RESUMEN
The yeast (Saccharomyces cerevisiae) plasma membrane (PM) is organised into specific subdomains that regulate surface membrane proteins. Surface transporters actively uptake nutrients in particular regions of the PM where they are also susceptible to substrate-induced endocytosis. However, transporters also diffuse into distinct subdomains termed eisosomes, where they are protected from endocytosis. Although most nutrient transporter populations are downregulated in the vacuole following glucose starvation, a small pool is retained in eisosomes to provide efficient recovery from starvation. We find the core eisosome subunit Pil1, a Bin, Amphiphysin and Rvs (BAR) domain protein required for eisosome biogenesis, is phosphorylated primarily by the kinase Pkh2. In response to acute glucose starvation, Pil1 is rapidly dephosphorylated. Enzyme localisation and activity screens suggest that the phosphatase Glc7 is the primary enzyme responsible for Pil1 dephosphorylation. Defects in Pil1 phosphorylation, achieved by depletion of GLC7 or expression of phospho-ablative or phospho-mimetic mutants, correlate with reduced retention of transporters in eisosomes and inefficient starvation recovery. We propose that precise post-translational control of Pil1 modulates nutrient transporter retention within eisosomes, depending on extracellular nutrient levels, to maximise recovery following starvation.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Glucosa/metabolismoRESUMEN
A major hallmark of Alzheimer's disease is the misfolding and aggregation of the amyloid- ß peptide (Aß). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble Aß oligomers as being orders of magnitude more harmful. Techniques capable of characterizing oligomer stoichiometry and assembly are thus critical for a deeper understanding of the earliest stages of neurodegeneration and for rationally testing next-generation oligomer inhibitors. While the fluorescence response of extrinsic fluorescent probes such as Thioflavin-T have become workhorse tools for characterizing large Aß aggregates in solution, it is widely accepted that these methods suffer from many important drawbacks, including an insensitivity to oligomeric species. Here, we integrate several biophysics techniques to gain new insight into oligomer formation at the single-molecule level. We showcase single-molecule stepwise photobleaching of fluorescent dye molecules as a powerful method to bypass many of the traditional limitations, and provide a step-by-step guide to implementing the technique in vitro. By collecting fluorescence emission from single Aß(1-42) peptides labelled at the N-terminal position with HiLyte Fluor 555 via wide-field total internal reflection fluorescence (TIRF) imaging, we demonstrate how to characterize the number of peptides per single immobile oligomer and reveal heterogeneity within sample populations. Importantly, fluorescence emerging from Aß oligomers cannot be easily investigated using diffraction-limited optical microscopy tools. To assay oligomer activity, we also demonstrate the implementation of another biophysical method involving the ratiometric imaging of Fura-2-AM loaded cells which quantifies the rate of oligomer-induced dysregulation of intracellular Ca2+ homeostasis. We anticipate that the integrated single-molecule biophysics approaches highlighted here will develop further and in principle may be extended to the investigation of other protein aggregation systems under controlled experimental conditions.
Asunto(s)
Fotoblanqueo , Enfermedad de Alzheimer , Péptidos beta-Amiloides , Colorantes Fluorescentes , Humanos , Fragmentos de Péptidos , Agregado de ProteínasRESUMEN
The presence of the neuronal-specific N1-Src splice variant of the C-Src tyrosine kinase is conserved through vertebrate evolution, suggesting an important role in complex nervous systems. Alternative splicing involving an N1-Src-specific microexon leads to a 5 or 6 aa insertion into the SH3 domain of Src. A prevailing model suggests that N1-Src regulates neuronal differentiation via cytoskeletal dynamics in the growth cone. Here we investigated the role of n1-src in the early development of the amphibian Xenopus tropicalis, and found that n1-src expression is regulated in embryogenesis, with highest levels detected during the phases of primary and secondary neurogenesis. In situ hybridization analysis, using locked nucleic acid oligo probes complementary to the n1-src microexon, indicates that n1-src expression is highly enriched in the open neural plate during neurula stages and in the neural tissue of adult frogs. Given the n1-src expression pattern, we investigated a possible role for n1-src in neurogenesis. Using splice site-specific antisense morpholino oligos, we inhibited n1-src splicing, while preserving c-src expression. Differentiation of neurons in the primary nervous system is reduced in n1-src-knockdown embryos, accompanied by a severely impaired touch response in later development. These data reveal an essential role for n1-src in amphibian neural development and suggest that alternative splicing of C-Src in the developing vertebrate nervous system evolved to regulate neurogenesis.SIGNIFICANCE STATEMENT The Src family of nonreceptor tyrosine kinases acts in signaling pathways that regulate cell migration, cell adhesion, and proliferation. Srcs are also enriched in the brain, where they play key roles in neuronal development and neurotransmission. Vertebrates have evolved a neuron-specific splice variant of C-Src, N1-Src, which differs from C-Src by just 5 or 6 aa. N1-Src is poorly understood and its high similarity to C-Src has made it difficult to delineate its function. Using antisense knockdown of the n1-src microexon, we have studied neuronal development in the Xenopus embryo in the absence of n1-src, while preserving c-src Loss of n1-src causes a striking absence of primary neurogenesis, implicating n1-src in the specification of neurons early in neural development.
Asunto(s)
Células-Madre Neurales/citología , Células-Madre Neurales/enzimología , Neurogénesis/fisiología , Neuronas/citología , Neuronas/enzimología , Xenopus/fisiología , Familia-src Quinasas/metabolismo , Animales , Diferenciación Celular/fisiología , Activación Enzimática , Masculino , Isoformas de Proteínas , Xenopus/anatomía & histología , Familia-src Quinasas/genéticaRESUMEN
MicroRNAs (miRNAs) are short non-coding RNAs that silence mRNAs. They are generated following transcription and cleavage by the DROSHA/DGCR8 and DICER/TRBP/PACT complexes. Although it is known that components of the miRNA biogenesis machinery can be phosphorylated, it remains poorly understood how these events become engaged during physiological cellular activation. We demonstrate that S6 kinases can phosphorylate the extended C-terminal domain of TRBP and interact with TRBP in situ in primary cells. TRBP serines 283/286 are essential for S6K-mediated TRBP phosphorylation, optimal expression of TRBP, and the S6K-TRBP interaction in human primary cells. We demonstrate the functional relevance of this interaction in primary human dermal lymphatic endothelial cells (HDLECs). Angiopoietin-1 (ANG1) can augment miRNA biogenesis in HDLECs through enhancing TRBP phosphorylation and expression in an S6K2-dependent manner. We propose that the S6K2/TRBP node controls miRNA biogenesis in HDLECs and provides a molecular link between the mTOR pathway and the miRNA biogenesis machinery.
Asunto(s)
Células Endoteliales/metabolismo , Regulación de la Expresión Génica , MicroARNs/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Angiopoyetina 1/farmacología , Línea Celular , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
Mint/X11 is one of the four neuronal trafficking adaptors that interact with amyloid precursor protein (APP) and are linked with its cleavage to generate ß-amyloid peptide, a key player in the pathology of Alzheimer's disease. How APP switches between adaptors at different stages of the secretory pathway is poorly understood. Here, we show that tyrosine phosphorylation of Mint1 regulates the destination of APP. A canonical SH2-binding motif ((202) YEEI) was identified in the N-terminus of Mint1 that is phosphorylated on tyrosine by C-Src and recruits the active kinase for sequential phosphorylation of further tyrosines (Y191 and Y187). A single Y202F mutation in the Mint1 N-terminus inhibits C-Src binding and tyrosine phosphorylation. Previous studies observed that co-expression of wild-type Mint1 and APP causes accumulation of APP in the trans-Golgi. Unphosphorylatable Mint1 (Y202F) or pharmacological inhibition of Src reduced the accumulation of APP in the trans-Golgi of heterologous cells. A similar result was observed in cultured rat hippocampal neurons where Mint1(Y202F) permitted the trafficking of APP to more distal neurites than the wild-type protein. These data underline the importance of the tyrosine phosphorylation of Mint1 as a critical switch for determining the destination of APP. The regulation of amyloid precursor protein (APP) trafficking is poorly understood. We have discovered that the APP adapter, Mint1, is phosphorylated by C-Src kinase. Mint1 causes APP accumulation in the trans-Golgi network, whereas inhibition of Src or mutation of Mint1-Y202 permits APP recycling. The phosphorylation status of Mint1 could impact on the pathological trafficking of APP in Alzheimer's disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Tirosina/metabolismo , Familia-src Quinasas/metabolismo , Red trans-Golgi/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Femenino , Células HeLa , Humanos , Masculino , Ratones , Proteínas del Tejido Nervioso/genética , Fosforilación/fisiología , Transporte de Proteínas/fisiología , Ratas , Ratas Wistar , Tirosina/genética , Familia-src Quinasas/genética , Red trans-Golgi/genéticaRESUMEN
Parkinson's disease (PD) is associated with loss of dopaminergic signalling, and affects not just movement, but also vision. As both mammalian and fly visual systems contain dopaminergic neurons, we investigated the effect of LRRK2 mutations (the most common cause of inherited PD) on Drosophila electroretinograms (ERGs). We reveal progressive loss of photoreceptor function in flies expressing LRRK2-G2019S in dopaminergic neurons. The photoreceptors showed elevated autophagy, apoptosis and mitochondrial disorganization. Head sections confirmed extensive neurodegeneration throughout the visual system, including regions not directly innervated by dopaminergic neurons. Other PD-related mutations did not affect photoreceptor function, and no loss of vision was seen with kinase-dead transgenics. Manipulations of the level of Drosophila dLRRK suggest G2019S is acting as a gain-of-function, rather than dominant negative mutation. Increasing activity of the visual system, or of just the dopaminergic neurons, accelerated the G2019S-induced deterioration of vision. The fly visual system provides an excellent, tractable model of a non-autonomous deficit reminiscent of that seen in PD, and suggests that increased energy demand may contribute to the mechanism by which LRRK2-G2019S causes neurodegeneration.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Proteínas de Drosophila/genética , Expresión Génica , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Proteínas Serina-Treonina Quinasas/genética , Degeneración Retiniana/genética , Animales , Apoptosis/genética , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/patología , Electrorretinografía , Femenino , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Mutación , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patologíaRESUMEN
Parkinson's disease (PD) is characterized by movement disorders, including bradykinesia. Analysis of inherited, juvenile PD, identified several genes linked via a common pathway to mitochondrial dysfunction. In this study, we demonstrate that the larva of the Drosophila parkin mutant faithfully models the locomotory and metabolic defects of PD and is an excellent system for investigating their inter-relationship. parkin larvae displayed a marked bradykinesia that was caused by a reduction in both the frequency of peristalsis and speed of muscle contractions. Rescue experiments confirmed that this phenotype was due to a defect in the nervous system and not in the muscle. Furthermore, recordings of motoneuron activity in parkin larvae revealed reduced bursting and a striking reduction in evoked and miniature excitatory junction potentials, suggesting a neuronal deficit. This was supported by our observations in parkin larvae that the resting potential was depolarized, oxygen consumption and ATP concentration were drastically reduced while lactate was increased. These findings suggest that neuronal mitochondrial respiration is severely compromised and there is a compensatory switch to glycolysis for energy production. parkin mutants also possessed overgrown neuromuscular synapses, indicative of oxidative stress, which could be rescued by overexpression of parkin or scavengers of reactive oxygen species (ROS). Surprisingly, scavengers of ROS did not rescue the resting membrane potential and locomotory phenotypes. We therefore propose that mitochondrial dysfunction in parkin mutants induces Parkinsonian bradykinesia via a neuronal energy deficit and resulting synaptic failure, rather than as a consequence of downstream oxidative stress.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Drosophila/fisiología , Metabolismo Energético , Neuronas/fisiología , Estrés Oxidativo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/fisiología , Adenosina Trifosfato/metabolismo , Animales , Catalasa/metabolismo , Drosophila/genética , Drosophila/metabolismo , Glucólisis , Larva/fisiología , Locomoción , Potenciales de la Membrana , Mitocondrias/metabolismo , Contracción Muscular , Neuronas/metabolismo , Consumo de Oxígeno , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Potenciales SinápticosRESUMEN
Intracellular Ca2+ signaling and Na+ homeostasis are inextricably linked via ion channels and co-transporters, with alterations in the concentration of one ion having profound effects on the other. Evidence indicates that intracellular Na+ concentration ([Na+ ]i ) is elevated in breast tumors, and that aberrant Ca2+ signaling regulates numerous key cancer hallmark processes. The present study therefore aimed to determine the effects of Na+ depletion on intracellular Ca2+ handling in metastatic breast cancer cell lines. The relationship between Na+ and Ca2+ was probed using fura-2 and SBFI fluorescence imaging and replacement of extracellular Na+ with equimolar N-methyl-D-glucamine (0Na+ /NMDG) or choline chloride (0Na+ /ChoCl). In triple-negative MDA-MB-231 and MDA-MB-468 cells and Her2+ SKBR3 cells, but not ER+ MCF-7 cells, 0Na+ /NMDG and 0Na+ /ChoCl resulted in a slow, sustained depletion in [Na+ ]i that was accompanied by a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+ ]i ). Application of La3+ in nominal Ca2+ -free conditions had no effect on this response, ruling out reverse-mode NCX activity and Ca2+ entry channels. Moreover, the Na+ -linked [Ca2+ ]i increase was independent of membrane potential hyperpolarization (NS-1619), but was inhibited by pharmacological blockade of IP3 receptors (2-APB), phospholipase C (PLC, U73122) or following depletion of endoplasmic reticulum Ca2+ stores (cyclopiazonic acid). Thus, Na+ is linked to PLC/IP3 -mediated activation of endoplasmic reticulum Ca2+ release in metastatic breast cancer cells and this may have an important role in breast tumors where [Na+ ]i is perturbed.
Asunto(s)
Neoplasias de la Mama , Señalización del Calcio , Humanos , Femenino , Señalización del Calcio/fisiología , Sodio/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Canales Iónicos/metabolismo , Calcio/metabolismoRESUMEN
Presynaptic long term potentiation of synaptic transmission activates silent synapses and potentiates existing active synapses. We sought to visualise these two processes by studying the cAMP-dependent protein kinase (PKA) potentiation of presynaptic vesicle cycling in cultured cerebellar granule neurons.Using FM dyes to label the pool of recycling synaptic vesicles,we found that trains of electrical stimulation which do not potentiate already active synapses are sufficient to rapidly activate a discrete population comprising silent and very low activity synapses. Silent synapse activation required PKA activity and conversely, active synapses could be silenced by PKA inhibition. Surprisingly, the recycling pool of synaptic vesicles in recently activated synapses was larger than in already active synapses and equivalent to synapses treated with forskolin. Imaging of synaptic vesicle cycling and cytosolic Ca(2+) in individual nerve terminals confirmed that silent synapses have evoked Ca(2+) transients comparable to those of active synapses. Furthermore, across populations of active synapses, changes in Ca(2+) influx did not correlate with changes in the size of the pool of recycling synaptic vesicles. Finally, we found that stimulation of synapsin phosphorylation, but not RIM1α, by PKA was frequency dependent and long lasting. These data are consistent with the idea that PKA regulates synaptic vesicle recycling downstream of Ca(2+) influx and that this pathway is highly active in recently activated synapses.
Asunto(s)
Cerebelo/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neuronas/enzimología , Transmisión Sináptica , Análisis de Varianza , Animales , Señalización del Calcio , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Estimulación Eléctrica , Activación Enzimática , Activadores de Enzimas/farmacología , Proteínas de Unión al GTP/metabolismo , Cinética , Aprendizaje , Memoria , Microscopía Fluorescente , Vías Nerviosas/enzimología , Plasticidad Neuronal , Neuronas/efectos de los fármacos , Fosforilación , Terminales Presinápticos/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Sinapsinas/metabolismo , Potenciales Sinápticos , Transmisión Sináptica/efectos de los fármacos , Vesículas Sinápticas/enzimologíaRESUMEN
Reactive oxygen species (ROS) are generated during physiological bouts of synaptic activity and as a consequence of pathological conditions in the central nervous system. How neurons respond to and distinguish between ROS in these different contexts is currently unknown. In Drosophila mutants with enhanced JNK activity, lower levels of ROS are observed and these animals are resistant to both changes in ROS and changes in synapse morphology induced by oxidative stress. In wild type flies, disrupting JNK-AP-1 signalling perturbs redox homeostasis suggesting JNK activity positively regulates neuronal antioxidant defense. We validated this hypothesis in mammalian neurons, finding that JNK activity regulates the expression of the antioxidant gene Srxn-1, in a c-Jun dependent manner. We describe a conserved 'adaptive' role for neuronal JNK in the maintenance of redox homeostasis that is relevant to several neurodegenerative diseases.
Asunto(s)
Antioxidantes , Proteínas Quinasas JNK Activadas por Mitógenos , Animales , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas , Neuronas/metabolismo , Estrés Oxidativo , Especies Reactivas de OxígenoRESUMEN
Bulk endocytosis in central nerve terminals is activated by strong stimulation; however, the speed at which it is initiated and for how long it persists is still a matter of debate. To resolve this issue, we performed a characterization of bulk endocytic retrieval using action potential trains of increasing intensity. Bulk endocytosis was monitored by the loading of the fluorescent dyes FM2-10 and FM1-43, uptake of tetramethylrhodamine-dextran (40 kDa), or morphological analysis of uptake of the fluid-phase marker horseradish peroxidase. When neuronal cultures were subjected to mild stimulation (200 action potentials at 10 Hz), bulk endocytosis was not observed using any of our assay systems. However, when more intense trains of action potentials (400 or 800 action potentials at 40 and 80 Hz, respectively) were applied to neurons, bulk endocytosis was activated immediately, with the majority of bulk endocytosis complete by the end of stimulation. This contrasts with single synaptic vesicle endocytosis, the majority of which occurred after stimulation was terminated. Thus, bulk endocytosis is a fast event that is triggered during strong stimulation and provides the nerve terminal with an appropriate mechanism to meet the demands of synaptic vesicle retrieval during periods of intense synaptic vesicle exocytosis.
Asunto(s)
Endocitosis/fisiología , Terminales Presinápticos/fisiología , Membranas Sinápticas/fisiología , Transmisión Sináptica/fisiología , Vesículas Sinápticas/fisiología , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Estimulación Eléctrica , Colorantes Fluorescentes , Peroxidasa de Rábano Silvestre , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Membranas Sinápticas/ultraestructura , Vesículas Sinápticas/ultraestructura , Factores de TiempoRESUMEN
The stimulated dephosphorylation of the dephosphin group of endocytic proteins by calcineurin and their subsequent rephosphorylation by cyclin-dependent kinase 5 (cdk5) is required for synaptic vesicle (SV) retrieval in central nerve terminals. However, the specific endocytic pathway(s) controlled by these enzymes is unknown. To address this issue, we combined functional and morphological assays of endocytosis in primary neuronal cultures with pharmacological and molecular ablation of calcineurin and cdk5 activity. During strong stimulation, inhibition of calcineurin or cdk5 blocked uptake of the activity-dependent membrane marker FM1-43, but not the more hydrophilic FM2-10. However, FM2-10 uptake-measured poststimulation was sensitive to cdk5 and calcineurin inhibition, indicating that a slow form of endocytosis persists after termination of stimulation. In parallel EM studies, inhibition of cdk5 during strong stimulation greatly reduced horseradish peroxidase labeling of plasma membrane-derived nerve terminal endosomes, but not SVs. Furthermore, during mild stimulation, FM1-43 uptake was unaffected by cdk5 inhibition and the SV membrane was exclusively retrieved via a single SV route, suggesting that recruitment of the endosomal route of membrane retrieval is activity dependent. Thus, we propose that the calcineurin/cdk5-dependent phosphorylation cycle of the dephosphins specifically controls a slow endocytic pathway that proceeds via endosomal intermediates and is activated by strong physiological stimulation in central nerve terminals.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/fisiología , Endocitosis/fisiología , Sinapsis/enzimología , Vesículas Sinápticas/enzimología , Animales , Células Cultivadas , Endosomas/enzimología , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
A major goal of learning and memory research is to correlate the function of molecules with the behaviour of organisms. The beautiful laminar structure of the cerebellar cortex lends itself to the study of synaptic plasticity, because its clearly defined patterns of neurons and their synapses form circuits that have been implicated in simple motor behaviour paradigms. The best understood in terms of molecular mechanism is the parallel fibre-Purkinje cell synapse, where presynaptic long-term potentiation and postsynaptic long-term depression and potentiation finely tune cerebellar output. Our understanding of these forms of plasticity has mostly come from the electrophysiological and behavioural analysis of knockout mutant mice, but more recently the knock-in of synaptic molecules with mutated phosphorylation sites and binding domains has provided more detailed insights into the signalling events. The present review details the major forms of plasticity in the cerebellar cortex, with particular attention to the membrane trafficking and intracellular signalling responsible. This overview of the current literature suggests it will not be long before the involvement of the cerebellum in certain motor behaviours is fully explained in molecular terms.
Asunto(s)
Corteza Cerebelosa/fisiología , Plasticidad Neuronal/fisiología , Transducción de Señal/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Conducta/fisiología , Corteza Cerebelosa/citología , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Memoria/fisiología , Fibras Nerviosas/metabolismoRESUMEN
Silicon photonic biosensors are being widely researched as they combine high performance with the potential for low-cost mass-manufacturing. Sensing is typically performed in an aqueous environment and it is assumed that the sensor is chemically stable, as silicon is known to etch in strong alkaline solutions but not in liquids with a pH close to 7. Here, we show that silicon can be affected surprisingly strongly by typical cell culture media, and we observe etch rates of up to 2 nm/hour. We then demonstrate that a very thin (< 10 nm) layer of thermal oxide is sufficient to suppress the etching process and provide the long-term stability required for monitoring cells and related biological processes over extended periods of time. We also show that employing an additional pH buffering compound in the culture medium can significantly reduce the etch rate.
RESUMEN
In the mammalian brain the ubiquitous tyrosine kinase, C-Src, undergoes splicing to insert short sequences in the SH3 domain to yield N1- and N2-Src. We and others have previously shown that the N-Srcs have altered substrate specificity and kinase activity compared to C-Src. However, the exact functions of the N-Srcs are unknown and it is likely that N-Src signalling events have been misattributed to C-Src because they cannot be distinguished by conventional Src inhibitors that target the kinase domain. By screening a peptide phage display library, we discovered a novel ligand (PDN1) that targets the unique SH3 domain of N1-Src and inhibits N1-Src in cells. In cultured neurons, PDN1 fused to a fluorescent protein inhibited neurite outgrowth, an effect that was mimicked by shRNA targeting the N1-Src microexon. PDN1 also inhibited L1-CAM-dependent neurite elongation in cerebellar granule neurons, a pathway previously shown to be disrupted in Src-/- mice. PDN1 therefore represents a novel tool for distinguishing the functions of N1-Src and C-Src in neurons and is a starting point for the development of a small molecule inhibitor of N1-Src.
Asunto(s)
Molécula L1 de Adhesión de Célula Nerviosa/fisiología , Neuritas/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismo , Empalme Alternativo , Animales , Proteína Tirosina Quinasa CSK , Ligandos , Ratones , Neuritas/fisiología , Dominios Homologos src , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genéticaRESUMEN
Advanced biomedical diagnostic technologies fulfill an important role in improving health and well-being in society. A large number of excellent technologies have already been introduced and have given rise to the "lab-on-a-chip" paradigm. Most of these technologies, however, require additional instrumentation for interfacing and readout, so they are often confined to the laboratory and are not suitable for use in the field or in wider clinical practice. Other technologies require a light coupling element, such as a grating coupler or a fiber coupler, which complicates packaging. Here, we introduce a novel biosensor based on a chirped guided-mode resonant grating. The chirped grating combines the sensing function with the readout function by translating spectral information into spatial information that is easily read out with a simple CMOS camera. We demonstrate a refractive index sensitivity of 137 nm/RIU and an extrapolated limit of detection of 267 pM for the specific binding of an immunoglobulin G antibody. The chirped guided-mode resonance approach introduces a new degree of freedom for sensing biomedical information that combines high sensitivity with autonomous operation. We estimate that the cost of components is U.S. $10 or less when mass manufactured, so the technology has the potential to truly transform point-of-care applications.
RESUMEN
The human brain is estimated to contain trillions of synaptic nerve terminals. These are the connections between neurons that are responsible for transmitting information and are modified as a result of learning. A valuable tool for studying synapses is the isolated nerve terminal, or synaptosome, which is obtained by homogenizing the brain in such a way that individual synapses pinch off to form metabolically active compartments that can recapitulate neurotransmitter release. This protocol describes the stepwise fractionation of rat brain tissue to yield synaptosomes and synaptic vesicles, which can be used in many different experimental approaches to study the structure and protein composition of the synapse and even dissect the molecular mechanisms of neurotransmission.
Asunto(s)
Química Encefálica , Encéfalo/citología , Fraccionamiento Celular/métodos , Vesículas Sinápticas/química , Vesículas Sinápticas/fisiología , Sinaptosomas/química , Sinaptosomas/fisiología , Animales , Ratas , Vesículas Sinápticas/metabolismo , Sinaptosomas/metabolismoRESUMEN
Alongside rodent brain slices and primary neuronal cultures, synaptosomes (isolated nerve terminals) have been an important model system for studying the molecular mechanisms of synaptic function in the brain. Synaptosomes were first prepared in the late 1950s by Whittaker and colleagues and were instrumental in studying synaptic structure and defining the functional components of the synapse, including the identity of the major neurotransmitters and their uptake mechanisms. Synaptosomes can also be stimulated to release neurotransmitters and were used to discover a number of regulatory signaling pathways that fine-tune synaptic transmission. In the past decade, landmark proteomic studies of synaptosomes and synaptic vesicle preparations have further dissected the protein composition of the synapse. This introduction briefly describes the history of the synaptosome preparation and highlights how it continues to be relevant as our focus in the neuroscience community centers on synaptic dysfunction in aging and neurological disease.
Asunto(s)
Encéfalo/fisiología , Fraccionamiento Celular/métodos , Emparejamiento Cromosómico/fisiología , Sinaptosomas/metabolismo , Animales , Modelos Biológicos , RoedoresRESUMEN
N2-Src is a poorly understood neuronal splice variant of the ubiquitous C-Src tyrosine kinase, containing a 17 amino acid insert in its Src homology 3 (SH3) domain. To characterise the properties of N2-Src we directly compared its SH3 domain specificity and kinase activity with C- and N1-Src in vitro. N2- and N1-Src had a similar low affinity for the phosphorylation of substrates containing canonical C-Src SH3 ligands and synaptophysin, an established neuronal substrate for C-Src. N2-Src also had a higher basal kinase activity than N1- and C-Src in vitro and in cells, which could be explained by weakened intramolecular interactions. Therefore, N2-Src is a highly active kinase that is likely to phosphorylate alternative substrates to C-Src in the brain.
Asunto(s)
Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Empalme del ARN , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular Tumoral , Ligandos , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas pp60(c-src)/química , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Ratas , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Although the ultrastructure of the schistosome esophageal gland was described >35 years ago, its role in the processing of ingested blood has never been established. The current study was prompted by our identification of MEG-4.1 expression in the gland and the observation of erythrocyte uncoating in the posterior esophagus. METHODOLOGY/PRINCIPAL FINDINGS: The salient feature of the posterior esophagus, characterized by confocal and electron microscopy, is the enormous increase in membrane surface area provided by the plate-like extensions and basal invaginations of the lining syncytium, with unique crystalloid vesicles releasing their contents between the plates. The feeding process was shown by video microscopy to be divided into two phases, blood first accumulating in the anterior lumen before passing as a bolus to the posterior. There it streamed around a plug of material revealed by confocal microscopy as tethered leucocytes. These were present in far larger numbers than predicted from the volume of the lumen, and in varying states of damage and destruction. Intact erythrocytes were detected in the anterior esophagus but not observed thereafter, implying that their lysis occurred rapidly as they enter the posterior. Two further genes, MEGs 4.2 and 14, were shown to be expressed exclusively in the esophageal gland. Bioinformatics predicted that MEGs 4.1 and 4.2 possessed a common hydrophobic region with a shared motif, while antibodies to SjMEG-4.1 showed it was bound to leucocytes in the esophageal lumen. It was also predicted that MEGs 4.1 and 14 were heavily O-glycosylated and this was confirmed for the former by 2D-electrophoresis and Western blotting. CONCLUSIONS/SIGNIFICANCE: The esophageal gland and its products play a central role in the processing of ingested blood. The binding of host antibodies in the esophageal lumen shows that some constituents are antibody targets and could provide a new source of vaccine candidates.