RESUMEN
ß-III-Spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 L253P mutation in the cytoskeletal protein ß-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small-molecule actin-binding modulators of the L253P mutant ß-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P ß-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay for high-throughput compatibility, we first confirmed that our 50% FRET signal was responsive to swinholide A, an actin-severing compound, and that this yielded excellent assay quality with a Z' value > 0.77. Second, we screened a 2684-compound library of US Food and Drug Administration-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous cosedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a spinocerebellar ataxia type 5-specific therapeutic. Furthermore, our validated FRET-based in vitro high-throughput screening platform is poised for screening large compound libraries for ß-III-spectrin ABD modulators.
Asunto(s)
Actinas , Espectrina , Ataxias Espinocerebelosas , Humanos , Actinas/genética , Actinas/metabolismo , Descubrimiento de Drogas , Neuronas/metabolismo , Espectrina/metabolismo , Ataxias Espinocerebelosas/tratamiento farmacológico , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/metabolismoRESUMEN
Numerous diseases are linked to mutations in the actin-binding domains (ABDs) of conserved cytoskeletal proteins, including ß-III-spectrin, α-actinin, filamin, and dystrophin. A ß-III-spectrin ABD mutation (L253P) linked to spinocerebellar ataxia type 5 (SCA5) causes a dramatic increase in actin binding. Reducing actin binding of L253P is thus a potential therapeutic approach for SCA5 pathogenesis. Here, we validate a high-throughput screening (HTS) assay to discover potential disrupters of the interaction between the mutant ß-III-spectrin ABD and actin in live cells. This assay monitors FRET between fluorescent proteins fused to the mutant ABD and the actin-binding peptide Lifeact, in HEK293-6E cells. Using a specific and high-affinity actin-binding tool compound, swinholide A, we demonstrate HTS compatibility with an excellent Z'-factor of 0.67 ± 0.03. Screening a library of 1280 pharmacologically active compounds in 1536-well plates to determine assay robustness, we demonstrate high reproducibility across plates and across days. We identified nine Hits that reduced FRET between Lifeact and ABD. Four of those Hits were found to reduce Lifeact cosedimentation with actin, thus establishing the potential of our assay for detection of actin-binding modulators. Concurrent to our primary FRET assay, we also developed a high-throughput compatible counter screen to remove undesirable FRET Hits. Using the FRET Hits, we show that our counter screen is sensitive to undesirable compounds that cause cell toxicity or ABD aggregation. Overall, our FRET-based HTS platform sets the stage to screen large compound libraries for modulators of ß-III-spectrin, or disease-linked spectrin-related proteins, for therapeutic development.
Asunto(s)
Actinas/metabolismo , Sitios de Unión/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Proteínas Recombinantes de Fusión/metabolismo , Espectrina/metabolismo , Actinas/química , Actinas/genética , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Cinética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Toxinas Marinas/farmacología , Modelos Biológicos , Modelos Moleculares , Mutación , Fármacos Neuroprotectores/farmacología , Unión Proteica/efectos de los fármacos , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Reproducibilidad de los Resultados , Espectrina/química , Espectrina/genética , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/metabolismo , Ataxias Espinocerebelosas/patología , Proteína Fluorescente RojaRESUMEN
A spinocerebellar ataxia type 5 (SCA5) L253P mutation in the actin-binding domain (ABD) of ß-III-spectrin causes high-affinity actin binding and decreased thermal stability in vitro. Here we show in mammalian cells, at physiological temperature, that the mutant ABD retains high-affinity actin binding. Significantly, we provide evidence that the mutation alters the mobility and recruitment of ß-III-spectrin in mammalian cells, pointing to a potential disease mechanism. To explore this mechanism, we developed a Drosophila SCA5 model in which an equivalent mutant Drosophila ß-spectrin is expressed in neurons that extend complex dendritic arbors, such as Purkinje cells, targeted in SCA5 pathogenesis. The mutation causes a proximal shift in arborization coincident with decreased ß-spectrin localization in distal dendrites. We show that SCA5 ß-spectrin dominantly mislocalizes α-spectrin and ankyrin-2, components of the endogenous spectrin cytoskeleton. Our data suggest that high-affinity actin binding by SCA5 ß-spectrin interferes with spectrin-actin cytoskeleton dynamics, leading to a loss of a cytoskeletal mechanism in distal dendrites required for dendrite stabilization and arbor outgrowth.
Asunto(s)
Citoesqueleto/genética , Dendritas/genética , Mutación/genética , Plasticidad Neuronal/genética , Espectrina/genética , Ataxias Espinocerebelosas/genética , Animales , Ancirinas/genética , Células Cultivadas , Drosophila/genética , Drosophila/fisiología , Células HEK293 , Humanos , Neuronas/fisiología , Unión Proteica/genética , Células de Purkinje/fisiologíaRESUMEN
Degradation of cellular material by autophagy is essential for cell survival and homeostasis, and requires intracellular transport of autophagosomes to encounter acidic lysosomes through unknown mechanisms. Here, we identify the PX-domain-containing kinesin Klp98A as a new regulator of autophagosome formation, transport and maturation in Drosophila. Depletion of Klp98A caused abnormal clustering of autophagosomes and lysosomes at the cell center and reduced the formation of starvation-induced autophagic vesicles. Reciprocally, overexpression of Klp98A redistributed autophagic vesicles towards the cell periphery. These effects were accompanied by reduced autophagosome-lysosome fusion and autophagic degradation. In contrast, depletion of the conventional kinesin heavy chain caused a similar mislocalization of autophagosomes without perturbing their fusion with lysosomes, indicating that vesicle fusion and localization are separable and independent events. Klp98A-mediated fusion required the endolysosomal GTPase Rab14, which interacted and colocalized with Klp98A, and required Klp98A for normal localization. Thus, Klp98A coordinates the movement and fusion of autophagic vesicles by regulating their positioning and interaction with the endolysosomal compartment.
Asunto(s)
Autofagosomas/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Cinesinas/fisiología , Lisosomas/fisiología , Proteínas de Unión al GTP rab/fisiología , Animales , Autofagia , Línea Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Unión Proteica , Transporte de Proteínas , Proteolisis , Vesículas Transportadoras/metabolismoRESUMEN
Neuronal function depends on the retrograde relay of growth and survival signals from the synaptic terminal, where the neuron interacts with its targets, to the nucleus, where gene transcription is regulated. Activation of the Bone Morphogenetic Protein (BMP) pathway at the Drosophila larval neuromuscular junction results in nuclear accumulation of the phosphorylated form of the transcription factor Mad in the motoneuron nucleus. This in turn regulates transcription of genes that control synaptic growth. How BMP signaling at the synaptic terminal is relayed to the cell body and nucleus of the motoneuron to regulate transcription is unknown. We show that the BMP receptors are endocytosed at the synaptic terminal and transported retrogradely along the axon. Furthermore, this transport is dependent on BMP pathway activity, as it decreases in the absence of ligand or receptors. We further demonstrate that receptor traffic is severely impaired when Dynein motors are inhibited, a condition that has previously been shown to block BMP pathway activation. In contrast to these results, we find no evidence for transport of phosphorylated Mad along the axons, and axonal traffic of Mad is not affected in mutants defective in BMP signaling or retrograde transport. These data support a model in which complexes of activated BMP receptors are actively transported along the axon towards the cell body to relay the synaptogenic signal, and that phosphorylated Mad at the synaptic terminal and cell body represent two distinct molecular populations.
Asunto(s)
Transporte Axonal/fisiología , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/metabolismo , Neuronas Motoras/metabolismo , Terminales Presinápticos/metabolismo , Animales , Dineínas Axonemales/metabolismo , Axones/metabolismo , Receptores de Proteínas Morfogenéticas Óseas/genética , Proteínas de Unión al ADN/metabolismo , Drosophila , Proteínas de Drosophila/genética , Endosomas/genética , Endosomas/metabolismo , Neuronas Motoras/citología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
Vitrification-based cryopreservation is a promising approach to achieving long-term storage of biological systems for maintaining biodiversity, healthcare, and sustainable food production. Using the "cryomesh" system achieves rapid cooling and rewarming of biomaterials, but further improvement in cooling rates is needed to increase biosystem viability and the ability to cryopreserve new biosystems. Improved cooling rates and viability are possible by enabling conductive cooling through cryomesh. Conduction-dominated cryomesh improves cooling rates from twofold to tenfold (i.e., 0.24 to 1.2 × 105 °C min-1 ) in a variety of biosystems. Higher thermal conductivity, smaller mesh wire diameter and pore size, and minimizing the nitrogen vapor barrier (e.g., vertical plunging in liquid nitrogen) are key parameters to achieving improved vitrification. Conduction-dominated cryomesh successfully vitrifies coral larvae, Drosophila embryos, and zebrafish embryos with improved outcomes. Not only a theoretical foundation for improved vitrification in µm to mm biosystems but also the capability to scale up for biorepositories and/or agricultural, aquaculture, or scientific use are demonstrated.
Asunto(s)
Vitrificación , Pez Cebra , Animales , Criopreservación , Frío , NitrógenoRESUMEN
Drosophila melanogaster cellularization is a dramatic form of cytokinesis in which a membrane furrow simultaneously encapsulates thousands of cortical nuclei of the syncytial embryo to generate a polarized cell layer. Formation of this cleavage furrow depends on Golgi-based secretion and microtubules. During cellularization, specific Golgi move along microtubules, first to sites of furrow formation and later to accumulate within the apical cytoplasm of the newly forming cells. Here we show that Golgi movements and furrow formation depend on cytoplasmic dynein. Furthermore, we demonstrate that Lava lamp (Lva), a golgin protein that is required for cellularization, specifically associates with dynein, dynactin, cytoplasmic linker protein-190 (CLIP-190) and Golgi spectrin, and is required for the dynein-dependent targeting of the secretory machinery. The Lva domains that bind these microtubule-dependent motility factors inhibit Golgi movement and cellularization in a live embryo injection assay. Our results provide new evidence that golgins promote dynein-based motility of Golgi membranes.
Asunto(s)
Citocinesis/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Aparato de Golgi/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Complejo Dinactina , Embrión no Mamífero/citología , Desarrollo Embrionario/fisiología , Femenino , Aparato de Golgi/ultraestructura , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Espectrina/metabolismoRESUMEN
Recent structural studies of ß-III-spectrin and related cytoskeletal proteins revealed N-terminal sequences that directly bind actin. These sequences are variable in structure, and immediately precede a conserved actin-binding domain composed of tandem calponin homology domains (CH1 and CH2). Here we investigated in Drosophila the significance of the ß-spectrin N-terminus, and explored its functional interaction with a CH2-localized L253P mutation that underlies the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5). We report that pan-neuronal expression of an N-terminally truncated ß-spectrin fails to rescue lethality resulting from a ß-spectrin loss-of-function allele, indicating that the N-terminus is essential to ß-spectrin function in vivo. Significantly, N-terminal truncation rescues neurotoxicity and defects in dendritic arborization caused by L253P. In vitro studies show that N-terminal truncation eliminates L253P-induced high-affinity actin binding, providing a mechanistic basis for rescue. These data suggest that N-terminal sequences may be useful therapeutic targets for small molecule modulation of the aberrant actin binding associated with SCA5 ß-spectrin and spectrin-related disease proteins.
Asunto(s)
Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Neuronas/metabolismo , Espectrina/metabolismo , Ataxias Espinocerebelosas/metabolismo , Animales , Animales Modificados Genéticamente , Sitios de Unión , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Masculino , Mutación , Plasticidad Neuronal , Neuronas/patología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Espectrina/genética , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/patologíaRESUMEN
The localization of specific mRNAs can establish local protein gradients that generate and control the development of cellular asymmetries. While all evidence underscores the importance of the cytoskeleton in the transport and localization of RNAs, we have limited knowledge of how these events are regulated. Using a visual screen for motile proteins in a collection of GFP protein trap lines, we identified the Drosophila IGF-II mRNA-binding protein (Imp), an ortholog of Xenopus Vg1 RNA binding protein and chicken zipcode-binding protein. In Drosophila, Imp is part of a large, RNase-sensitive complex that is enriched in two polarized cell types, the developing oocyte and the neuron. Using time-lapse confocal microscopy, we establish that both dynein and kinesin contribute to the transport of GFP-Imp particles, and that regulation of transport in egg chambers appears to differ from that in neurons. In Drosophila, loss-of-function Imp mutations are zygotic lethal, and mutants die late as pharate adults. Imp has a function in Drosophila oogenesis that is not essential, as well as functions that are essential during embryogenesis and later development. Germline clones of Imp mutations do not block maternal mRNA localization or oocyte development, but overexpression of a specific Imp isoform disrupts dorsal/ventral polarity. We report here that loss-of-function Imp mutations, as well as Imp overexpression, can alter synaptic terminal growth. Our data show that Imp is transported to the neuromuscular junction, where it may modulate the translation of mRNA targets. In oocytes, where Imp function is not essential, we implicate a specific Imp domain in the establishment of dorsoventral polarity.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Oogénesis/fisiología , Terminales Presinápticos/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Transporte Biológico Activo , Tipificación del Cuerpo , Cartilla de ADN/genética , Drosophila/genética , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/genética , Femenino , Expresión Génica , Genes de Insecto , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Mutación , Oogénesis/genética , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms.
Asunto(s)
Citoplasma/enzimología , Dineínas/clasificación , Terminología como Asunto , Animales , HumanosRESUMEN
In Drosophila, the asymmetric localization of specific mRNAs to discrete regions within the developing oocyte determines the embryonic axes. The microtubule motors dynein and kinesin are required for the proper localization of the determinant ribonucleoprotein (RNP) complexes, but the mechanisms that account for RNP transport to and within the oocyte are not well understood. In this work, we focus on the transport of RNA complexes containing bicoid (bcd), an anterior determinant. We show in live egg chambers that, within the nurse cell compartment, dynein actively transports green fluorescent protein-tagged Exuperantia, a cofactor required for bcd RNP localization. Surprisingly, the loss of kinesin I activity elevates RNP motility in nurse cells, whereas disruption of dynein activity inhibits RNP transport. Once RNPs are transferred through the ring canal to the oocyte, they no longer display rapid, linear movements, but they are distributed by cytoplasmic streaming and gradually disassemble. By contrast, bcd mRNA injected into oocytes assembles de novo into RNP particles that exhibit rapid, dynein-dependent transport. We speculate that after delivery to the oocyte, RNP complexes may disassemble and be remodeled with appropriate accessory factors to ensure proper localization.
Asunto(s)
Drosophila melanogaster , Oocitos/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Transporte Biológico/fisiología , Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Proteínas del Huevo/genética , Proteínas del Huevo/metabolismo , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Sustancias Macromoleculares , Masculino , Microtúbulos/metabolismo , Oocitos/citología , Ovario/anatomía & histología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Animal cytokinesis relies on membrane addition as well as acto-myosin-based constriction. Recycling endosome (RE)-derived vesicles are a key source of this membrane. Rab11, a small GTPase associated with the RE and involved in vesicle targeting, is required for elongation of the cytokinetic furrow. In the early Drosophila embryo, Nuclear-fallout (Nuf), a Rab11 effector, promotes vesicle-mediated membrane delivery and actin organization at the invaginating furrow. Although Rab11 maintains a relatively constant localization at the microtubule-organizing center (MTOC), Nuf is present at the MTOC only during the phases of the cell cycle in which furrow invagination occurs. We demonstrate that Nuf protein levels remain relatively constant throughout the cell cycle, suggesting that Nuf is undergoing cycles of concentration and dispersion from the MTOC. Microtubules, but not microfilaments, are required for proper MTOC localization of Nuf and Rab11. The MTOC localization of Nuf also relies on Dynein. Immunoprecipitation experiments demonstrate that Nuf and Dynein physically interact. In accord with these findings, and in contrast to previous reports, we demonstrate that microtubules are required for proper metaphase furrow formation. We propose that the cell cycle-regulated, Dynein-dependent recruitment of Nuf to the MTOC influences the timing of RE-based vesicle delivery to the invaginating furrows.
Asunto(s)
Ciclo Celular , Proteínas de Drosophila/metabolismo , Dineínas/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Anafase , Animales , Centrosoma/metabolismo , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Inmunoprecipitación , Microtúbulos/metabolismo , Profase , Unión Proteica , Transporte de Proteínas , TelofaseRESUMEN
Cytokinesis requires a dramatic remodeling of the cortical cytoskeleton as well as membrane addition. The Drosophila pericentrosomal protein, Nuclear-fallout (Nuf), provides a link between these two processes. In nuf-derived embryos, actin remodeling and membrane recruitment during the initial stages of metaphase and cellular furrow formation are disrupted. Nuf is a homologue of arfophilin-2, an ADP ribosylation factor effector that binds Rab11 and influences recycling endosome (RE) organization. Here, we show that Nuf is an important component of the RE, and that these phenotypes are a consequence of Nuf activities at the RE. Nuf exhibits extensive colocalization with Rab11, a key RE component. GST pull-downs and the presence of a conserved Rab11-binding domain in Nuf demonstrate that Nuf and Rab11 physically associate. In addition, Nuf and Rab11 are mutually required for their localization to the RE. Embryos with reduced levels of Rab11 produce membrane recruitment and actin remodeling defects strikingly similar to nuf-derived embryos. These analyses support a common role for Nuf and Rab11 at the RE in membrane trafficking and actin remodeling during the initial stages of furrow formation.
Asunto(s)
Actinas/metabolismo , División Celular/fisiología , Proteínas de Drosophila , Proteínas Nucleares/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Centrosoma/metabolismo , Drosophila/embriología , Drosophila/fisiología , Genes Reporteros , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rab/genéticaRESUMEN
INTRODUCTION: The microtubule motor protein kinesin-5 is well known to establish the bipolar spindle by outward sliding of antiparallel interpolar microtubules. In yeast, kinesin-5 also facilitates chromosome alignment "congression" at the spindle equator by preferentially depolymerizing long kinetochore microtubules (kMTs). The motor protein kinesin-8 has also been linked to chromosome congression. Therefore, we sought to determine whether kinesin-5 or kinesin-8 facilitates chromosome congression in insect spindles. METHODS: RNAi of the kinesin-5 Klp61F and kinesin-8 Klp67A were performed separately in Drosophila melanogaster S2 cells to test for inhibited chromosome congression. Klp61F RNAi, Klp67A RNAi, and control metaphase mitotic spindles expressing fluorescent tubulin and fluorescent Cid were imaged, and their fluorescence distributions were compared. RESULTS: RNAi of Klp61F with a weak Klp61F knockdown resulted in longer kMTs and less congressed kinetochores compared to control over a range of conditions, consistent with kinesin-5 length-dependent depolymerase activity. RNAi of the kinesin-8 Klp67A revealed that kMTs relative to the spindle lengths were not longer compared to control, but rather that the spindles were longer, indicating that Klp67A acts preferentially as a length-dependent depolymerase on interpolar microtubules without significantly affecting kMT length and chromosome congression. CONCLUSIONS: This study demonstrates that in addition to establishing the bipolar spindle, kinesin-5 regulates kMT length to facilitate chromosome congression in insect spindles. It expands on previous yeast studies, and it expands the role of kinesin-5 to include kMT assembly regulation in eukaryotic mitosis.
RESUMEN
Variations in subunit composition and modification have been proposed to regulate the multiple functions of cytoplasmic dynein. Here, we examine the role of the Drosophila ortholog of tctex-1, the 14-kDa dynein light chain. We show that the 14-kDa light chain is a bona fide component of Drosophila cytoplasmic dynein and use P element excision to generate flies that completely lack this dynein subunit. Remarkably, the null mutant is viable and the only observed defect is complete male sterility. During spermatid differentiation, the 14-kDa light chain is required for the localization of a nuclear "cap" of cytoplasmic dynein and for proper attachment between the sperm nucleus and flagellar basal body. Our results provide evidence that the function of the 14-kDa light chain in Drosophila is distinct from other dynein subunits and is not required for any essential functions in early development or in the adult organism.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/fisiología , Dineínas/metabolismo , Dineínas/fisiología , Espermátides/ultraestructura , Espermatogénesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dineínas Citoplasmáticas , Análisis Mutacional de ADN , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestructura , Dineínas/análisis , Dineínas/genética , Fertilidad/genética , Masculino , Datos de Secuencia Molecular , Mutagénesis Insercional , Espermátides/química , Espermatogénesis/genética , Testículo/química , Testículo/metabolismo , Testículo/ultraestructuraRESUMEN
Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of the Drosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice.
Asunto(s)
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Dineínas/química , Dineínas/metabolismo , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Sitios de Unión , Citoplasma/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Dineínas/genética , Femenino , Genes de Insecto , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/genética , Mutagénesis Sitio-Dirigida , Oogénesis , Estructura Terciaria de Proteína , Rayos Ultravioleta , VanadatosRESUMEN
Autophagy plays an essential role in the cellular homeostasis of neurons, facilitating the clearance of cellular debris. This clearance process is orchestrated through the assembly, transport, and fusion of autophagosomes with lysosomes for degradation. The motor protein dynein drives autophagosome motility from distal sites of assembly to sites of lysosomal fusion. In this study, we identify the scaffold protein CKA (connector of kinase to AP-1) as essential for autophagosome transport in neurons. Together with other core components of the striatin-interacting phosphatase and kinase (STRIPAK) complex, we show that CKA associates with dynein and directly binds Atg8a, an autophagosomal protein. CKA is a regulatory subunit of PP2A, a component of the STRIPAK complex. We propose that the STRIPAK complex modulates dynein activity. Consistent with this hypothesis, we provide evidence that CKA facilitates axonal transport of dense core vesicles and autophagosomes in a PP2A-dependent fashion. In addition, CKA-deficient flies exhibit PP2A-dependent motor coordination defects. CKA function within the STRIPAK complex is crucial to prevent transport defects that may contribute to neurodegeneration.
Asunto(s)
Autofagosomas/enzimología , Transporte Axonal , Axones/enzimología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Complejos Multiproteicos/metabolismo , Proteína Fosfatasa 2/metabolismo , Vesículas Secretoras/enzimología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Dineínas/genética , Dineínas/metabolismo , Genotipo , Microscopía Fluorescente , Complejos Multiproteicos/genética , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , Terminales Presinápticos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Fosfatasa 2/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , TransfecciónRESUMEN
Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the cytoskeletal protein ß-III-spectrin. Previously, a SCA5 mutation resulting in a leucine-to-proline substitution (L253P) in the actin-binding domain (ABD) was shown to cause a 1000-fold increase in actin-binding affinity. However, the structural basis for this increase is unknown. Here, we report a 6.9 Å cryo-EM structure of F-actin complexed with the L253P ABD. This structure, along with co-sedimentation and pulsed-EPR measurements, demonstrates that high-affinity binding caused by the CH2-localized mutation is due to opening of the two CH domains. This enables CH1 to bind actin aided by an unstructured N-terminal region that becomes α-helical upon binding. This helix is required for association with actin as truncation eliminates binding. Collectively, these results shed light on the mechanism by which ß-III-spectrin, and likely similar actin-binding proteins, interact with actin, and how this mechanism can be perturbed to cause disease.
Asunto(s)
Actinas/metabolismo , Mutación Missense , Espectrina/química , Espectrina/genética , Sitios de Unión , Microscopía por Crioelectrón , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Espectrina/metabolismoRESUMEN
Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein ß-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of ß-III-spectrin. We report that the L253P substitution in the isolated ß-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 µM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P ß-III-spectrin is a likely driver of neurodegeneration.
Asunto(s)
Mutación , Espectrina/genética , Espectrina/metabolismo , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Estabilidad Proteica , Espectrina/química , TermodinámicaRESUMEN
Proper neuronal function critically depends on efficient intracellular transport and disruption of transport leads to neurodegeneration. Molecular pathways that support or regulate neuronal transport are not fully understood. A greater understanding of these pathways will help reveal the pathological mechanisms underlying disease. Drosophila melanogaster is the premier model system for performing large-scale genetic functional screens. Here we describe methods to carry out primary and secondary genetic screens in Drosophila aimed at identifying novel gene products and pathways that impact neuronal intracellular transport. These screens are performed using whole animal or live cell imaging of intact neural tissue to ensure integrity of neurons and their cellular environment. The primary screen is used to identify gross defects in neuronal function indicative of a disruption in microtubule-based transport. The secondary screens, conducted in both motoneurons and dendritic arborization neurons, will confirm the function of candidate gene products in intracellular transport. Together, the methodologies described here will support labs interested in identifying and characterizing gene products that alter intracellular transport in Drosophila.