify a 14-spoked arrangement of GCP proteins and γ-tubulins in a partially flexible open left-handed spiral with a uniform sequence of GCP variants. By forming specific interactions with other GCP proteins, the GCP6-specific insertion domain acts as a scaffold for the assembly of the γ-TuRC. Unexpectedly, we identify actin as a bona fide structural component of the γ-TuRC with functional relevance in microtubule nucleation. The spiral geometry of γ-TuRC is suboptimal for microtubule nucleation and a controlled conformational rearrangement of the γ-TuRC is required for its activation. Collectively, our cryo-electron microscopy reconstructions provide detailed insights into the molecular organization, assembly and activation mechanism of vertebrate γ-TuRC, and will serve as a framework for the mechanistic understanding of fundamental biological processes associated with microtubule nucleation, such as meiotic and mitotic spindle formation and centriole biogenesis4.
Assuntos
Microscopia Crioeletrônica , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Xenopus , Actinas/química , Actinas/metabolismo , Actinas/ultraestrutura , Animais , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Modelos Moleculares , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestruturaRESUMO
CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which lacks minus-end specificity. Here we report near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes, which show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit different footprints on microtubules. NMR experiments show that both HsCKK and NgCKK are remarkably rigid. However, whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. Thus, in contrast to many MAPs, the HsCKK domain can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition.
Assuntos
Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/ultraestrutura , Naegleria/ultraestrutura , Tubulina (Proteína)/ultraestrutura , Microscopia Crioeletrônica , Humanos , Espectroscopia de Ressonância Magnética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Modelos Moleculares , Naegleria/metabolismo , Ligação Proteica , Domínios Proteicos , Tubulina (Proteína)/metabolismoRESUMO
Selective autophagy sequesters cytoplasmic cargo for lysosomal degradation via the binding of autophagy receptors to Atg8 (autophagy-related 8) family proteins on the autophagic membrane. The sole yeast Atg8 gene has six mAtg8 (mammalian Atg8) homologs, including the MAP1LC3 (microtubule-associated protein-1 light chain 3) family and the GABA receptor-associated proteins. Selective autophagy receptors interact with two conserved hydrophobic pockets (termed the W-site and L-site) of mATG8 proteins through a linear motif called the LC3-interacting region (LIR) with the general composition (W/F/Y)XX(I/L/V). To address a lack in our knowledge regarding LIR peptide specificity toward each mATG8 homolog, here we used competitive time-resolved FRET to sensitively and quantitatively characterize the interactions between LIRs and mAtg8. We report that 14 representative LIR-containing peptides display differential binding affinities toward the mAtg8 proteins and identified the LIR domain peptide of TP53INP1 as exhibiting high affinity for all six mATG8 proteins. Using peptide truncation studies, we found that both N- and C-terminal acidic residues, as well as the C-terminal Cys residue of the TP53INP1 LIR peptide, are required for its high-affinity binding to LC3A and LC3B, whereas binding to the GABARAP subfamily proteins was facilitated by residues either N-terminal or C-terminal to the core motif. Finally, we used NMR chemical shift perturbation analysis to gain molecular insights into these findings. Collectively, our results may aid in the development of molecules that selectively disrupt specific mATG8-LIR interactions to dissect the biological roles of the six mATG8 homologs for potential therapeutic applications.
Assuntos
Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/ultraestrutura , Proteínas Associadas aos Microtúbulos/metabolismo , Motivos de Aminoácidos , Animais , Autofagia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodos , Proteínas Associadas aos Microtúbulos/ultraestrutura , Mitofagia , Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/fisiologiaRESUMO
Misfolding of the microtubule-binding protein tau into filamentous aggregates is characteristic of many neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Determining the structures and dynamics of these tau fibrils is important for designing inhibitors against tau aggregation. Tau fibrils obtained from patient brains have been found by cryo-electron microscopy to adopt disease-specific molecular conformations. However, in vitro heparin-fibrillized 2N4R tau, which contains all four microtubule-binding repeats (4R), was recently found to adopt polymorphic structures. Here we use solid-state NMR spectroscopy to investigate the global fold and dynamics of heparin-fibrillized 0N4R tau. A single set of 13C and 15N chemical shifts was observed for residues in the four repeats, indicating a single ß-sheet conformation for the fibril core. This rigid core spans the R2 and R3 repeats and adopts a hairpin-like fold that has similarities to but also clear differences from any of the polymorphic 2N4R folds. Obtaining a homogeneous fibril sample required careful purification of the protein and removal of any proteolytic fragments. A variety of experiments and polarization transfer from water and mobile side chains indicate that 0N4R tau fibrils exhibit heterogeneous dynamics: Outside the rigid R2-R3 core, the R1 and R4 repeats are semirigid even though they exhibit ß-strand character and the proline-rich domains undergo large-amplitude anisotropic motions, whereas the two termini are nearly isotropically flexible. These results have significant implications for the structure and dynamics of 4R tau fibrils in vivo.
Assuntos
Doença de Alzheimer/genética , Citoesqueleto/ultraestrutura , Proteínas Associadas aos Microtúbulos/química , Proteínas tau/química , Doença de Alzheimer/patologia , Sequência de Aminoácidos/genética , Microscopia Crioeletrônica , Citoesqueleto/química , Citoesqueleto/patologia , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/química , Microtúbulos/genética , Ressonância Magnética Nuclear Biomolecular , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Ligação Proteica/genética , Conformação Proteica em Folha beta/genética , Domínios Proteicos/genética , Estrutura Secundária de Proteína , Proteínas tau/genética , Proteínas tau/ultraestruturaRESUMO
End-binding (EB) proteins associate with the growing tips of microtubules (MTs)and modulate their dynamics directly and indirectly, by recruiting essential factors to fine-tune MTs for their many essential roles in cells. Previously EB proteins have been shown to recognize a stabilizing GTP/GDP-Pi cap at the tip of growing MTs, but information about additional EB-binding zones on MTs has been limited. In this work, we studied fluorescence intensity profiles of one of the three mammalian EB-proteins, EB3, fused with red fluorescent protein (RFP). The distribution of EB3 on MTs in mouse fibroblasts frequently deviated from single exponential decay and exhibited secondary peaks. Those secondary peaks, which we refer to as EB3-islands, were detected on 56% comets of growing MTs and were encountered once per 44 s of EB3-RFP comet growth time with about 5 s half-lifetime. The majority of EB3-islands in the vicinity of MT tips was stationary and originated from EB3 comets moving with the growing MT tips. Computational modeling of the decoration of dynamic MT tips by EB3 suggested that the EB3-islands could not be explained simply by a stochastic first-order GTP hydrolysis/phosphate release. We speculate that additional protein factors contribute to EB3 residence time on MTs in cells, likely affecting MT dynamics.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/metabolismo , Animais , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Proteínas Luminescentes , Camundongos , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/fisiologia , Células NIH 3T3 , Ligação Proteica/fisiologia , Proteína Vermelha FluorescenteRESUMO
Microtubules form from longitudinally and laterally assembling tubulin α-ß dimers. The assembly induces strain in tubulin, resulting in cycles of microtubule catastrophe and regrowth. This 'dynamic instability' is governed by GTP hydrolysis that renders the microtubule lattice unstable, but it is unclear how. We used a human microtubule nucleating and stabilizing neuronal protein, doublecortin, and high-resolution cryo-EM to capture tubulin's elusive hydrolysis intermediate GDPâ¢Pi state, alongside the prehydrolysis analog GMPCPP state and the posthydrolysis GDP state with and without an anticancer drug, Taxol. GTP hydrolysis to GDPâ¢Pi followed by Pi release constitutes two distinct structural transitions, causing unevenly distributed compressions of tubulin dimers, thereby tightening longitudinal and loosening lateral interdimer contacts. We conclude that microtubule catastrophe is triggered because the lateral contacts can no longer counteract the strain energy stored in the lattice, while reinforcement of the longitudinal contacts may support generation of force.