Me gustaría agradecer a Christopher Evans y Darren Burke, del Centro para el Estudio Integral del Comportamiento Animal (CISAB).

Jacky Dragons Jacky lagartos (A. muricatus) se llevaron a cabo en instalaciones climatizadas en cautiverio. Recintos fueron construidos de terrarios de cristal fabricados de 60 cm 3. Recintos se llenaron también con sustrato de arena Sydney, follaje nativo y natural, perchas de madera. Recintos fueron colocados en la parte superior de los carros móviles que permite a los sujetos que se coloca en el centro de todos los monitores de visualización. Los lagartos fueron sometidos a un 14L: 10D ciclo de luz durante el verano las condiciones simuladas, y se les proporcionó dos lámparas de calor (125W) y ultravioleta adecuada (UV) iluminación (300 W) durante la primera media hora de luz del día. Lagartos también recibieron agua ad libitum, los grillos alimentados (Achetus domesticus) espolvoreados con vitaminas durante las sesiones no experimental, y de manera rutinaria para los parásitos desparasitados con dosis diluidas de ivermectina. Maquinaria para la reproducción Tres equipos de reproducción de vídeo se utilizan para la presentación del estímulo (ver Figura 1). El equipo central se compone de un iMac (Apple Computer Inc.) que contiene estímulos punto. Los dos equipos adicionales consisten en una iMac con Final Cut Pro HD para la presentación del estímulo que contiene la reproducción de los estímulos (es decir, invertebrados). La señal digital es enviada a una avanzada de vídeo digital (Canopus © gt ADVC110, Melbourne, Victoria, Australia) para la señal digital a analógica para la reproducción de vídeo y se muestra en un Trintron Sony (Sony Corporation, Shinagawa, Tokio, Japón) monitor (de la serie Nos. PVM-14M2A/PVM-14L2/PVM-14N5A). A Canon (modelo n º MV650i) videocámara digital con zoom óptico de 22X (Cannon Inc., Japón) se monta directamente sobre la zona de pruebas y las respuestas de los registros a los estímulos directamente a Sony (Sony Corporation, Shinagawa, Tokio, Japón) videograbadora ( No. de serie SLV-EZ717AS). Las imágenes grabadas se envían a un visor en color (NEC, N º de serie 1892, Kanagawa, Japón). En 7,5 segundos desde el inicio de la pantalla de puntos, una marca de sonido generado electrónicamente se envía a un ADVC110 Canopus ® y graba en VHS. La reproducción de vídeo se ajusta para PAL-DV estándar (5:1 de compresión, la resolución horizontal 575 líneas, 25 cuadros s-1). De puntos aleatorios Kinematograms (RDK) RDK se pueden desarrollar utilizando VPixx V1.79 (VPixx V1.79, VPixx Technologies Inc. 2002). Estímulos de diseño con un campo de 30 x 20 grados, se llenan de 200 puntos, con cada punto de medición de 5 x 5 píxeles. Crear estímulos puntos con ocho variaciones en la velocidad combinada con ocho niveles de coherencia y de dos direcciones, la generación de 128 diferentes estímulos. Los parámetros de velocidad se han diseñado en grados por segundo (° / s). Las variaciones de velocidad fueron de 0,5, 2,5, 5, 10, 20, 40, 80 y 160 º / s. Porcentajes coherencia fueron 0, 2,5, 5, 10, 20, 40, 80 y 100%, ya sea en un movimiento direccional izquierda o derecha. La exportación cada secuencia como Motion JPEG-B de archivos. Sin embargo, usted puede diseñar el RDK que una serie de características. Cada secuencia es un total de 15 s de duración. La secuencia comienza con 2,5 s de la coherencia del 0% emparejado con la variable de la velocidad de prueba. Este período se inicia el ensayo en curso. Esto es seguido por 5 s de la variación en la combinación de velocidad y coherencia. La secuencia se completa con 7,5 s de 100% en la dirección apropiada. Importar todos los formatos Motion JPEG-B y editar secuencias de Apple Final Cut Pro HD (Apple Computers Inc.). Añadir un marcador de sonido en cada intervalo. Este marcador sólo es audible a VHS, y se utiliza como una señal de la hora de puntuar el comportamiento cuando los cambios en la presentación del estímulo se producen. Exportado cada una de las 128 secuencias de películas como Final Cut Pro. Reforzadores primarios - gusanos de la harina Sujetos a cabo en un programa de refuerzo de cinco ensayos aleatorios por ensayo de bloque (16 ensayos), durante el experimento. Los reforzadores primarios son gusanos de la harina (Tenebrio molitor). Reforzadores secundarios - Invertebrados animados Reforzadores secundarios han de ser emparejado con el reforzador primario. Creado refuerzos biológicamente significativos, tales como tres tipos de invertebrados de animación por ordenador: 1) cricket, 2) los ácaros, arañas y 3). Crea animaciones utilizando Lightwave v8.3 (NewTek Inc., San Antonio, Texas, EE.UU.) durante 15 s de duración, con la inicial de 7,5 s de fondo todavía, y seguido por 7,5 s de los invertebrados en la pantalla adecuada. PhoTOGRAFÍA un fondo de vegetación natural para la importación como un archivo JPEG en el clip eah. De exportación de cada secuencia en una secuencia de imágenes JPEG individuales (375 cuadros), y comprimirlo en un archivo de película con Final Cut Pro HD para reproducción de vídeo. Diseño de 50 clips único para evitar la habituación al paradigma de condicionamiento. Procedimiento General Temas entre los monitores de visualización. Selección aleatoria de presentación de estímulos RDK para cada tema. Mostrar los estímulos en bloques de 16 ensayos de más de ocho días. Cada ensayo es de 15 s con intervalos entre 30 a 60 diferentes s. Segmento de cada ensayo en el intervalo de 2,5 s de 0% de la coherencia reflexiva de la prueba de velocidad, 5 s de las pruebas de velocidad y coherencia, y seguido por 7,5 s del 100% en la dirección adecuada coincidir con la velocidad. Durante este período crítico de 5 s, los sujetos están obligados a responder. Registro de las medidas dependientes como la orientación hacia un monitor de la izquierda o la derecha y la latencia de respuesta. Use sólo cinco ensayos aleatorios con un reforzador primario y reforzar cada ensayo con un invertebrado de animación. Al final de cada ensayo de bloque, eliminar recintos sujeto de la zona de pruebas, e introducir otro recinto.

<ol>
	<li>Burghardt, G. M., Gans, C., Tinkle, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+processes+in+reptiles.">Learning processes in reptiles.</a> Biology of the reptilia. 7, 555-681 (1977).</li><li>VPixx, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Instruction Manual. VPixx Techniologies Inc. , 96 (2002).</li></ol>

El financiamiento fue apoyado por el Premio de Postgrado CISAB y el Departamento de Psicología de la Universidad Macquarie, y el Consejo Australiano de Investigación. La investigación fue realizada dentro de las pautas éticas de la Universidad de Macquarie Comité de Ética Animal (2003/014) y Nueva Gales del Sur Parques Nacionales y Vida Silvestre de Servicios (S11024) de Australia.

Prueba de la capacidad sensorial de especies de lagartos ha sido difícil y ha sido en gran medida tanto un proceso tedioso y arduo. Estas dificultades provienen de encontrar la correcta refuerzo o el establecimiento de un protocolo de funcionamiento. En este caso, he establecido un protocolo de éxito en el que poner a prueba la percepción del movimiento en lagartos. Este procedimiento también puede ser aplicable en las pruebas de otras características visuales, así como otros mecanismos sensoriales. Además, este procedimiento puede s...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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		<tbody>
		
<tr>
<td>iMac</td>
<td>Computer</td>
<td>Apple Computers, Inc.</td>
<td>&nbsp;</td>
<td>Three systems</td>
</tr>
<tr>
<td>Canopus&#174;</td>
<td>Digital-to-analog converter</td>
<td>Canopus&#174;</td>
<td>ADVC110</td>
<td>&nbsp;</td>
<tr>
<td>Sony Trintron</td>
<td>Colour viewfinder</td>
<td>Sony Corporation</td>
<td>PVM-14M2A, PVM-14L2, PVM-14N5A</td>
<td>Three monitors</td>
</tr>
<tr>
<td>Canon</td>
<td>Digital camcorder</td>
<td>Cannon Inc.</td>
<td>MV650i</td>
<td>Records behavior</td>
</tr>
<tr>
<td>NEC</td>
<td>Colour viewfinder</td>
<td>NEC</td>
<td>1892</td>
<td>View responses</td>
</tr>
<tr>
<td>Sony VCR</td>
<td>VHS recorder</td>
<td>Sony Corporation</td>
<td>SLV-EZ717AS</td>
<td>Record behavior to VHS</td>
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<td>VHS tape</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Generic VHS</td>
<td>Recording media</td>
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<td>VPixx</td>
<td>Software program</td>
<td>VPixx Technologies, Inc.</td>
<td>v1.79</td>
<td>To create RDKs</td>
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<td>Final Cut Pro</td>
<td>Software program</td>
<td>Apple Computer, Inc.</td>
<td>HD</td>
<td>To create playback sequences</td>
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<td>Lightwave&#174; 3D</td>
<td>Software program</td>
<td>NewTek Inc.</td>
<td>v8.3</td>
<td>To create secondary reinforcers</td>
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<td>Mealworm</td>
<td>Tenebrio molitor</td>
<td>Pisces Enterprises LTD</td>
<td>&nbsp;</td>
<td>Primary reinforcer</td>
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<td>Cricket</td>
<td>Achetus domesticus</td>
<td>Pisces Enterprises LTD</td>
<td>&nbsp;</td>
<td>Food for husbandry</td>
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<td>Terrariums</td>
<td>Glass</td>
<td>Crystal Palace Aquariums</td>
<td>&nbsp;</td>
<td>Enclosure material for lizards (60 x 60 x 60 cm)</td>
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<td>Globes</td>
<td>Spotone</td>
<td>Phillips</td>
<td>&nbsp;</td>
<td>125W, Heat lamps for lizards</td>
</tr>
<tr>
<td>Ultraviolet globes</td>
<td>Ultra-Vitalux</td>
<td>Osram</td>
<td>&nbsp;</td>
<td> 300W, UV lamps for lizards</td>
</tr>
<tr>
<td>Herptivite</td>
<td>&nbsp;</td>
<td>Rep-Cal Research Labs</td>
<td>&nbsp;</td>
<td>Vitamins: beta carotene, calcium, and vitamin D3</td>
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testing visual sensitivity to the speed and direction of motion in lizards

Pruebas de sensibilidad visual de cualquier especie se proporciona información básica sobre el comportamiento, la evolución y la ecología. Sin embargo, las pruebas de características del sistema visual proporcionan más evidencia empírica para aplicaciones funcionales. Investigación sobre el sistema sensorial proporciona información acerca de la capacidad sensorial de aprendizaje, y la capacidad de la memoria, y establece el comportamiento conocido de referencia en el que medir las desviaciones (Burghardt, 1977). Sin embargo, a diferencia de los sistemas de mamíferos o aves, las pruebas para el aprendizaje y la memoria en una especie de reptil es difícil. Además, utilizando un paradigma operante como una medida de la capacidad psicofísica sensorial es también tan difícil. Históricamente, las especies de reptiles han respondido mal a los ensayos de condicionamiento debido a problemas relacionados con la motivación, la fisiología, el metabolismo y las características básicas biológicas. Aquí, demuestran un paradigma operante utiliza una especie de lagarto nuevo modelo, el dragón de Jacky (Amphibolurus muricatus) y describir cómo probar la sensibilidad periférica a la velocidad y las características sobresalientes del movimiento. Este método utiliza un enfoque innovador para el aprendizaje y la evaluación de la capacidad sensorial de los lagartos. Yo empleo el uso de puntos aleatorios kinematograms (RDK) para medir la sensibilidad a la velocidad, y manipular el nivel de intensidad de la señal cambiando la proporción de puntos que se mueven en una dirección coherente. RDK no representan un estímulo biológicamente significativo, se involucra el sistema visual, y es una herramienta clásica psicofísico utilizado para medir la sensibilidad de los seres humanos y otros animales. Aquí, RDK se muestran a los lagartos con tres sistemas de reproducción de vídeo. Los lagartos son para seleccionar la dirección (izquierda o derecha) en los que perciben los puntos que se mueven. La selección de la dirección apropiada se ve reforzada por los estímulos biológicamente importantes presas, simulado por computadora-animated invertebrados.

Watch this Scientific Journal Video about Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards at JoVE.com

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Pruebas de sensibilidad visual de las lagartijas con un paradigma de condicionamiento operante que utiliza la reproducción de vídeo de puntos aleatorios kinematograms e invertebrados generadas por ordenador.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

testing-visual-sensitivity-to-speed-direction-motion

Research

JoVE Journal

Biology

11.4K vistas.  Macquarie University. Pruebas de sensibilidad visual de las lagartijas con un paradigma de condicionamiento operante que utiliza la reproducción de vídeo de puntos aleatorios kinematograms e invertebrados generadas por ordenador.

Video: Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

Prueba de las barreras fisiológicas a la transmisión del virus en los áfidos con microinyección

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

Investigación

Biología

Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

Pruebas del comportamiento olfativo en el ratón adulto

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Una sola célula grabaciones de succión de los conos del ratón

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Usando la respuesta de optocinético para estudiar la función visual del pez cebra

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Inyección de insulina y extracción hemolinfa para medir la sensibilidad a la insulina en adultos Drosophila melanogaster</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Diseño y Análisis Del Comportamiento De Preferencia De Temperatura Y Su Ritmo Circadiano En Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Validación de un modelo de ratón para interrumpir LINC Complejos en una celda de manera específica

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Una interfaz gráfica de usuario para el seguimiento asistido por software de la concentración de proteínas en protones dinámicos celulares

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

Aplicación de la microscopía de resolución súper alta velocidad velocidad en vivo cilio primario

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

Protocolos para las pruebas de la toxicidad de la novela química insecticida para mosquitos

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...