RESUMEN
T-cells massively restructure their internal architecture upon reaching an antigen-presenting cell (APC) to form the immunological synapse (IS), a cell-cell interface necessary for efficient elimination of the APC. This reorganization occurs through tight coordination of cytoskeletal processes: actin forms a peripheral ring, and dynein motors translocate the centrosome toward the IS. A recent study proposed that centrosome translocation involves a microtubule (MT) bundle that connects the centrosome perpendicularly to dynein at the synapse center: the "stalk." The synapse center, however, is actin-depleted, while actin was assumed to anchor dynein. We propose that dynein is attached to mobile membrane anchors, and investigate this model with computer simulations. We find that dynein organizes into a cluster in the synapse when translocating the centrosome, aligning MTs into a stalk. By implementing both a MT-capture-shrinkage and a MT-sliding mechanism, we explicitly demonstrate that this organization occurs in both systems. However, results obtained with MT-sliding dynein are more robust and display a stalk morphology consistent with our experimental data obtained with expansion microscopy. Thus, our simulations suggest that actin organization in T-cells during activation defines a specific geometry in which MT-sliding dynein can self-organize into a cluster and cause stalk formation.
Asunto(s)
Dineínas/metabolismo , Dineínas/fisiología , Linfocitos T/metabolismo , Actinas/metabolismo , Células Presentadoras de Antígenos/metabolismo , Centrosoma/metabolismo , Centrosoma/fisiología , Simulación por Computador , Citoesqueleto/metabolismo , Humanos , Sinapsis Inmunológicas/metabolismo , Células Jurkat , Microtúbulos/metabolismo , Modelos Teóricos , Linfocitos T/fisiologíaRESUMEN
The formation of an epithelial tube is a fundamental process for organogenesis. During Drosophila embryonic salivary gland (SG) invagination, Folded gastrulation (Fog)-dependent Rho-associated kinase (Rok) promotes contractile apical myosin formation to drive apical constriction. Microtubules (MTs) are also crucial for this process and are required for forming and maintaining apicomedial myosin. However, the underlying mechanism that coordinates actomyosin and MT networks still remains elusive. Here, we show that MT-dependent intracellular trafficking regulates apical constriction during SG invagination. Key components involved in protein trafficking, such as Rab11 and Nuclear fallout (Nuf), are apically enriched near the SG invagination pit in a MT-dependent manner. Disruption of the MT networks or knockdown of Rab11 impairs apicomedial myosin formation and apical constriction. We show that MTs and Rab11 are required for apical enrichment of the Fog ligand and the continuous distribution of the apical determinant protein Crumbs (Crb) and the key adherens junction protein E-Cadherin (E-Cad) along junctions. Targeted knockdown of crb or E-Cad in the SG disrupts apical myosin networks and results in apical constriction defects. Our data suggest a role of MT- and Rab11-dependent intracellular trafficking in regulating actomyosin networks and cell junctions to coordinate cell behaviors during tubular organ formation.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila/embriología , Microtúbulos/fisiología , Glándulas Salivales/embriología , Proteínas de Unión al GTP rab/fisiología , Citoesqueleto de Actina/fisiología , Actomiosina/fisiología , Animales , Transporte Biológico , Cadherinas/fisiología , Proteínas de Drosophila/genética , Dineínas/fisiología , Gastrulación , Técnicas de Silenciamiento del Gen , Uniones Intercelulares/fisiología , Miosinas/fisiología , Proteínas Nucleares/fisiología , Proteínas de Unión al GTP rab/genéticaRESUMEN
Dynein light chain (DLC) proteins are an important component of dynein complexes, which are widely distributed in plants and animals and involved in a variety of cellular processes. The functions of DLC genes in plant chilling stress remain unclear. In this study, we isolated a DLC gene from tomato, designated SlLC6D. Promoter analysis revealed many cis-elements involved in abiotic stress in the SlLC6D promoter. Expression of SlLC6D was induced by heat and salt stress, and inhibited by polyethylene glycol and chilling stress. Knockdown of SlLC6D in tomato exhibited low relative electrolyte leakage, malondialdehyde content, and reactive oxygen species (ROS) accumulation under chilling stress. The content of proline and activities of superoxide dismutase and peroxidase in knockdown lines were higher than in the wild type and overexpression lines during chilling stress. The high transcript abundances of three cold-responsive genes were detected in knockdown lines in response to chilling stress. Seedling growth of knockdown lines was significantly higher than that of the wild type and overexpression lines under chilling stress. These results suggest that SlLC6D is a negative regulator of chilling stress tolerance, possibly by regulating ROS contents and the ICE1-CBF-COR pathway.
Asunto(s)
Dineínas/genética , Genes de Plantas/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Respuesta al Choque por Frío , Secuencia Conservada/genética , Dineínas/metabolismo , Dineínas/fisiología , Genes de Plantas/fisiología , Solanum lycopersicum/metabolismo , Solanum lycopersicum/fisiología , Malondialdehído/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiología , Prolina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Proteins interacting with G protein-coupled receptors (GPCRs) can modulate signal transduction of these receptors. However, the regulatory mechanisms of the interacting proteins are diverse and largely unknown. We have previously shown that Tctex-1 (or DYNLT1) can interact with the parathyroid hormone receptor (PTHR). In the present study, we investigated the role of Tctex-1 in the PTHR signaling and found that Tctex-1 augmented the PTHR-mediated Gs/adenylyl cyclase (AC) pathway by activating AC regardless of the binding to PTHR. Furthermore, Tctex-1 directly bound to AC type 6. These data demonstrate a novel mechanism underlying GPCR/Gs signaling regulated by Tctex-1.
Asunto(s)
Adenilil Ciclasas/metabolismo , Dineínas/metabolismo , Dineínas/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Células 3T3 , Animales , Células HEK293 , Humanos , Ratones , Unión Proteica , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/fisiologíaRESUMEN
Proliferating animal cells are able to orient their mitotic spindles along their interphase cell axis, setting up the axis of cell division, despite rounding up as they enter mitosis. This has previously been attributed to molecular memory and, more specifically, to the maintenance of adhesions and retraction fibers in mitosis [1-6], which are thought to act as local cues that pattern cortical Gαi, LGN, and nuclear mitotic apparatus protein (NuMA) [3, 7-18]. This cortical machinery then recruits and activates Dynein motors, which pull on astral microtubules to position the mitotic spindle. Here, we reveal a dynamic two-way crosstalk between the spindle and cortical motor complexes that depends on a Ran-guanosine triphosphate (GTP) signal [12], which is sufficient to drive continuous monopolar spindle motion independently of adhesive cues in flattened human cells in culture. Building on previous work [1, 12, 19-23], we implemented a physical model of the system that recapitulates the observed spindle-cortex interactions. Strikingly, when this model was used to study spindle dynamics in cells entering mitosis, the chromatin-based signal was found to preferentially clear force generators from the short cell axis, so that cortical motors pulling on astral microtubules align bipolar spindles with the interphase long cell axis, without requiring a fixed cue or a physical memory of interphase shape. Thus, our analysis shows that the ability of chromatin to pattern the cortex during the process of mitotic rounding is sufficient to translate interphase shape into a cortical pattern that can be read by the spindle, which then guides the axis of cell division.
Asunto(s)
Dineínas/fisiología , Mecanotransducción Celular , Microtúbulos/fisiología , Mitosis , Huso Acromático/fisiología , Células HeLa , Humanos , Transducción de SeñalRESUMEN
One of the characteristic features of different classes of neurons that is vital for their proper functioning within neuronal networks is the shape of their dendritic arbors. To properly develop dendritic trees, neurons need to accurately control the intracellular transport of various cellular cargo (e.g., mRNA, proteins, and organelles). Microtubules and motor proteins (e.g., dynein and kinesins) that move along microtubule tracks play an essential role in cargo sorting and transport to the most distal ends of neurons. Equally important are motor adaptors, which may affect motor activity and specify cargo that is transported by the motor. Such transport undergoes very dynamic fine-tuning in response to changes in the extracellular environment and synaptic transmission. Such regulation is achieved by the phosphorylation of motors, motor adaptors, and cargo, among other mechanisms. This review focuses on the contribution of the dynein-dynactin complex, kinesins, their adaptors, and the phosphorylation of these proteins in the formation of dendritic trees by maturing neurons. We primarily review the effects of the motor activity of these proteins in dendrites on dendritogenesis. We also discuss less anticipated mechanisms that contribute to dendrite growth, such as dynein-driven axonal transport and non-motor functions of kinesins.
Asunto(s)
Dendritas , Complejo Dinactina/fisiología , Dineínas/fisiología , Cinesinas/fisiología , Neuronas Motoras/fisiología , Animales , Humanos , Neurogénesis/fisiología , FosforilaciónRESUMEN
How cells position their organelles is a fundamental biological question. During Drosophila embryonic muscle development, multiple nuclei transition from being clustered together to splitting into two smaller clusters to spreading along the myotube's length. Perturbations of microtubules and motor proteins disrupt this sequence of events. These perturbations do not allow intuiting which molecular forces govern the nuclear positioning; we therefore used computational screening to reverse-engineer and identify these forces. The screen reveals three models. Two suggest that the initial clustering is due to nuclear repulsion from the cell poles, while the third, most robust, model poses that this clustering is due to a short-ranged internuclear attraction. All three models suggest that the nuclear spreading is due to long-ranged internuclear repulsion. We test the robust model quantitatively by comparing it with data from perturbed muscle cells. We also test the model using agent-based simulations with elastic dynamic microtubules and molecular motors. The model predicts that, in longer mammalian myotubes with a large number of nuclei, the spreading stage would be preceded by segregation of the nuclei into a large number of clusters, proportional to the myotube length, with a small average number of nuclei per cluster.
Asunto(s)
Núcleo Celular/fisiología , Drosophila melanogaster/embriología , Microtúbulos/metabolismo , Animales , Transporte Biológico , Núcleo Celular/metabolismo , Análisis por Conglomerados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Dineínas/fisiología , Cinesinas/metabolismo , Cinesinas/fisiología , Microtúbulos/fisiología , Modelos Biológicos , Desarrollo de Músculos/fisiología , Fibras Musculares Esqueléticas/metabolismo , Miosinas/metabolismoRESUMEN
Non-centrosomal microtubule-organizing centres (ncMTOCs) have a variety of roles that are presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization and function. Here we report a perinuclear MTOC in Drosophila fat body cells that is anchored by the Nesprin homologue Msp300 at the cytoplasmic surface of the nucleus. Msp300 recruits the microtubule minus-end protein Patronin, a calmodulin-regulated spectrin-associated protein (CAMSAP) homologue, which functions redundantly with Ninein to further recruit the microtubule polymerase Msps-a member of the XMAP215 family-to assemble non-centrosomal microtubules and does so independently of the widespread microtubule nucleation factor γ-Tubulin. Functionally, the fat body ncMTOC and the radial microtubule arrays that it organizes are essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture that regulates microtubules, serving vital functions.
Asunto(s)
Membrana Basal/metabolismo , Núcleo Celular , Centro Organizador de los Microtúbulos/fisiología , Actinas/fisiología , Animales , Membrana Celular , Núcleo Celular/ultraestructura , Centrosoma , Drosophila/metabolismo , Drosophila/ultraestructura , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Dineínas/fisiología , Endosomas/metabolismo , Cuerpo Adiposo/metabolismo , Cuerpo Adiposo/ultraestructura , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Centro Organizador de los Microtúbulos/ultraestructura , Microtúbulos/fisiología , Proteínas Musculares/metabolismo , Tubulina (Proteína)/fisiologíaRESUMEN
Using a geometric feedback model of the flagellar axoneme accounting for dynein motor kinetics, we study elastohydrodynamic phase synchronization in a pair of spontaneously beating filaments with waveforms ranging from sperm to cilia and Chlamydomonas. Our computations reveal that both in-phase and antiphase synchrony can emerge for asymmetric beats while symmetric waveforms go in phase, and elucidate the mechanism for phase slips due to biochemical noise. Model predictions agree with recent experiments and illuminate the crucial roles of hydrodynamics and mechanochemical feedback in synchronization.
Asunto(s)
Flagelos/fisiología , Modelos Biológicos , Fenómenos Biomecánicos , Chlamydomonas/fisiología , Cilios/fisiología , Dineínas/fisiología , Humanos , Hidrodinámica , Masculino , Espermatozoides/fisiologíaRESUMEN
Primary cilia, which protrude from the surfaces of most human cells, function as cellular antennae that receive extracellular signals. To serve as antennae, primary cilia contain unique proteins, such as G-protein-coupled receptors and ion channels. Defects in the assembly and functions of primary cilia cause hereditary disorders with a wide range of symptoms, including cystic kidney and retinal degeneration. The assembly and maintenance of cilia depend on protein trafficking mediated by the intraflagellar transport (IFT) machinery, which contains three protein complexes (IFT-A, IFT-B, and BBSome) and two motor proteins (kinesin-2 and dynein-2 complex) and is composed of more than 40 subunits in total. We recently revealed the interaction between the kinesin-2 and IFT-B complexes and overall architecture of the dynein-2 complex by taking advantage of the visible immunoprecipitation (VIP) assay. In addition, we clarified the roles of dynein-2 subunits using gene knockout cell lines established using the CRISPR/Cas9 system. This review focuses on recent advances in the architectures and functions of two motor proteins underlying the IFT machinery.
Asunto(s)
Cilios/fisiología , Dineínas/fisiología , Flagelos/fisiología , Cinesinas/fisiología , Línea Celular , Técnicas de Inactivación de Genes , Humanos , Inmunoprecipitación , Transporte de ProteínasRESUMEN
Primary cilium is a membrane-protruding sensory organelle, which is organized from a basal body in G0/G1 phase cells. The resorption of primary cilia under specific growth factor stimuli is coupled to cell cycle re-entry and cell proliferation, and the proliferative function is vital for the organization of organs at an embryonic stage. In fact, abnormalities in ciliogenesis and/or cilium-derived signaling lead to malformation of various organs, such as the brain, eyes, nose, ear, heart, lung, liver, kidney, and bones. The inborn genetic disorders are collectively called ãciliopathyã. However, the pathogenesis of the ciliopathies has largely remained unexplained, especially little is known about the cellular machinery that controls the ciliary resorption. Tctex-1 (t-complex testis expressed-1), one of the light chains of cytoplasmic dynein complex, regulates intracellular trafficking along microtubule. Tctex-1 phosphorylated at Thr94 is, in turn, free from the dynein complex to execute dynein-free functions. This review summarizes the current situation of the mechanisms of ciliary resorption with a central focus on the role of phosphorylated Tctex-1.
Asunto(s)
Proliferación Celular , Cilios/fisiología , Transducción de Señal , Dineínas/fisiología , HumanosRESUMEN
The flagellated eukaryote Trypanosoma brucei alternates between the insect vector and the mammalian host and proliferates through an unusual mode of cell division. Cell division requires flagellum motility-generated forces, but flagellum motility exerts distinct effects between different life cycle forms. Motility is required for the final cell abscission of the procyclic form in the insect vector, but is necessary for the initiation of cell division of the bloodstream form in the mammalian host. The underlying mechanisms remain elusive. Here we carried out functional analyses of a flagellar axonemal inner-arm dynein complex in the bloodstream form and investigated its mechanistic role in cytokinesis initiation. We showed that the axonemal inner-arm dynein heavy chain TbIAD5-1 and TbCentrin3 form a complex, localize to the flagellum, and are required for viability in the bloodstream form. We further demonstrated the interdependence between TbIAD5-1 and TbCentrin3 for maintenance of protein stability. Finally, we showed that depletion of TbIAD5-1 and TbCentrin3 arrested cytokinesis initiation and disrupted the localization of multiple cytokinesis initiation regulators. These findings identified the essential role of an axonemal inner-arm dynein complex in cell division, and provided molecular insights into the flagellum motility-mediated cytokinesis initiation in the bloodstream form of T. brucei.
Asunto(s)
Dineínas Axonemales/metabolismo , Proteínas Contráctiles/metabolismo , Citocinesis/fisiología , Proteínas Protozoarias/metabolismo , Dineínas Axonemales/fisiología , Axonema/metabolismo , División Celular/genética , División Celular/fisiología , Línea Celular , Movimiento Celular , Proteínas Contráctiles/genética , Proteínas Contráctiles/fisiología , Dineínas/metabolismo , Dineínas/fisiología , Flagelos/metabolismo , Flagelos/fisiología , Estadios del Ciclo de Vida , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Interferencia de ARN , Trypanosoma brucei brucei/metabolismoRESUMEN
Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD) and it also causes severe neurologic complications in infected children. The interactions between some viruses and the host mitochondria are crucial for virus replication and pathogenicity. In this study, it was observed that EV-A71 infection resulted in a perinuclear redistribution of the mitochondria. The mitochondria rearrangement was found to require the microtubule network, the dynein complex and a low cytosolic calcium concentration. Subsequently, the EV-A71 non-structural protein 2BC was identified as the viral protein capable of inducing mitochondria clustering. The protein was found localized on mitochondria and interacted with the mitochondrial Rho GTPase 1 (RHOT1) that is a key protein required for attachment between the mitochondria and the motor proteins, which are responsible for the control of mitochondria movement. Additionally, suppressing mitochondria clustering by treating cells with nocodazole, EHNA, thapsigargin or A23187 consistently inhibited EV-A71 replication, indicating that mitochondria recruitment played a crucial role in the EV-A71 life cycle. This study identified a novel function of the EV-A71 2BC protein and provided a potential model for the regulation of mitochondrial motility in EV-A71 infection.
Asunto(s)
Enterovirus Humano A/fisiología , Interacciones Microbiota-Huesped , Mitocondrias/metabolismo , Replicación Viral , Citosol/química , Citosol/virología , Dineínas/fisiología , Células HeLa , Humanos , Microtúbulos/fisiología , Proteínas Mitocondriales/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Following injury, axons of the peripheral nervous system have retained the capacity for regeneration. While it is well established that injury signals require molecular motors for their transport from the injury site to the nucleus, whether kinesin and dynein motors play additional roles in peripheral nerve regeneration is not well understood. Here we use genetic mutants of motor proteins in a zebrafish peripheral nerve regeneration model to visualize and define in vivo roles for kinesin and dynein. We find that both kinesin-1 and dynein are required for zebrafish peripheral nerve regeneration. While loss of kinesin-1 reduced the overall robustness of axonal regrowth, loss of dynein dramatically impaired axonal regeneration and also reduced injury-induced Schwann cell remodeling. Chimeras between wild type and dynein mutant embryos demonstrate that dynein function in neurons is sufficient to promote axonal regrowth. Finally, by simultaneously monitoring actin and microtubule dynamics in regenerating axons we find that dynein appears dispensable to initiate axonal regrowth, but is critical to stabilize microtubules, thereby sustaining axonal regeneration. These results reveal two previously unappreciated roles for dynein during peripheral nerve regeneration, initiating injury induced Schwann cell remodeling and stabilizing axonal microtubules to sustain axonal regrowth.
Asunto(s)
Dineínas/fisiología , Regeneración Nerviosa/fisiología , Nervios Periféricos/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Axones/fisiología , Axones/ultraestructura , Dineínas/genética , Cinesinas/genética , Cinesinas/fisiología , Mutación , Regeneración Nerviosa/genética , Traumatismos de los Nervios Periféricos/genética , Traumatismos de los Nervios Periféricos/patología , Traumatismos de los Nervios Periféricos/fisiopatología , Células de Schwann/citología , Células de Schwann/fisiología , Pez Cebra/genética , Pez Cebra/fisiología , Proteínas de Pez Cebra/genéticaRESUMEN
Bidirectional cargo transport in neurons can be explained by two models: the "tug-of-war model" for short-range transport, in which several kinesin and dynein motors are bound to the same cargo but travel in opposing directions, and by the "motor coordination model" for long-range transport, in which small adaptors or the cargo itself activates or deactivates opposing motors. Direct interactions between the major axonal transporter kinesin-3 UNC-104(KIF1A) and the dynein/dynactin complex remains unknown. In this study, we dissected and evaluated the interaction sites between UNC-104 and dynein as well as between UNC-104 and dynactin using yeast two-hybrid assays. We found that the DYLT-1(Tctex) subunit of dynein binds near the coiled coil 3 (CC3) of UNC-104, and that the DYRB-1(Roadblock) subunit binds near the CC2 region of UNC-104. Regarding dynactin, we specifically revealed strong interactions between DNC-6(p27) and the FHA-CC3 stretch of UNC-104, as well as between the DNC-5(p25) and the CC2-CC3 region of UNC-104. Motility analysis of motors and cargo in the nervous system of Caenorhabditis elegans revealed impaired transport of UNC-104 and synaptic vesicles in dynein and dynactin mutants (or in RNAi knockdown animals). Further, in these mutants UNC-104 clustering along axons was diminished. Interestingly, when dynamic UNC-104 motors enter a stationary UNC-104 cluster their dwelling times are increased in dynein mutants (suggesting that dynein may act as an UNC-104 activator). In summary, we provide novel insights on how UNC-104 interacts with the dynein/dynactin complex and how UNC-104 driven axonal transport depends on dynein/dynactin in C. elegans neurons.
Asunto(s)
Transporte Axonal/fisiología , Proteínas de Caenorhabditis elegans/fisiología , Complejo Dinactina/fisiología , Dineínas/fisiología , Proteínas del Tejido Nervioso/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , Animales , Transporte Axonal/genética , Axones/metabolismo , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Ensayos de Migración Celular , Complejo Dinactina/genética , Dineínas/genética , Cinesinas , Proteínas Asociadas a Microtúbulos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Vesículas Sinápticas/metabolismoRESUMEN
The microtubule cytoskeleton supports diverse cellular morphogenesis and migration processes during brain development. Mutations in tubulin genes are associated with severe human brain malformations known as 'tubulinopathies'; however, it is not understood how molecular-level changes in microtubule subunits lead to brain malformations. In this study, we demonstrate that missense mutations affecting arginine at position 402 (R402) of TUBA1A α-tubulin selectively impair dynein motor activity and severely and dominantly disrupt cortical neuronal migration. TUBA1A is the most commonly affected tubulin gene in tubulinopathy patients, and mutations altering R402 account for 30% of all reported TUBA1A mutations. We show for the first time that ectopic expression of TUBA1A-R402C and TUBA1A-R402H patient alleles is sufficient to dominantly disrupt cortical neuronal migration in the developing mouse brain, strongly supporting a causal role in the pathology of brain malformation. To isolate the precise molecular impact of R402 mutations, we generated analogous R402C and R402H mutations in budding yeast α-tubulin, which exhibit a simplified microtubule cytoskeleton. We find that R402 mutant tubulins assemble into microtubules that support normal kinesin motor activity but fail to support the activity of dynein motors. Importantly, the level of dynein impairment scales with the expression level of the mutant in the cell, suggesting a 'poisoning' mechanism in which R402 mutant α-tubulin acts dominantly by populating microtubules with defective binding sites for dynein. Based on our results, we propose a new model for the molecular pathology of tubulinopathies that may also extend to other tubulin-related neuropathies.
Asunto(s)
Dineínas/fisiología , Lisencefalia/genética , Tubulina (Proteína)/genética , Animales , Encéfalo/metabolismo , Movimiento Celular/genética , Dineínas/genética , Lisencefalia/metabolismo , Ratones , Ratones Endogámicos C57BL , Microtúbulos/metabolismo , Mutación , Mutación Missense , Neurogénesis , Neuronas/metabolismo , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Tubulina (Proteína)/fisiologíaRESUMEN
Macromolecular binding is a complex process that involves sensing and approaching the binding partner, adopting the proper orientation, and performing the physical binding. We computationally investigated the role of E-hooks, which are intrinsically disordered regions (IDRs) at the C-terminus of tubulin, on dynein microtubule binding domain (MTBD) binding to the microtubule as a function of the distance between the MTBD and its binding site on the microtubule. Our results demonstrated that the contacts between E-hooks and the MTBD are dynamical; multiple negatively charted patches of amino acids on the E-hooks grab and release the same positively charged patches on the MTBD as it approaches the microtubule. Even when the distance between the MTBD and the microtubule was greater than the E-hook length, the E-hooks sensed and guided MTBD via long-range electrostatic interactions in our simulations. Moreover, we found that E-hooks exerted electrostatic forces on the MTBD that were distance dependent; the force pulls the MTBD toward the microtubule at long distances but opposes binding at short distances. This mechanism provides a "soft-landing" for the MTBD as it binds to the microtubule. Finally, our analysis of the conformational states of E-hooks in presence and absence of the MTBD indicates that the binding process is a mixture of the induced-fit and lock-and-key macromolecular binding hypotheses. Overall, this novel binding mechanism is termed "guided-soft-binding" and could have broad-reaching impacts on the understanding of how IDRs dock to structured proteins.
Asunto(s)
Dineínas/metabolismo , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/fisiología , Sitios de Unión , Biología Computacional/métodos , Dineínas/fisiología , Microtúbulos/metabolismo , Simulación de Dinámica Molecular , Unión Proteica , Dominios Proteicos , Electricidad EstáticaRESUMEN
Active intracellular transport of organelles relies on the coordinated activities of cytoplasmic dynein and kinesin, ATP-dependent microtubule motor proteins. While axonemal dynein was discovered during the mid-1960s, it was not until the mid-1980s that kinesin was discovered by Ron Vale and colleagues, as reported in 1985. Their research demonstrated that the newly identified protein, isolated from both squid axoplasm and bovine brain, was independently capable of driving microtubule gliding or organelle movement. These findings kicked off rapid progress in the fields of physiology and neuroscience, leading to the identification of the many members of the extended kinesin superfamily, as well as detailed explorations of their biophysical properties, cellular mechanisms of action, and roles in disease.
Asunto(s)
Cinesinas/fisiología , Animales , Transporte Biológico , Dineínas/fisiología , Humanos , Orgánulos/fisiologíaRESUMEN
After acute wound formation, the oxygen supply is reduced, which results in the formation of an acute hypoxic microenvironment; whether this hypoxic microenvironment enhances epidermal cell migration and the underlying regulatory molecular mechanism of this effect are unclear. In this study, HaCaT cells were maintained under hypoxic (1% oxygen) or normoxic conditions. Methods including immunofluorescence staining, wound scratch assays, transwell assays, Western blotting and high- and low-expression lentiviral vector transfection were utilized to observe the changes in cell migration, microtubule dynamics and the expression levels of microtubule-associated protein (MAP) 4 and the light chain protein DYNLT1 (Tctex-1). The possible mechanisms were studied and discussed. The results showed that epidermal cell migration was enhanced during early hypoxia. Further experiments revealed that MAP4 regulates microtubule dynamics and promotes epidermal cell migration through Tctex-1. MAP4 and Tctex-1 play important roles in regulating the migration of epidermal cells under hypoxia. This evidence will provide a basis for further revealing the cellular and molecular mechanisms of local wound hypoxia and for promoting wound healing.
Asunto(s)
Hipoxia de la Célula/fisiología , Movimiento Celular , Dineínas/fisiología , Queratinocitos/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Cicatrización de Heridas , Línea Celular , Microambiente Celular , Dineínas/genética , Humanos , Proteínas Asociadas a Microtúbulos/genética , MicrotúbulosRESUMEN
Myosin motors power movements on actin filaments, whereas dynein and kinesin motors power movements on microtubules. The mechanisms of these motor proteins differ, but, in all cases, ATP hydrolysis and subsequent release of the hydrolysis products drives a cycle of interactions with the track (either an actin filament or a microtubule), resulting in force generation and directed movement.