RESUMEN
Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes (mei2-/linup- or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice. We next genetically engineered a PfLARC2 parasite strain that was devoid of extraneous DNA and produced cryopreserved PfSPZ-LARC2. PfSPZ-LARC2 liver stages replicated robustly in liver-humanized mice but displayed severe defects in late liver stage differentiation and did not form liver stage merozoites. This resulted in complete abrogation of parasite transition to viable blood stage infection. Therefore, PfSPZ-LARC2 is the next-generation vaccine strain expected to unite the safety profile of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac.
Asunto(s)
Vacunas contra la Malaria , Malaria Falciparum , Parásitos , Animales , Ratones , Plasmodium falciparum/genética , Malaria Falciparum/prevención & control , Eliminación de Gen , Vacunas contra la Malaria/genética , Vacunas Atenuadas/genética , Esporozoítos/genéticaRESUMEN
Plasmodium sporozoites invade hepatocytes and transform into liver stages within a parasitophorous vacuole (PV). The parasites then grow and replicate their genome to form exoerythrocytic merozoites that infect red blood cells. We report that the human malaria parasite Plasmodium falciparum (Pf) expresses a C-type ATP-binding cassette transporter, Pf ABCC2, which marks the transition from invasive sporozoite to intrahepatocytic early liver stage. Using a humanized mouse infection model, we show that Pf ABCC2 localizes to the parasite plasma membrane in early and mid-liver stage parasites but is not detectable in late liver stages. Pf abcc2 - sporozoites invade hepatocytes, form a PV, and transform into liver stage trophozoites but cannot transition to exoerythrocytic schizogony and fail to transition to blood stage infection. Thus, Pf ABCC2 is an expression marker for early phases of parasite liver infection and plays an essential role in the successful initiation of liver stage replication.
RESUMEN
Meloxicam is a widely used nonsteroidal anti-inflammatory drug (NSAID) that is used to treat pain and some inflammatory disorders. One rare side effect is drug-induced thrombocytopenia (DITP), which can result in hemorrhage and death if not treated urgently. This diagnosis can be missed if mistaken for other conditions such as ITP, which is problematic since the mainstay of treatment is medication cessation. We present a case of a 42-year-old Hispanic female who was recently started on meloxicam and presented with petechiae, ecchymosis, and a platelet count of 2 (normal 150-350 K/mm3).
RESUMEN
Whole-sporozoite vaccines engender sterilizing immunity against malaria in animal models and importantly, in humans. Gene editing allows for the removal of specific parasite genes, enabling generation of genetically attenuated parasite (GAP) strains for vaccination. Using rodent malaria parasites, we have previously shown that late liver stage-arresting replication-competent (LARC) GAPs confer superior protection when compared with early liver stage-arresting replication-deficient GAPs and radiation-attenuated sporozoites. However, generating a LARC GAP in the human malaria parasite Plasmodium falciparum (P. falciparum) has been challenging. Here, we report the generation and characterization of a likely unprecedented P. falciparum LARC GAP generated by targeted gene deletion of the Mei2 gene: P. falciparum mei2-. Robust exoerythrocytic schizogony with extensive cell growth and DNA replication was observed for P. falciparum mei2- liver stages in human liver-chimeric mice. However, P. falciparum mei2- liver stages failed to complete development and did not form infectious exoerythrocytic merozoites, thereby preventing their transition to asexual blood stage infection. Therefore, P. falciparum mei2- is a replication-competent, attenuated human malaria parasite strain with potentially increased potency, useful for vaccination to protect against P. falciparum malaria infection.
Asunto(s)
Vacunas contra la Malaria/farmacología , Malaria Falciparum/prevención & control , Malaria/prevención & control , Parásitos/efectos de los fármacos , Esporozoítos/patogenicidad , Animales , Humanos , Hígado/inmunología , Malaria/parasitología , Malaria Falciparum/tratamiento farmacológico , Parásitos/inmunología , Parásitos/patogenicidad , Plasmodium falciparum/genética , Plasmodium yoelii/inmunología , Vacunación/métodos , Vacunas Atenuadas/inmunologíaRESUMEN
Central nervous system (CNS) involvement occurs in about 1% of all tuberculosis (TB) cases, classically presenting as a meningitis. Intracerebral tuberculomas are a much rarer manifestation. We describe the case of a young black male who presented with new-onset seizure. Cerebral computerized tomography from an outside hospital reportedly showed findings concerning for septic emboli. Brain magnetic resonance imaging at our institution confirmed the presence of multiple, peripherally enhancing lesions in the right frontal and temporal lobes, cerebellum, and pons. Thoracentesis was performed for a concomitant pleural effusion, which contained elevated levels of adenosine deaminase and ultimately grew Mycobacterium tuberculosis. After ruling out other causes, we reached a diagnosis of CNS TB manifesting as cerebral tuberculomas. The patient was initiated on a course of rifampin, isoniazid, pyrazinamide, and ethambutol for two months, followed by rifampin and isoniazid to complete at least twelve months of antimicrobial therapy. We present this case to highlight this unusual manifestation of CNS TB and review the challenges in diagnosis.
RESUMEN
Within the liver, Plasmodium sporozoites traverse cells searching for a "suitable" hepatocyte, invading these cells through a process that results in the formation of a parasitophorous vacuole (PV), within which the parasite undergoes intracellular replication as a liver stage. It was previously established that two members of the Plasmodium s48/45 protein family, P36 and P52, are essential for productive invasion of host hepatocytes by sporozoites as their simultaneous deletion results in growth-arrested parasites that lack a PV. Recent studies point toward a pathway of entry possibly involving the interaction of P36 with hepatocyte receptors EphA2, CD81, and SR-B1. However, the relationship between P36 and P52 during sporozoite invasion remains unknown. Here we show that parasites with a single P52 or P36 gene deletion each lack a PV after hepatocyte invasion, thereby pheno-copying the lack of a PV observed for the P52/P36 dual gene deletion parasite line. This indicates that both proteins are equally important in the establishment of a PV and act in the same pathway. We created a Plasmodium yoelii P36mCherry tagged parasite line that allowed us to visualize the subcellular localization of P36 and found that it partially co-localizes with P52 in the sporozoite secretory microneme organelles. Furthermore, through co-immunoprecipitation studies in vivo, we determined that P36 and P52 form a protein complex in sporozoites, indicating a concerted function for both proteins within the PV formation pathway. However, upon sporozoite stimulation, only P36 was released as a secreted protein while P52 was not. Our results support a model in which the putatively glycosylphosphatidylinositol (GPI)-anchored P52 may serve as a scaffold to facilitate the interaction of secreted P36 with the host cell during sporozoite invasion of hepatocytes.
Asunto(s)
Hepatocitos/parasitología , Malaria/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Esporozoítos/metabolismo , Animales , Antígenos CD36/metabolismo , Culicidae , Citoplasma/metabolismo , Femenino , Eliminación de Gen , Técnicas de Inactivación de Genes , Glicosilfosfatidilinositoles , Hepatocitos/patología , Ratones , Ratones Endogámicos BALB C , Plasmodium yoelii/metabolismo , Receptor EphA2/metabolismo , Glándulas Salivales/parasitología , Glándulas Salivales/patologíaRESUMEN
Chronic pain is increasingly recognized as a public health problem. We assessed the effectiveness of a multi-modal, interprofessional educational approach aimed at empowering healthcare professionals to make deliberative changes, especially in opiate prescribing practices. Education activities included enduring webcasts, regional interprofessional roundtable events, and state-level conference presentations within targeted Kentucky and West Virginia regions of the United States. Over 1,000 participants accessed the various activities. For the live events, the largest groups reached included nurses (38.1%), nurse practitioners (31.2%), and physicians (22.1%). In addition to our reach, higher levels of educational effectiveness were measured, specifically, learner's intentions to change practice patterns, confidence in meeting patient's needs, and knowledge of pain management guidelines. The majority of the conference (58%) and roundtable (69%) participants stated they intend to make a practice change in one or more areas of chronic pain patient management in post-event evaluation. Differences in pre- and post-activity responses on the measures of confidence and knowledge, with additional comparison to a control population who were not in attendance, were analyzed using non-parametric tests of significance. While neither activity produced significant changes in confidence from pre-activity, participants were more confident post-activity than their control group peers. There were significant changes in knowledge for both live event and webcast participants. Impactful chronic pain continuing the education that emphasizes collaborative care is greatly needed; these results show that the approaches taken here can impact learner's knowledge and confidence, and hold potential for creating change in how opioid prescribing is managed.
Asunto(s)
Analgésicos Opioides/uso terapéutico , Actitud del Personal de Salud , Dolor Crónico/tratamiento farmacológico , Educación Médica Continua , Trastornos Relacionados con Opioides/prevención & control , Pautas de la Práctica en Medicina , Analgésicos Opioides/efectos adversos , Competencia Clínica/normas , Humanos , Encuestas y Cuestionarios , Estados UnidosRESUMEN
Malaria eradication necessitates new tools to fight the evolving and complex Plasmodium pathogens. These tools include prophylactic drugs that eliminate Plasmodium liver stages and consequently prevent clinical disease, decrease transmission, and reduce the propensity for resistance development. Currently, the identification of these drugs relies on in vitro P. falciparum liver stage assays or in vivo causal prophylaxis assays using rodent malaria parasites; there is no method to directly test in vivo liver stage activity of candidate antimalarials against the human malaria-causing parasite P. falciparum. Here, we use a liver-chimeric humanized mouse (FRG huHep) to demonstrate in vivo P. falciparum liver stage development and describe the efficacy of clinically used and candidate antimalarials with prophylactic activity. We show that daily administration of atovaquone-proguanil (ATQ-PG; ATQ, 30 mg/kg, and PG, 10 mg/kg) protects 5 of 5 mice from liver stage infection, consistent with the use in humans as a causal prophylactic drug. Single-dose primaquine (60 mg/kg) has similar activity to that observed in humans, demonstrating the activity of this drug (and its active metabolites) in FRG huHep mice. We also show that DSM265, a selective Plasmodial dihydroorotate dehydrogenase inhibitor with causal prophylactic activity in humans, reduces liver stage burden in FRG huHep mice. Finally, we measured liver stage-to-blood stage transition of the parasite, the ultimate readout of prophylactic activity and measurement of infective capacity of parasites in the liver, to show that ATQ-PG reduces blood stage patency to below the limit of quantitation by quantitative PCR (qPCR). The FRG huHep model, thus, provides a platform for preclinical evaluation of drug candidates for liver stage causal prophylactic activity, pharmacokinetic/pharmacodynamics studies, and biological studies to investigate the mechanism of action of liver stage active antimalarials.
Asunto(s)
Antimaláricos/farmacología , Hígado/efectos de los fármacos , Hígado/parasitología , Plasmodium falciparum/efectos de los fármacos , Animales , Atovacuona/farmacología , Modelos Animales de Enfermedad , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos , Malaria Falciparum/tratamiento farmacológico , Ratones , Proguanil/farmacología , Pirimidinas/farmacología , Triazoles/farmacologíaRESUMEN
Virtually all functions of the nervous system rely upon synapses, the sites of communication between neurons and between neurons and other cells. Synapses are complex structures, each one comprising hundreds of different types of molecules working in concert. They are organized by adhesive and scaffolding molecules that align presynaptic vesicular release sites, namely, active zones, with postsynaptic neurotransmitter receptors, thereby allowing rapid and reliable intercellular communication. Most synapses are relatively small, and acting alone exerts little effect on their postsynaptic partners. Some, however, are much larger and stronger, reliably driving the postsynaptic cell to its action potential threshold, acting essentially as electrical relays of excitation. These large synapses are among the best understood, and two of these are the subject of this review, namely, the vertebrate neuromuscular junction and the calyx of Held synapse in the mammalian auditory pathway of the brain stem. Both synapses undergo through a complex and well-coordinated maturation process, during which time the molecular elements and the biophysical properties of the secretory machinery are continuously adjusted to the synapse size and to the functional requirements. We here review the morphological and functional changes occurring during postnatal maturation, noting particular similarities and differences between these two large synapses.
Asunto(s)
Sinapsis/fisiología , Sinapsis/ultraestructura , Envejecimiento/fisiología , Animales , Calcio/metabolismo , Endocitosis , Exocitosis , HumanosRESUMEN
The neuromuscular junction (NMJ) is the original model synapse, and while others have emerged, especially as models of plasticity involving coincidence detectors, the NMJ continues to provide useful new information. It remains, for example, one of the best understood synapses in terms of the relationship between structure and function. In particular, the advent of new tools for fluorescence imaging has allowed the processes of vesicle exocytosis, endocytosis, and receptor activation to be spatially mapped in considerable detail. Here, we will focus on the spatial properties of transmitter release at the presynaptic motor terminal at the mouse NMJ. The preparation offers several experimental advantages, such as easy accessibility, a nearly planar, unobstructed view of several hundred square microns of synaptic membrane, as well as a highly stereotyped, consistent structure of a fully differentiated adult mammalian synapse.
Asunto(s)
Exocitosis/fisiología , Terminales Presinápticos/metabolismo , Animales , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Microscopía Fluorescente , Unión Neuromuscular/metabolismo , Compuestos de Piridinio/química , Compuestos de Amonio Cuaternario/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Vesículas Sinápticas/metabolismoRESUMEN
The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. FM dyes reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. This protocol describes the imaging of synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Nerve terminals are stimulated (electrically or by depolarization with high K(+)) in the presence of dye, their vesicles are then allowed to recycle, and finally dye is washed from the chamber. In neuromuscular junction (NMJ) preparations, movements of the muscle must be inhibited if imaging during stimulation is desired (e.g., by application of curare, a potent acetylcholine receptor inhibitor). The main characteristics of FM dyes are also reviewed here, as are recent FM dye monitoring techniques that have been used to investigate the kinetics of synaptic vesicle fusion.
Asunto(s)
Técnicas Citológicas/métodos , Colorantes Fluorescentes/metabolismo , Manejo de Especímenes/métodos , Vesículas Sinápticas/metabolismo , Animales , Colorantes Fluorescentes/química , Colorantes Fluorescentes/clasificación , Humanos , Compuestos de Piridinio/química , Compuestos de Piridinio/clasificación , Compuestos de Piridinio/metabolismoRESUMEN
The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. The FM dyes have a unique set of properties that allows them to selectively label recycling vesicles: They reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. FM dyes can also be used as endocytic markers in electron microscopy (EM) through a procedure termed photoconversion (or photooxidation), as described here. Fluorescent dye molecules generate free radicals (reactive oxygen species) when subjected to strong illumination. These short-lived radicals readily oxidize any molecules found in the immediate vicinity of the fluorophore. When photoconversion of FM dyes is performed while the preparation is bathing in diaminobenzidine (DAB), a dark brown precipitate forms after the DAB is oxidized. Thus, illumination turns FM-labeled organelles into dark electron-dense ones. The technique results in a substantial increase in the resolution of FM dye labeling studies (with the obvious caveat that it is restricted to fixed preparations).
Asunto(s)
Colorantes Fluorescentes/metabolismo , Microscopía Electrónica/métodos , Coloración y Etiquetado/métodos , Vesículas Sinápticas/metabolismo , Animales , Colorantes Fluorescentes/química , Colorantes Fluorescentes/clasificación , Humanos , Oxidación-Reducción , Compuestos de Piridinio/química , Compuestos de Piridinio/clasificación , Compuestos de Piridinio/metabolismo , Vesículas Sinápticas/ultraestructuraRESUMEN
We observed endocytosis in real time in stimulated frog motor nerve terminals by imaging the growth of large membrane infoldings labeled with a low concentration of FM dye. The spatial and temporal information made available by these experiments allowed us to image several new aspects of this synaptic vesicle recycling pathway. Membrane infoldings appeared near synaptic vesicle clusters and grew rapidly during long-duration, high-frequency stimulation. In some cases, we observed large, elongated infoldings growing laterally into the terminal. We used these observations to calculate infolding growth rates. A decrease in stimulation frequency caused a decrease in growth rates, but the overall length of these structures was unaffected by frequency changes. Attempts to wash the dye from these infoldings after stimulation were unsuccessful, demonstrating that the fluorescent structures had been endocytosed. We also used this technique to trigger and image infoldings during repeated, short trains. We found that membrane uptake occurred repeatedly at individual endocytosis sites, but only during a portion of the total number of trains delivered to the terminal. Finally, we showed that phosphatidylinositol 3-kinase, but not actin, was involved in this endocytosis pathway. The ability to monitor many individual bulk endocytosis sites in real time should allow for new types of endocytosis measurements and could reveal novel and unexpected mechanisms for coordinating membrane recovery during synaptic activity.
Asunto(s)
Endocitosis/fisiología , Colorantes Fluorescentes , Neuronas Motoras/fisiología , Terminaciones Nerviosas/fisiología , Terminales Presinápticos/fisiología , Animales , Neuronas Motoras/química , Neuronas Motoras/citología , Terminaciones Nerviosas/química , Técnicas de Cultivo de Órganos , Terminales Presinápticos/química , Rana pipiens , Vesículas Sinápticas/química , Vesículas Sinápticas/fisiologíaRESUMEN
Synchronous neurotransmitter release is a highly regulated process that takes place at specializations at the presynaptic membrane called active zones (AZs). The relationships between AZs, quantal release, and vesicle replenishment are not well understood in a mature synapse. We have measured the number, distribution, and other properties of AZs in mouse motor nerve terminals and combined these observations with electrophysiological estimates of the size of the readily releasable pool (RRP) of synaptic vesicles. On average, we counted 850 AZs per terminal. Assuming two primary docked vesicles per AZ, we predict a total of â¼1700 vesicles optimally positioned for exocytosis. Electrophysiological estimates of the size of the RRP, using a simple kinetic model that assumes exponential depletion of the initial pool and refilling by recruitment, gave an average value of 1730 quanta during 100 Hz stimulation, in satisfying agreement with the morphology. At lower stimulus frequencies, however, the model revealed that the estimated RRP size is smaller, suggesting that not all AZs participate in release at low stimulation frequencies.
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Unión Neuromuscular/citología , Unión Neuromuscular/fisiología , Terminales Presinápticos/fisiología , Vesículas Sinápticas/fisiología , Animales , Fenómenos Biofísicos , Biofisica , Proteínas del Citoesqueleto/metabolismo , Estimulación Eléctrica/métodos , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Femenino , Técnicas In Vitro , Masculino , Ratones , Potenciales Postsinápticos Miniatura/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuropéptidos/metabolismo , Neurotransmisores/metabolismoRESUMEN
A series of graphitized carbon blacks have been studied using argon and nitrogen adsorption at their boiling points. Analysis of adsorption isotherms was performed with nonlocal density functional theory (NLDFT) accounting for the Axilrod-Teller equation to describe the effect of nonadditivity of the gas-solid interaction. In our previous study [Ustinov, E. A. J. Chem. Phys. 2010, 132, 194703] we have shown that the nonadditivity effect decreases the attractive component of Ar-Ar interaction in the first molecular layer adjacent to the graphite surface by about 23%. This is a source of a large error (up to 40%) when a standard NLDFT is applied to fitting the low-temperature Ar adsorption isotherm on a graphitized carbon black. A new approach that incorporates the Axilrod-Teller equation into the standard NLDFT diminishes the relative error from 40 to 4%, which suggests that the nonadditivity correction should not be ignored in most adsorption systems including crystalline and amorphous solids. The present study is an extension of our approach to N(2) adsorption isotherms at 77.3 K on graphitized carbon blacks. We show that the approach allows to reliably determine the gas-solid molecular parameters, the gas-solid nonadditivity coefficient, the Henry coefficient, and the specific surface area. The surface areas of different carbon blacks determined with the N(2) at 77.35 K and Ar at 87.29 K are very close to each other, though in the former case the values proved to be slightly smaller presumably due to nonspherical shape of the nitrogen molecule. A comparison with the Brunauer, Emmett, and Teller method is provided.
RESUMEN
The spatial relationship of exocytosis and endocytosis in motor nerve terminals has been explored, with varied results, mostly in fixed preparations and without direct information on the utilization of each exocytic site. We sought to determine these spatial properties in real time using synaptopHluorin (spH) and FM4-64. Earlier we showed that nerve stimulation elicits the appearance of spH fluorescence hot spots, which mark preferred sites of exocytosis. Here we show that nerve stimulation in the presence of the styryl dye FM4-64 evokes hot spots of FM4-64 fluorescence. Their size, density, and rate of appearance are similar to the spH hot spots, but their rate of disappearance after stimulation was much slower (t(1/2) approximately 9 min vs approximately 10 s for spH hot spots), consistent with FM4-64 spots identifying bulk endocytosis and subsequent slow intracellular dispersion of nascent vesicles. Simultaneous imaging of both fluorophores revealed a strong colocalization of spH and FM4-64 spots, but only during high (100 Hz) stimulation. At 40 Hz stimulation, exocytic and endocytic spots did not colocalize. Our results are consistent with the hypothesis that hot spots of endocytosis, possibly in the form of bulk uptake, occur at or very near highly active exocytic sites during high-frequency stimulation.
Asunto(s)
Endocitosis/fisiología , Exocitosis/fisiología , Neuronas Motoras/fisiología , Terminales Presinápticos/fisiología , Animales , Biofisica/métodos , Estimulación Eléctrica/métodos , Endocitosis/genética , Potenciales Evocados Motores/genética , Potenciales Evocados Motores/fisiología , Exocitosis/genética , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Placa-Clamp , Probabilidad , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Vesículas Sinápticas/metabolismo , Factores de TiempoRESUMEN
We monitored the spatial distribution of exo- and endocytosis at 37 degrees C in mouse motor nerve terminals expressing synaptopHluorin (spH), confirming and extending earlier work at room temperature, which had revealed fluorescent 'hot spots' appearing in repeatable locations during tetanic stimulation. We also tested whether hot spots appeared during mild stimulation. Averaged responses from single shocks showed a clear fluorescence jump, but revealed no sign of hot spots; instead, fluorescence rose uniformly across the terminal. Only after 5-25 stimuli given at high frequency did hot spots appear, suggesting a novel initiation mechanism. Experiments showed that about half of the surface spH molecules were mobile, and that spH movement occurred out of hot spots, demonstrating their origin as exocytic sources, not endocytic sinks. Taken together, our results suggest that synaptic vesicles exocytose equally throughout the terminal with mild stimulation, but preferentially exocytose at specific, repeatable locations during tetanic stimulation.
Asunto(s)
Exocitosis/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Neuronas Motoras/fisiología , Terminales Presinápticos/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Vesículas Sinápticas/fisiología , Animales , Difusión , Estimulación Eléctrica , Endocitosis/fisiología , Recuperación de Fluorescencia tras Fotoblanqueo , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Membranas Sinápticas/fisiología , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
When pituitary lactotroph granules undergo exocytosis in the presence of FM1-43, their cores absorb dye and fluoresce brightly. We report that different granules fluoresce with different colors, despite being stained with a single fluorescent dye; emission spectra from individual granules show up to a 25 nm difference between the greenest and reddest granules. We found a correlation between granule color and average fluorescence intensity, suggesting that granule color depends upon dye concentration. We confirmed this in two ways: by increasing FM dye concentration in granules, which red shifted granule color, and by partially photobleaching the FM dye in granules, which green shifted granule color. Increasing stimulation intensity (by increasing KCl concentration) increased the proportion of red granules, indicating that granules exocytosing during intense stimulation bound more dye. This, perhaps, reflects differences in granule core maturation and condensation in which mature granules with condensed cores bind more FM dye but require more intense stimulation to be released. Concentration-dependent color shifts of FM dyes may be useful for monitoring aggregation processes occurring on a size scale smaller than the optical limit.
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Colorantes Fluorescentes/química , Lactotrofos/ultraestructura , Microscopía Fluorescente/métodos , Compuestos de Piridinio/química , Compuestos de Amonio Cuaternario/química , Vesículas Secretoras/ultraestructura , Coloración y Etiquetado/métodos , Animales , Células Cultivadas , Ratas , Ratas Sprague-DawleyRESUMEN
We measured synaptic vesicle mobility using fluorescence recovery after photobleaching of FM 1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] stained mouse motor nerve terminals obtained from wild-type (WT) and synapsin triple knock-out (TKO) mice at room temperature and physiological temperature. Vesicles were mobile in resting terminals at physiological temperature but virtually immobile at room temperature. Mobility was increased at both temperatures by blocking phosphatases with okadaic acid, decreased at physiological temperature by blocking kinases with staurosporine, and unaffected by disrupting actin filaments with latrunculin A or reducing intracellular calcium concentration with BAPTA-AM. Synapsin TKO mice showed reduced numbers of synaptic vesicles and reduced FM 1-43 staining intensity. Synaptic transmission, however, was indistinguishable from WT, as was synaptic vesicle mobility under all conditions tested. Thus, in TKO mice, and perhaps WT mice, a phospho-protein different from synapsin but otherwise of unknown identity is the primary regulator of synaptic vesicle mobility.
Asunto(s)
Neuronas Motoras/metabolismo , Terminales Presinápticos/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Calcio/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Recuperación de Fluorescencia tras Fotoblanqueo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/ultraestructura , Ácido Ocadaico/farmacología , Técnicas de Cultivo de Órganos , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Fosfotransferasas/antagonistas & inhibidores , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/ultraestructura , Compuestos de Piridinio , Compuestos de Amonio Cuaternario , Estaurosporina/farmacología , Sinapsinas/genética , Temperatura , Tiazolidinas/farmacologíaRESUMEN
We monitored presynaptic exocytosis and vesicle recycling at neuromuscular junctions of transgenic mice expressing synaptopHluorin (spH), using simultaneous optical and electrophysiological recordings. Synaptic transmission was indistinguishable from that in wild-type controls. Fluorescence rose during and decayed monotonically after stimulus trains to the nerve, with amplitudes and decay times increasing with the amount of stimulation. The relatively large size of synaptic terminals allowed us to examine the spatial profile of fluorescence changes. We identified hot spots of exocytosis, which were stable with repeated trains. Photobleach experiments showed that spH freshly exposed by nerve stimulation was not preferentially retrieved by compensatory endocytosis; instead, most retrieved spH preexisted in the surface membrane. Finally, we compared fluorescence and electrical [summed end-plate potentials (EPPs)] estimates of exocytosis, which diverged during repeated trains, as fluorescence exceeded summed EPPs, although the average amplitude of miniature EPPs was unchanged. This might reflect exocytosis of spH-containing, acetylcholine-free ("empty") vesicles or other organelles during intense stimulation.