RESUMEN
The kinesin-3 motor KIF13B functions in endocytosis, vesicle transport and regulation of ciliary length and signaling. Direct binding of the membrane-associated guanylate kinase (MAGUK) DLG1 to the MAGUK-binding stalk domain of KIF13B relieves motor autoinhibition and promotes microtubule plus-end-directed cargo transport. Here, we characterize angiomotin (AMOT) isoform 2 (p80, referred to as Ap80) as a novel KIF13B interactor that promotes binding of another MAGUK, the polarity protein and Crumbs complex component PALS1, to KIF13B. Live-cell imaging analysis indicated that Ap80 is concentrated at and recruits PALS1 to the base of the primary cilium, but is not a cargo of KIF13B itself. Consistent with a ciliary function for Ap80, its depletion led to elongated primary cilia and reduced agonist-induced ciliary accumulation of SMO, a key component of the Hedgehog signaling pathway, whereas Ap80 overexpression caused ciliary shortening. Our results suggest that Ap80 activates KIF13B cargo binding at the base of the primary cilium to regulate ciliary length, composition and signaling.
Asunto(s)
Angiomotinas , Proteínas de la Membrana , Cilios/metabolismo , Guanilato-Quinasas , Proteínas Hedgehog/metabolismo , Proteínas de la Membrana/metabolismo , Isoformas de ProteínasRESUMEN
Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, migration, and morphogenesis. EB1 and its homologs are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general "microtubule tip localization signal" (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process.
Asunto(s)
Proteínas Asociadas a Microtúbulos/química , Microtúbulos/química , Señales de Clasificación de Proteína , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Alineación de SecuenciaRESUMEN
MOTIVATION: Biological studies of dynamic processes in living cells often require accurate particle tracking as a first step toward quantitative analysis. Although many particle tracking methods have been developed for this purpose, they are typically based on prior assumptions about the particle dynamics, and/or they involve careful tuning of various algorithm parameters by the user for each application. This may make existing methods difficult to apply by non-expert users and to a broader range of tracking problems. Recent advances in deep-learning techniques hold great promise in eliminating these disadvantages, as they can learn how to optimally track particles from example data. RESULTS: Here, we present a deep-learning-based method for the data association stage of particle tracking. The proposed method uses convolutional neural networks and long short-term memory networks to extract relevant dynamics features and predict the motion of a particle and the cost of linking detected particles from one time point to the next. Comprehensive evaluations on datasets from the particle tracking challenge demonstrate the competitiveness of the proposed deep-learning method compared to the state of the art. Additional tests on real-time-lapse fluorescence microscopy images of various types of intracellular particles show the method performs comparably with human experts. AVAILABILITY AND IMPLEMENTATION: The software code implementing the proposed method as well as a description of how to obtain the test data used in the presented experiments will be available for non-commercial purposes from https://github.com/yoyohoho0221/pt_linking. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Aprendizaje Profundo , Algoritmos , Humanos , Microscopía Fluorescente , Redes Neurales de la Computación , Programas InformáticosRESUMEN
During cytokinesis, the antiparallel array of microtubules forming the central spindle organizes the midbody, a structure that anchors the ingressed cleavage furrow and guides the assembly of abscission machinery. Here, we identified a role for the flavoprotein monooxygenase MICAL3, an actin disassembly factor, in organizing midbody-associated protein complexes. By combining cell biological assays with cross-linking mass spectrometry, we show that MICAL3 is recruited to the central spindle and the midbody through a direct interaction with the centralspindlin component MKLP1. Knock-out of MICAL3 leads to an increased frequency of cytokinetic failure and a delayed abscission. In a mechanism independent of its enzymatic activity, MICAL3 targets the adaptor protein ELKS and Rab8A-positive vesicles to the midbody, and the depletion of ELKS and Rab8A also leads to cytokinesis defects. We propose that MICAL3 acts as a midbody-associated scaffold for vesicle targeting, which promotes maturation of the intercellular bridge and abscission.
Asunto(s)
Citocinesis/fisiología , Oxigenasas de Función Mixta/metabolismo , Huso Acromático/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Oxigenasas de Función Mixta/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Huso Acromático/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Neuronal responses to Ca2+-signals are provided by EF-hand-type neuronal Ca2+-sensor (NCS) proteins, which have similar core domains containing Ca2+-binding and target-recognizing sites. NCS proteins vary in functional specificity, probably depending on the structure and conformation of their non-conserved C-terminal segments. Here, we investigated the role of the C-terminal segment in guanylate cyclase activating protein-2, GCAP2, an NCS protein controlling the Ca2+-dependent regulation of photoreceptor guanylate cyclases. We obtained two chimeric proteins by exchanging C-terminal segments between GCAP2 and its photoreceptor homolog recoverin, a Ca2+-sensor controlling rhodopsin kinase (RK) activity. The exchange affected neither the structural integrity of GCAP2 and recoverin nor the Ca2+-sensitivity of GCAP2. Intrinsic fluorescence, circular dichroism, biochemical studies and hydrophobic dye probing revealed Ca2+-dependent conformational transition of the C-terminal segment of GCAP2 occurring in the molecular environment of both proteins. In Ca2+-GCAP2, the C-terminal segment was constrained and its replacement provided the protein with approximately two-fold inhibitory activity towards RK, suggesting that the segment contributes to specific target recognition by interfering with RK-binding. Upon Ca2+-release, it became less constrained and more available for phosphorylation by cyclic nucleotide-dependent protein kinase. The transition from the Ca2+-bound to the apo-state exposed hydrophobic sites in GCAP2, and was associated with its activating function without affecting its dimerization. The released C-terminal segment participated further in photoreceptor membrane binding making it sensitive to phosphorylation. Thus, the C-terminal segment in GCAP2 confers target selectivity, facilitates membrane binding and provides sensitivity of the membrane localization of the protein to phosphorylation by signaling kinases.
Asunto(s)
Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Proteínas Activadoras de la Guanilato-Ciclasa/metabolismo , Guanilato Ciclasa/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Recoverina/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcio/metabolismo , Señalización del Calcio , Bovinos , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Regulación de la Expresión Génica , Guanilato Ciclasa/genética , Proteínas Activadoras de la Guanilato-Ciclasa/química , Proteínas Activadoras de la Guanilato-Ciclasa/genética , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Recoverina/química , Recoverina/genética , Alineación de SecuenciaRESUMEN
Membrane and secretory trafficking are essential for proper neuronal development. However, the molecular mechanisms that organize secretory trafficking are poorly understood. Here, we identify Bicaudal-D-related protein 1 (BICDR-1) as an effector of the small GTPase Rab6 and key component of the molecular machinery that controls secretory vesicle transport in developing neurons. BICDR-1 interacts with kinesin motor Kif1C, the dynein/dynactin retrograde motor complex, regulates the pericentrosomal localization of Rab6-positive secretory vesicles and is required for neural development in zebrafish. BICDR-1 expression is high during early neuronal development and strongly declines during neurite outgrowth. In young neurons, BICDR-1 accumulates Rab6 secretory vesicles around the centrosome, restricts anterograde secretory transport and inhibits neuritogenesis. Later during development, BICDR-1 expression is strongly reduced, which permits anterograde secretory transport required for neurite outgrowth. These results indicate an important role for BICDR-1 as temporal regulator of secretory trafficking during the early phase of neuronal differentiation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Centrosoma/ultraestructura , Proteínas del Citoesqueleto/metabolismo , Neuronas/patología , Proteínas de Unión al GTP rab/metabolismo , Animales , Encéfalo/metabolismo , Células COS , Diferenciación Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Humanos , Riñón/metabolismo , Cinesinas/química , Modelos Biológicos , Neuronas/metabolismo , Pez CebraRESUMEN
Development, polarization, structural integrity, and plasticity of neuronal cells critically depend on the microtubule network and its dynamic properties. SLAIN1 and SLAIN2 are microtubule plus-end tracking proteins that have been recently identified as regulators of microtubule dynamics. SLAINs are targeted to microtubule tips through an interaction with the core components of microtubule plus-end tracking protein network, End Binding family members. SLAINs promote persistent microtubule growth by recruiting the microtubule polymerase ch-TOG to microtubule plus-ends. Here, we show that SLAIN1/2 and ch-TOG-proteins are highly enriched in brain and are expressed throughout mouse brain development. Disruption of the SLAIN-ch-TOG complex in cultured primary rat hippocampal neurons by RNA interference-mediated knockdown and a dominant-negative approach perturbs microtubule growth by increasing catastrophe frequency and inhibits axon extension during neuronal development. Our study shows that proper control of microtubule dynamics is important for axon elongation in developing neurons.
Asunto(s)
Axones/fisiología , Hipocampo/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas/fisiología , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Células Cultivadas , Femenino , Hipocampo/embriología , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , RatasRESUMEN
The microtubule (MT) plus-end-tracking protein EB1 is required for assembly of primary cilia in mouse fibroblasts, but the mechanisms involved and the roles of the related proteins EB2 and EB3 in ciliogenesis are unknown. Using protein depletion experiments and expression of dominant-negative constructs we show here that EB1 and EB3, but not EB2, are required for assembly of primary cilia in cultured cells. Electron microscopy and live imaging showed that cells lacking EB1 or EB3 are defective in MT minus-end anchoring at the centrosome and/or basal body, and possess abnormally short cilia stumps surrounded by vesicles. Further, GST pull-down assays, mass spectrometry and immunoprecipitation indicated that EB1 and EB3 interact with proteins implicated in MT minus-end anchoring or vesicular trafficking to the cilia base, suggesting that EB1 and EB3 promote ciliogenesis by facilitating such trafficking. In addition, we show that EB3 is localized to the tip of motile cilia in bronchial epithelial cells and affects the formation of centriole-associated rootlet filaments. Collectively, our findings indicate that EBs affect biogenesis of cilia by several centrosome-related mechanisms and support the idea that different EB1-EB3 dimer species have distinct functions within cells.
Asunto(s)
Centrosoma/metabolismo , Cilios/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Células Cultivadas , Centrosoma/ultraestructura , Cilios/ultraestructura , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Inmunoprecipitación , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genéticaRESUMEN
BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D, an evolutionarily conserved adaptor between microtubule motors and their cargo that was previously shown to link vesicles and mRNP complexes to the dynein motor. Here, we identified a G2-specific role for BICD2 in the relative positioning of the nucleus and centrosomes in dividing cells. By combining mass spectrometry, biochemical and cell biological approaches, we show that the nuclear pore complex (NPC) component RanBP2 directly binds to BICD2 and recruits it to NPCs specifically in G2 phase of the cell cycle. BICD2, in turn, recruits dynein-dynactin to NPCs and as such is needed to keep centrosomes closely tethered to the nucleus prior to mitotic entry. When dynein function is suppressed by RNA interference-mediated depletion or antibody microinjection, centrosomes and nuclei are actively pushed apart in late G2 and we show that this is due to the action of kinesin-1. Surprisingly, depletion of BICD2 inhibits both dynein and kinesin-1-dependent movements of the nucleus and cytoplasmic NPCs, demonstrating that BICD2 is needed not only for the dynein function at the nuclear pores but also for the antagonistic activity of kinesin-1. Our study demonstrates that the nucleus is subject to opposing activities of dynein and kinesin-1 motors and that BICD2 contributes to nuclear and centrosomal positioning prior to mitotic entry through regulation of both dynein and kinesin-1.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Centrosoma/metabolismo , Dineínas/metabolismo , Cinesinas/metabolismo , Proteínas de la Membrana/metabolismo , Mitosis/fisiología , Poro Nuclear/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Núcleo Celular/ultraestructura , Complejo Dinactina , Humanos , Cinesinas/genética , Proteínas de la Membrana/genética , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Huso Acromático/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling the formation of interphase microtubule arrays and mitotic spindles have been extensively studied, the biochemical mechanisms responsible for generating stable microtubule cores of centrioles and cilia are poorly understood. Here, we used in vitro reconstitution assays to investigate microtubule-stabilizing properties of CSPP1, a centrosome and cilia-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome. We found that CSPP1 preferentially binds to polymerizing microtubule ends that grow slowly or undergo growth perturbations and, in this way, resembles microtubule-stabilizing compounds such as taxanes. Fluorescence microscopy and cryo-electron tomography showed that CSPP1 is deposited in the microtubule lumen and inhibits microtubule growth and shortening through two separate domains. CSPP1 also specifically recognizes and stabilizes damaged microtubule lattices. These data help to explain how CSPP1 regulates the elongation and stability of ciliary axonemes and other microtubule-based structures.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Asociadas a Microtúbulos , Microtúbulos , Centriolos/metabolismo , Centrosoma/metabolismo , Citoesqueleto/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , HumanosRESUMEN
Constitutive exocytosis delivers newly synthesized proteins, lipids, and other molecules from the Golgi apparatus to the cell surface. This process is mediated by vesicles, which bud off the trans-Golgi network, move along cytoskeletal filaments, and fuse with the plasma membrane. Here, we show that the small GTPase Rab6 marks exocytotic vesicles and, together with the microtubule plus-end-directed motor kinesin-1, stimulates their processive microtubule-based transport to the cell periphery. Furthermore, Rab6 directs targeting of secretory vesicles to plasma-membrane sites enriched in the cortical protein ELKS, a known Rab6 binding partner. Our data demonstrate that although Rab6 is not essential for secretion, it controls the organization of exocytosis within the cellular space.
Asunto(s)
Exocitosis , Vesículas Transportadoras/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Transporte Biológico , Biomarcadores/metabolismo , Línea Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Perros , Dineínas/metabolismo , Células HeLa , Humanos , Cinesinas/metabolismo , Fusión de Membrana , Proteínas del Tejido Nervioso/metabolismo , RatasRESUMEN
NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin-kinase complex provided insight into the protein-protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation-π interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.
Asunto(s)
Quinasa 1 del Receptor Acoplado a Proteína-G/química , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Dominios y Motivos de Interacción de Proteínas/fisiología , Recoverina/química , Recoverina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/fisiología , Animales , Sitios de Unión/genética , Bovinos , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Dominios y Motivos de Interacción de Proteínas/genética , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Recoverina/genética , Homología de Secuencia de AminoácidoRESUMEN
Microtubules are cytoskeletal polymers that separate chromosomes during mitosis and serve as rails for intracellular transport and organelle positioning. Manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubules are currently lacking. Here, we describe a light-activated system for localized recruitment of the microtubule-severing enzyme katanin. This system, named opto-katanin, uses targeted illumination with blue light to induce rapid, localized, and reversible microtubule depolymerization. This tool allows precise clearing of a subcellular region of microtubules while preserving the rest of the microtubule network, demonstrating that regulation of katanin recruitment to microtubules is sufficient to control its severing activity. The tool is not toxic in the absence of blue light and can be used to disassemble both dynamic and stable microtubules in primary neurons as well as in dividing cells. We show that opto-katanin can be used to locally block vesicle transport and to clarify the dependence of organelle morphology and dynamics on microtubules. Specifically, our data indicate that microtubules are not required for the maintenance of the Golgi stacks or the tubules of the endoplasmic reticulum but are needed for the formation of new membrane tubules. Finally, we demonstrate that this tool can be applied to study the contribution of microtubules to cell mechanics by showing that microtubule bundles can exert forces constricting the nucleus.
Asunto(s)
Adenosina Trifosfatasas , Optogenética , Katanina/genética , Katanina/metabolismo , Adenosina Trifosfatasas/metabolismo , Microtúbulos/metabolismo , MitosisRESUMEN
Stromal interaction molecule 1 (STIM1) is a transmembrane protein that is essential for store-operated Ca(2+) entry, a process of extracellular Ca(2+) influx in response to the depletion of Ca(2+) stores in the endoplasmic reticulum (ER) (reviewed in [1-4]). STIM1 localizes predominantly to the ER; upon Ca(2+) release from the ER, STIM1 translocates to the ER-plasma membrane junctions and activates Ca(2+) channels (reviewed in [1-4]). Here, we show that STIM1 directly binds to the microtubule-plus-end-tracking protein EB1 and forms EB1-dependent comet-like accumulations at the sites where polymerizing microtubule ends come in contact with the ER network. Therefore, the previously observed tubulovesicular motility of GFP-STIM1 [5] is not a motor-based movement but a traveling wave of diffusion-dependent STIM1 concentration in the ER membrane. STIM1 overexpression strongly stimulates ER extension occurring through the microtubule "tip attachment complex" (TAC) mechanism [6, 7], a process whereby an ER tubule attaches to and elongates together with the EB1-positive end of a growing microtubule. Depletion of STIM1 and EB1 decreases TAC-dependent ER protrusion, indicating that microtubule growth-dependent concentration of STIM1 in the ER membrane plays a role in ER remodeling.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Calcio/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Molécula de Interacción Estromal 1RESUMEN
CLASPs are mammalian microtubule-stabilizing proteins that can mediate the interaction between distal microtubule ends and the cell cortex. Using mass spectrometry-based assays, we have identified two CLASP partners, LL5beta and ELKS. LL5beta and ELKS form a complex that colocalizes with CLASPs at the cortex of HeLa cells as well as at the leading edge of motile fibroblasts. LL5beta is required for cortical CLASP accumulation and microtubule stabilization in HeLa cells, while ELKS plays an accessory role in these processes. LL5beta is a phosphatidylinositol-3,4,5-triphosphate (PIP3) binding protein, and its recruitment to the cell cortex is influenced by PI3 kinase activity but does not require intact microtubules. Cortical clusters of LL5beta and ELKS do not overlap with focal adhesions but often form in their vicinity and can affect their size. We propose that LL5beta and ELKS can form a PIP3-regulated cortical platform to which CLASPs attach distal microtubule ends.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Proteínas Portadoras/genética , Línea Celular , Chlorocebus aethiops , Citoesqueleto/metabolismo , Eliminación de Gen , Células HeLa , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Complejos Multiproteicos , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Fracciones Subcelulares/metabolismo , Células 3T3 Swiss , TransfecciónRESUMEN
Many aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating this concept. However, despite the use of ATIs in plant research for many decades, the mechanism of ATI action has remained largely elusive. Using real-time live-cell microscopy, we show here that prominent ATIs such as 2,3,5-triiodobenzoic acid (TIBA) and 2-(1-pyrenoyl) benzoic acid (PBA) inhibit vesicle trafficking in plant, yeast, and mammalian cells. Effects on micropinocytosis, rab5-labeled endosomal motility at the periphery of HeLa cells and on fibroblast mobility indicate that ATIs influence actin cytoskeleton. Visualization of actin cytoskeleton dynamics in plants, yeast, and mammalian cells show that ATIs stabilize actin. Conversely, stabilizing actin by chemical or genetic means interferes with endocytosis, vesicle motility, auxin transport, and plant development, including auxin transport-dependent processes. Our results show that a class of ATIs act as actin stabilizers and advocate that actin-dependent trafficking of auxin transport components participates in the mechanism of auxin transport. These studies also provide an example of how the common eukaryotic process of actin-based vesicle motility can fulfill a plant-specific physiological role.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Ácidos Indolacéticos/antagonistas & inhibidores , Ácidos Indolacéticos/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Transporte Biológico , Línea Celular , Endocitosis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismoRESUMEN
CLIP-associating protein (CLASP) 1 and CLASP2 are mammalian microtubule (MT) plus-end binding proteins, which associate with CLIP-170 and CLIP-115. Using RNA interference in HeLa cells, we show that the two CLASPs play redundant roles in regulating the density, length distribution and stability of interphase MTs. In HeLa cells, both CLASPs concentrate on the distal MT ends in a narrow region at the cell margin. CLASPs stabilize MTs by promoting pauses and restricting MT growth and shortening episodes to this peripheral cell region. We demonstrate that the middle part of CLASPs binds directly to EB1 and to MTs. Furthermore, we show that the association of CLASP2 with the cell cortex is MT independent and relies on its COOH-terminal domain. Both EB1- and cortex-binding domains of CLASP are required to promote MT stability. We propose that CLASPs can mediate interactions between MT plus ends and the cell cortex and act as local rescue factors, possibly through forming a complex with EB1 at MT tips.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animales , Células COS , Chlorocebus aethiops , Citoesqueleto/metabolismo , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Mitosis/fisiología , Proteínas de Neoplasias , Proteínas del Tejido Nervioso/metabolismo , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Microtubule-dependent organization of membranous organelles occurs through motor-based pulling and by coupling microtubule dynamics to membrane remodeling. For example, tubules of endoplasmic reticulum (ER) can be extended by kinesin- and dynein-mediated transport and through the association with the tips of dynamic microtubules. The binding between ER and growing microtubule plus ends requires End Binding (EB) proteins and the transmembrane protein STIM1, which form a tip-attachment complex (TAC), but it is unknown whether these proteins are sufficient for membrane remodeling. Furthermore, EBs and their partners undergo rapid turnover at microtubule ends, and it is unclear how highly transient protein-protein interactions can induce load-bearing processive motion. Here, we reconstituted membrane tubulation in a minimal system with giant unilamellar vesicles, dynamic microtubules, an EB protein, and a membrane-bound protein that can interact with EBs and microtubules. We showed that these components are sufficient to drive membrane remodeling by three mechanisms: membrane tubulation induced by growing microtubule ends, motor-independent membrane sliding along microtubule shafts, and membrane pulling by shrinking microtubules. Experiments and modeling demonstrated that the first two mechanisms can be explained by adhesion-driven biased membrane spreading on microtubules. Optical trapping revealed that growing and shrinking microtubule ends can exert forces of â¼0.5 and â¼5 pN, respectively, through attached proteins. Rapidly exchanging molecules that connect membranes to dynamic microtubules can thus bear a sufficient load to induce membrane deformation and motility. Furthermore, combining TAC components and a membrane-attached kinesin in the same in vitro assays demonstrated that they can cooperate in promoting membrane tubule extension.
Asunto(s)
Retículo Endoplásmico/fisiología , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/fisiología , Cinesinas/metabolismo , Microtúbulos/metabolismoRESUMEN
During cytokinesis, signals from the anaphase spindle direct the formation and position of a contractile ring at the cell cortex [1]. The chromosomal passenger complex (CPC) participates in cytokinesis initiation by signaling from the spindle midzone and equatorial cortex [2], but the mechanisms underlying the anaphase-specific CPC localization are currently unresolved. Accumulation of the CPC at these sites requires the presence of microtubules and the mitotic kinesin-like protein 2, MKLP2 (KIF20A), a member of the kinesin-6 family [2-7], and this has led to the hypothesis that the CPC is transported along microtubules by MKLP2 [3-5, 7]. However, the structure of the MKLP2 motor domain with its extended neck-linker region suggests that this kinesin might not be able to drive processive transport [8, 9]. Furthermore, experiments in Xenopus egg extracts indicated that the CPC might be transported by kinesin-4, KIF4A [10]. Finally, CPC-MKLP2 complexes might be directly recruited to the equatorial cortex via association with actin and myosin II, independent of kinesin activity [4, 8]. Using microscopy-based assays with purified proteins, we demonstrate that MKLP2 is a processive plus-end directed motor that can transport the CPC along microtubules in vitro. In cells, strong suppression of MKLP2-dependent CPC motility by expression of an MKLP2 P-loop mutant perturbs CPC accumulation at both the spindle midzone and equatorial cortex, whereas a weaker inhibition of MKLP2 motor using Paprotrain mainly affects CPC localization to the equatorial cortex. Our data indicate that control of cytokinesis initiation by the CPC requires its directional MKLP2-dependent transport.
Asunto(s)
Anafase/fisiología , Citocinesis , Cinesinas/genética , Familia de Multigenes , Células HEK293 , Células HeLa , Humanos , Cinesinas/metabolismo , Transporte de ProteínasRESUMEN
Intracellular transport relies on multiple kinesins, but it is poorly understood which kinesins are present on particular cargos, what their contributions are and whether they act simultaneously on the same cargo. Here, we show that Rab6-positive secretory vesicles are transported from the Golgi apparatus to the cell periphery by kinesin-1 KIF5B and kinesin-3 KIF13B, which determine the location of secretion events. KIF5B plays a dominant role, whereas KIF13B helps Rab6 vesicles to reach freshly polymerized microtubule ends, to which KIF5B binds poorly, likely because its cofactors, MAP7-family proteins, are slow in populating these ends. Sub-pixel localization demonstrated that during microtubule plus-end directed transport, both kinesins localize to the vesicle front and can be engaged on the same vesicle. When vesicles reverse direction, KIF13B relocates to the middle of the vesicle, while KIF5B shifts to the back, suggesting that KIF5B but not KIF13B undergoes a tug-of-war with a minus-end directed motor.