RESUMEN
Developing programmable bacterial cell-cell adhesion is of significant interest due to its versatile applications. Current methods that rely on presenting cell adhesion molecules (CAMs) on bacterial surfaces are limited by the lack of a generalizable strategy to identify such molecules targeting bacterial membrane proteins in their natural states. Here, we introduce a whole-cell screening platform designed to discover CAMs targeting bacterial membrane proteins within a synthetic bacteria-displayed nanobody library. Leveraging the potency of the bacterial type IV secretion system-a contact-dependent DNA delivery nanomachine-we have established a positive feedback mechanism to selectively enrich for bacteria displaying nanobodies that target antigen-expressing cells. Our platform successfully identified functional CAMs capable of recognizing three distinct outer membrane proteins (TraN, OmpA, OmpC), demonstrating its efficacy in CAM discovery. This approach holds promise for engineering bacterial cell-cell adhesion, such as directing the antibacterial activity of programmed inhibitor cells toward target bacteria in mixed populations.
Asunto(s)
Adhesión Bacteriana , Moléculas de Adhesión Celular , Anticuerpos de Dominio Único , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/genética , Anticuerpos de Dominio Único/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Escherichia coli/metabolismo , Bacterias/metabolismoRESUMEN
Human Cep57 is a coiled-coil scaffold at the pericentriolar matrix (PCM), controlling centriole duplication and centrosome maturation for faithful cell division. Genetic truncation mutations of Cep57 are associated with the mosaic-variegated aneuploidy (MVA) syndrome. During interphase, Cep57 forms a complex with Cep63 and Cep152, serving as regulators for centrosome maturation. However, the molecular interplay of Cep57 with these essential scaffolding proteins remains unclear. Here, we demonstrate that Cep57 undergoes liquid-liquid phase separation (LLPS) driven by three critical domains (NTD, CTD, and polybasic LMN). In vitro Cep57 condensates catalyze microtubule nucleation via the LMN motif-mediated tubulin concentration. In cells, the LMN motif is required for centrosomal microtubule aster formation. Moreover, Cep63 restricts Cep57 assembly, expansion, and microtubule polymerization activity. Overexpression of competitive constructs for multivalent interactions, including an MVA mutation, leads to excessive centrosome duplication. In Cep57-depleted cells, self-assembly mutants failed to rescue centriole disengagement and PCM disorganization. Thus, Cep57's multivalent interactions are pivotal for maintaining the accurate structural and functional integrity of human centrosomes.
Asunto(s)
Proteínas de Ciclo Celular , Centriolos , Centrosoma , Microtúbulos , Humanos , Centrosoma/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Microtúbulos/metabolismo , Centriolos/metabolismo , Centriolos/genética , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Mutación , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Unión Proteica , Proteínas NuclearesRESUMEN
Efficiently delivering exogenous materials into primary neurons and neural stem cells (NSCs) has long been a challenge in neurobiology. Existing methods have struggled with complex protocols, unreliable reproducibility, high immunogenicity, and cytotoxicity, causing a huge conundrum and hindering in-depth analyses. Here, we establish a cutting-edge method for transfecting primary neurons and NSCs, named teleofection, by a two-step process to enhance the formation of biocompatible calcium phosphate (CaP) nanoparticles. Teleofection enables both nucleic acid and protein transfection into primary neurons and NSCs, eliminating the need for specialized skills and equipment. It can easily fine-tune transfection efficiency by adjusting the incubation time and nanoparticle quantity, catering to various experimental requirements. Teleofection's versatility allows for the delivery of different cargos into the same cell culture, whether simultaneously or sequentially. This flexibility proves invaluable for long-term studies, enabling the monitoring of neural development and synapse plasticity. Moreover, teleofection ensures the consistent and robust expression of delivered genes, facilitating molecular and biochemical investigations. Teleofection represents a significant advancement in neurobiology, which has promise to transcend the limitations of current gene delivery methods. It offers a user-friendly, cost-effective, and reproducible approach for researchers, potentially revolutionizing our understanding of brain function and development.
Asunto(s)
Nanopartículas , Células-Madre Neurales , Ácidos Nucleicos , Ácidos Nucleicos/metabolismo , Reproducibilidad de los Resultados , Células-Madre Neurales/metabolismo , Nanopartículas/química , Transfección , Fosfatos de Calcio/químicaRESUMEN
The transcription factor TATA-box binding protein (TBP) modulates gene expression in nuclei. This process requires the involvement of nuclear transport receptors, collectively termed karyopherin-ß (Kap-ß) in yeast, and various regulatory factors. In previous studies we showed that Kap114p, a Kap-ß that mediates nuclear import of yeast TBP (yTBP), modulates yTBP-dependent transcription. However, how Kap114p associates with yTBP to exert its multifaceted functions has remained elusive. Here, we employ single-particle cryo-electron microscopy to determine the structure of Kap114p in complex with the core domain of yTBP (yTBPC). Remarkably, Kap114p wraps around the yTBPC N-terminal lobe, revealing a structure resembling transcriptional regulators in complex with TBP, suggesting convergent evolution of the two protein groups for a common function. We further demonstrate that Kap114p sequesters yTBP away from promoters, preventing a collapse of yTBP dynamics required for yeast responses to environmental stress. Hence, we demonstrate that nuclear transport receptors represent critical elements of the transcriptional regulatory network.
Asunto(s)
Saccharomyces cerevisiae , Factores de Transcripción , Transporte Activo de Núcleo Celular , Proteína de Unión a TATA-Box/genética , Saccharomyces cerevisiae/genética , Microscopía por Crioelectrón , Factores de Transcripción/genética , Receptores Citoplasmáticos y Nucleares/genética , beta Carioferinas/genéticaRESUMEN
The centrosome, a non-membranous organelle, constrains various soluble molecules locally to execute its functions. As the centrosome is surrounded by various dense components, we hypothesized that it may be bordered by a putative diffusion barrier. After quantitatively measuring the trapping kinetics of soluble proteins of varying size at centrosomes by a chemically inducible diffusion trapping assay, we find that centrosomes are highly accessible to soluble molecules with a Stokes radius of less than 5.8 nm, whereas larger molecules rarely reach centrosomes, indicating the existence of a size-dependent diffusion barrier at centrosomes. The permeability of this barrier is tightly regulated by branched actin filaments outside of centrosomes and it decreases during anaphase when branched actin temporally increases. The actin-based diffusion barrier gates microtubule nucleation by interfering with γ-tubulin ring complex recruitment. We propose that actin filaments spatiotemporally constrain protein complexes at centrosomes in a size-dependent manner.
Asunto(s)
Microtúbulos , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Microtúbulos/metabolismo , Actinas/metabolismo , Centrosoma/metabolismo , Citoesqueleto de Actina/metabolismoRESUMEN
BACKGROUND: Pregnenolone (P5) is a neurosteroid that promotes microtubule polymerization. It also reduces stress and negative symptoms of schizophrenia, promotes memory, as well as recovery from spinal cord injury. P5 is the first substance in the steroid-synthetic pathway; it can be further metabolized into other steroids. Therefore, it is difficult to differentiate the roles of P5 versus its metabolites in the brain. To alleviate this problem, we synthesized and screened a series of non-metabolizable P5 derivatives for their ability to polymerize microtubules similar to P5. RESULTS: We identified compound #43 (3-beta-pregnenolone acetate), which increased microtubule polymerization. We showed that compound #43 modified microtubule dynamics in live cells, increased neurite outgrowth and changed growth cone morphology in mouse cerebellar granule neuronal culture. Furthermore, compound #43 promoted the formation of stable microtubule tracks in zebrafish developing cerebellar axons. CONCLUSIONS: We have developed compound #43, a nonmetabolized P5 analog, that recapitulates P5 functions in vivo and can be a new therapeutic candidate for the treatment of neurodevelopmental diseases.
RESUMEN
The primary cilium, a microtubule-based sensory organelle, undergoes cycles of assembly and disassembly that govern the cell cycle progression critical to cell proliferation and differentiation. Although cilia assembly has been studied extensively, the molecular mechanisms underlying cilia disassembly are less well understood. Here, we uncover a γ-tubulin ring complex (γ-TuRC)-dependent pathway that promotes cilia disassembly and thereby prevents cilia formation. We further demonstrate that Kif2A, a kinesin motor that bears microtubule-depolymerizing activity, is recruited to the cilium basal body in a γ-TuRC-dependent manner. Our mechanistic analyses show that γ-TuRC specifically recruits Kif2A via the GCP2 subunit and its binding partner Mzt2. Hence, despite the long-standing view that γ-TuRC acts mainly as a microtubule template, we illustrate that its functional heterogeneity at the basal body facilitates both microtubule nucleation and Kif2A recruitment-mediated regulation of ciliogenesis, ensuring cell cycle progression.
Asunto(s)
Proteínas Asociadas a Microtúbulos , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Cilios/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismoRESUMEN
The spindle is a dynamic intracellular structure self-organized from microtubules and microtubule-associated proteins. The spindle's bipolar morphology is essential for the faithful segregation of chromosomes during cell division, and it is robustly maintained by multifaceted mechanisms. However, abnormally shaped spindles, such as multipolar spindles, can stochastically arise in a cell population and cause chromosome segregation errors. The physical basis of how microtubules fail in bipolarization and occasionally favor nonbipolar assembly is poorly understood. Here, using live fluorescence imaging and quantitative shape analysis in Xenopus egg extracts, we find that spindles of varied shape morphologies emerge through nonrandom, bistable self-organization paths, one leading to a bipolar and the other leading to a multipolar phenotype. The bistability defines the spindle's unique morphological growth dynamics linked to each shape phenotype and can be promoted by a locally distorted microtubule flow that arises within premature structures. We also find that bipolar and multipolar spindles are stable at the steady-state in bulk but can infrequently switch between the two phenotypes. Our microneedle-based physical manipulation further demonstrates that a transient force perturbation applied near the assembled pole can trigger the phenotypic switching, revealing the mechanical plasticity of the spindle. Together with molecular perturbation of kinesin-5 and augmin, our data propose the physical and molecular bases underlying the emergence of spindle-shape variation, which influences chromosome segregation fidelity during cell division.
Asunto(s)
Cinesinas , Huso Acromático , Huso Acromático/metabolismo , Microtúbulos/metabolismo , Segregación Cromosómica , Proteínas Asociadas a Microtúbulos/metabolismo , MitosisRESUMEN
Many synaptic proteins form biological condensates via liquid-liquid phase separation (LLPS). Synaptopathy, a key feature of autism spectrum disorders (ASD), is likely relevant to the impaired phase separation and/or transition of ASD-linked synaptic proteins. Here, we report that LLPS and zinc-induced liquid-to-gel phase transition regulate the synaptic distribution and protein-protein interaction of cortactin-binding protein 2 (CTTNBP2), an ASD-linked protein. CTTNBP2 forms self-assembled condensates through its C-terminal intrinsically disordered region and facilitates SHANK3 co-condensation at dendritic spines. Zinc binds the N-terminal coiled-coil region of CTTNBP2, promoting higher-order assemblies. Consequently, it leads to reduce CTTNBP2 mobility and enhance the stability and synaptic retention of CTTNBP2 condensates. Moreover, ASD-linked mutations alter condensate formation and synaptic retention of CTTNBP2 and impair mouse social behaviors, which are all ameliorated by zinc supplementation. Our study suggests the relevance of condensate formation and zinc-induced phase transition to the synaptic distribution and function of ASD-linked proteins.
Asunto(s)
Trastorno Autístico , Animales , Trastorno Autístico/genética , Ratones , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Conducta Social , Zinc/metabolismoRESUMEN
Extensive structural and functional studies have been carried out in the field of nucleocytoplasmic transport. Nuclear transport factors, such as Importin-α/-ß, recognize nuclear localization signals (NLSs) on cargo, and together with the small GTPase Ran, facilitate their nuclear localization. However, it is now emerging that binding of nuclear transport factors to NLSs not only mediates nuclear transport but also contributes to a variety of cellular functions in eukaryotes. Here, we describe recent advances that reveal how NLSs facilitate diverse cellular functions beyond nuclear transport activity. We review separately NLS-mediated regulatory mechanisms at different levels of biological organization, including (a) assembly of higher-order structures; (b) cellular organelle dynamics; and (c) modulation of cellular stress responses and viral infections. Finally, we provide mechanistic insights into how NLSs can regulate such a broad range of functions via their structural and biochemical properties.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Señales de Localización Nuclear/genética , Virosis/genética , Proteína de Unión al GTP ran/genética , Humanos , Orgánulos/genética , Orgánulos/metabolismo , Estrés Fisiológico/genética , Virosis/virologíaRESUMEN
How γ-tubulin ring complex (γ-TuRC), a master template for microtubule nucleation, is spatially and temporally regulated for the assembly of new microtubule arrays remains unclear. Here, we report that an evolutionarily conserved microprotein, Mozart1 (Mzt1), regulates subcellular targeting and microtubule formation activity of γ-TuRC at different cell cycle stages. Crystal structures of protein complexes demonstrate that Mzt1 promiscuously interacts with the N-terminal domains of multiple γ-tubulin complex protein subunits in γ-TuRC via an intercalative binding mode. Genetic- and microscopy-based analyses show that promiscuous binding of Mzt1 in γ-TuRC controls specific subcellular localization of γ-TuRC to modulate microtubule nucleation and stabilization in fission yeast. Moreover, we find Mzt1-independent targeting of γ-TuRC to be crucial for mitotic spindle assembly, demonstrating the cell-cycle-dependent regulation and function of γ-TuRC. Our findings reveal a microprotein-mediated regulatory mechanism underlying microtubule cytoskeleton formation, whereby Mzt1 binding promiscuity confers localization specificity on the multi-protein complex γ-TuRC.
Asunto(s)
Evolución Molecular , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Tubulina (Proteína)/metabolismo , Secuencia Conservada , Humanos , Interfase , Proteínas Asociadas a Microtúbulos/química , Centro Organizador de los Microtúbulos/metabolismo , Mitosis , Modelos Biológicos , Unión Proteica , Dominios Proteicos , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/química , Eliminación de Secuencia , Soluciones , Cuerpos Polares del Huso/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
Microtubule organization depends on the γ-tubulin ring complex (γ-TuRC), a â¼2.3-MDa nucleation factor comprising an asymmetric assembly of γ-tubulin and GCP2-GCP6. However, it is currently unclear how the γ-TuRC-associated microproteins MZT1 and MZT2 contribute to the structure and regulation of the holocomplex. Here, we report cryo-EM structures of MZT1 and MZT2 in the context of the native human γ-TuRC. MZT1 forms two subcomplexes with the N-terminal α-helical domains of GCP3 or GCP6 (GCP-NHDs) within the γ-TuRC "lumenal bridge." We determine the X-ray structure of recombinant MZT1/GCP6-NHD and find it is similar to that within the native γ-TuRC. We identify two additional MZT/GCP-NHD-like subcomplexes, one of which is located on the outer face of the γ-TuRC and comprises MZT2 and GCP2-NHD in complex with a centrosomin motif 1 (CM1)-containing peptide. Our data reveal how MZT1 and MZT2 establish multi-faceted, structurally mimetic "modules" that can expand structural and regulatory interfaces in the γ-TuRC.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Tubulina (Proteína)/metabolismo , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/química , Modelos Moleculares , Complejos Multiproteicos/ultraestructura , Péptidos/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestructuraRESUMEN
Nuclear accessibility of transcription factors controls gene expression, co-regulated by Ran-dependent nuclear localization and a competitive regulatory network. Here, we reveal that nuclear import factor-facilitated transcriptional repression attenuates ribosome biogenesis under chronic salt stress. Kap114p, one of the karyopherin-ßs (Kap-ßs) that mediates nuclear import of yeast TATA-binding protein (yTBP), exhibits a yTBP-binding affinity four orders of magnitude greater than its counterparts and suppresses binding of yTBP with DNA. Our crystal structure of Kap114p reveals an extensively negatively charged concave surface, accounting for high-affinity basic-protein binding. KAP114 knockout in yeast leads to a high-salt growth defect, with transcriptomic analyses revealing that Kap114p modulates expression of genes associated with ribosomal biogenesis by suppressing yTBP binding to target promoters, a trans-repression mechanism we attribute to reduced nuclear Ran levels under salinity stress. Our findings reveal that Ran integrates the nuclear transport pathway and transcription regulatory network, allowing yeast to respond to environmental stresses.
Asunto(s)
Carioferinas , Proteínas de Saccharomyces cerevisiae , Núcleo Celular/genética , Núcleo Celular/metabolismo , Expresión Génica , Proteínas Nucleares/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , beta Carioferinas/genéticaRESUMEN
To facilitate proper mitotic cell partitioning, the Golgi disassembles by suppressing vesicle fusion. However, the underlying mechanism has not been characterized previously. Here, we report a Ran pathway-independent attenuation mechanism that allows Importin-α (a nuclear transport factor) to suppress the vesicle fusion mediated by p115 (a vesicular tethering factor) and is required for mitotic Golgi disassembly. We demonstrate that Importin-α directly competes with p115 for interaction with the Golgi protein GM130. This interaction, promoted by a phosphate moiety on GM130, is independent of Importin-ß and Ran. A GM130 K34A mutant, in which the Importin-α-GM130 interaction is specifically disrupted, exhibited abundant Golgi puncta during metaphase. Importantly, a mutant showing enhanced p115-GM130 interaction presented proliferative defects and G2/M arrest, demonstrating that Importin-α-GM130 binding modulates the Golgi disassembly that governs mitotic progression. Our findings illuminate that the Ran and kinase-phosphatase pathways regulate multiple aspects of mitosis coordinated by Importin-α (e.g. spindle assembly, Golgi disassembly).
Asunto(s)
Autoantígenos/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Matriz de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Metafase/fisiología , Proteínas de Transporte Vesicular/metabolismo , alfa Carioferinas/metabolismo , Autoantígenos/genética , Cristalografía por Rayos X , Puntos de Control de la Fase G2 del Ciclo Celular , Células HEK293 , Humanos , Fusión de Membrana , Proteínas de la Membrana/genética , Mitosis/fisiología , Fosforilación , Unión Proteica , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/metabolismoRESUMEN
Cardiovascular disease is the leading cause of morbidity and mortality in the world. Mutations in the FHL2 (Four and a half LIM domains protein 2) gene are associated with cardiomyopathy in patients. Here, we generated two homozygous knockout lines using CRISPR/Cas9-mediated ablation in a human embryonic stem cell (hESC) WA09 line. These knockout lines exhibit a normal karyotype without expressing FHL2 protein, while maintaining pluripotency and differentiation properties. These isogenic mutation lines will be provided as a disease model for cardiomyopathy studies and drug screening.
Asunto(s)
Sistemas CRISPR-Cas/fisiología , Células Madre Embrionarias Humanas/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas Musculares/genética , Factores de Transcripción/genética , Sistemas CRISPR-Cas/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Exones/genética , Técnicas de Inactivación de Genes , Hepatocitos/metabolismo , Humanos , CariotipoRESUMEN
Ran-guanosine triphosphatase orchestrates mitotic spindle assembly by modulation of the interaction between Importin-α/-ß and spindle assembly factors (SAFs). The inhibition of SAFs performed by importins needs to be done without much sequestration from abundant nuclear localization signal (NLS) -containing proteins. However, the molecular mechanisms that determine NLS-binding selectivity and that inhibit activity of Importin-ß-regulated SAFs (e.g., nuclear mitotic apparatus protein [NuMA]) remain undefined. Here, we present a crystal structure of the Importin-α-NuMA C terminus complex showing a novel binding pattern that accounts for selective NLS recognition. We demonstrate that, in the presence of Importin-α, Importin-ß inhibits the microtubule-binding function of NuMA. Further, we have identified a high-affinity microtubule-binding region that lies carboxyl-terminal to the NLS, which is sterically masked by Importin-ß on being bound by Importin-α. Our study provides mechanistic evidence of how Importin-α/-ß regulates the NuMA functioning required for assembly of higher-order microtubule structures, further illuminating how Ran-governed transport factors regulate diverse SAFs and accommodate various cell demands.
Asunto(s)
Antígenos Nucleares/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Huso Acromático/metabolismo , beta Carioferinas/metabolismo , Animales , Antígenos Nucleares/química , Antígenos Nucleares/genética , Proteínas de Ciclo Celular , Humanos , Microtúbulos/metabolismo , Modelos Moleculares , Complejos Multiproteicos , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Huso Acromático/química , Huso Acromático/genética , Relación Estructura-Actividad , Xenopus , alfa Carioferinas/metabolismo , beta Carioferinas/química , beta Carioferinas/genética , Proteína de Unión al GTP ran/metabolismoRESUMEN
Proper microtubule nucleation during cell division requires augmin, a microtubule-associated hetero-octameric protein complex. In current models, augmin recruits γ-tubulin, through the carboxyl terminus of its hDgt6 subunit to nucleate microtubules within spindles. However, augmin's biochemical complexity has restricted analysis of its structural organization and function. Here, we reconstitute human augmin and show that it is a Y-shaped complex that can adopt multiple conformations. Further, we find that a dimeric sub-complex retains in vitro microtubule-binding properties of octameric complexes, but not proper metaphase spindle localization. Addition of octameric augmin complexes to Xenopus egg extracts promotes microtubule aster formation, an activity enhanced by Ran-GTP. This activity requires microtubule binding, but not the characterized hDgt6 γ-tubulin-recruitment domain. Tetrameric sub-complexes induce asters, but activity and microtubule bundling within asters are reduced compared with octameric complexes. Together, our findings shed light on augmin's structural organization and microtubule-binding properties, and define subunits required for its function in organizing microtubule-based structures.
Asunto(s)
Proteínas Asociadas a Microtúbulos/química , Animales , Sistema Libre de Células , Escherichia coli , Humanos , Metafase , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/ultraestructura , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Unión Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Xenopus laevisRESUMEN
Diverse cellular processes require microtubules to be organized into distinct structures, such as asters or bundles. Within these dynamic motifs, microtubule-associated proteins (MAPs) are frequently under load, but how force modulates these proteins' function is poorly understood. Here, we combine optical trapping with TIRF-based microscopy to measure the force dependence of microtubule interaction for three nonmotor MAPs (NuMA, PRC1, and EB1) required for cell division. We find that frictional forces increase nonlinearly with MAP velocity across microtubules and depend on filament polarity, with NuMA's friction being lower when moving toward minus ends, EB1's lower toward plus ends, and PRC1's exhibiting no directional preference. Mathematical models predict, and experiments confirm, that MAPs with asymmetric friction can move directionally within actively moving microtubule pairs they crosslink. Our findings reveal how nonmotor MAPs can generate frictional resistance in dynamic cytoskeletal networks via micromechanical adaptations whose anisotropy may be optimized for MAP localization and function within cellular structures.
Asunto(s)
Antígenos Nucleares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Antígenos Nucleares/química , Fenómenos Biomecánicos , Proteínas de Ciclo Celular/química , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/química , Modelos Biológicos , Proteínas Asociadas a Matriz Nuclear/química , Pinzas ÓpticasRESUMEN
The heptameric coatomer complex forms the protein shell of membrane-bound vesicles that are involved in transport from the Golgi to the endoplasmatic reticulum and in intraGolgi trafficking. The heptamer can be dissected into a heterotetrameric F-subcomplex, which displays similarities to the adapter complex of the "inner" coat in clathrin-coated vesicles, and a heterotrimeric B-subcomplex, which is believed to form an "outer" coat with a morphology distinct from that of clathrin-coated vesicles. We have determined the crystal structure of the complex between the C-terminal domain (CTD) of alpha-COP and full-length epsilon-COP, two components of the B-subcomplex, at a 2.9 A resolution. The alpha-COP(CTD) x epsilon-COP heterodimer forms a rod-shaped structure, in which epsilon-COP adopts a tetratricopeptide repeat (TPR) fold that deviates substantially from the canonical superhelical conformation. The alpha-COP CTD adopts a U-shaped architecture that complements the TPR fold of epsilon-COP. The epsilon-COP TPRs form a circular bracelet that wraps around a protruding beta-hairpin of the alpha-COP CTD, thus interlocking the two proteins. The alpha-COP(CTD) x epsilon-COP complex forms heterodimers in solution, and we demonstrate biochemically that the heterodimer directly interacts with the Dsl1 tethering complex. These data suggest that the heterodimer is exposed on COPI vesicles, while the remaining part of the B-subcomplex oligomerizes underneath into a cage.
Asunto(s)
Proteína Coat de Complejo I/química , Proteína Coatómero/química , Saccharomyces cerevisiae/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Membrana Celular/metabolismo , Cristalografía por Rayos X/métodos , Dimerización , Escherichia coli/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Secuencias Repetitivas de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
Nuclear pore complexes (NPCs) function as selective gates for nucleocytoplasmic transport. Although the NPC was discovered more than half a century ago, our knowledge of NPC components in atomic detail has exploded only over the past few years. Recent structural, biochemical, and in vivo studies of NPC components, in particular the membrane-coating heptameric Nup84 complex, have shed light onto the NPC architecture as well as onto its dynamic nature. Striking similarities were revealed between the components of the NPC and of coat protein complexes in the endocytic and secretory pathways, supporting their common evolutionary origin in a progenitor protocoatomer. Here, we summarize these findings and discuss emerging concepts that underlie the molecular architecture and the dynamics of the NPC. We conclude that the uncovered principles are not limited to the NPC, but are likely to extend to other macromolecular assemblies.