Financiamiento proporcionado por la National Science Foundation (MF).

Introducción La OMT es una adaptación de un sistema de sujeción magnético [1] diseñado para incorporar un &quot;simulador de la pluma virtual&quot;. Los siguientes protocolos se explican cómo atar moscas correctamente (Parte 1) y proporcionar estrategias para la creación de los imanes (Parte 2) y la masa de flujo de un sistema regulado de entrega olor (Parte 3). La información descrita aquí está optimizado para este sistema en particular y puede variar para los componentes con otras especificaciones técnicas. Parte 1: moscas Tethering Los pasos para atar descritos aquí son similares a muchos estudios anteriores [2], pero adaptada para su uso en una OMT [3]. Inmovilización adecuada es crucial para asegurar las pruebas experimentales robusta y repetible. La importancia de la inmovilización cuidado no puede ser exagerada. <ol><li> Recoger las moscas 4-6 días después de la eclosión y matarlos de hambre durante 4-6 horas. Coloque una Kimwipe humedecido con agua en el vial sin alimentos para evitar la desecación. </li><li> La transferencia de un lote de moscas de una botella a un pequeño frasco y la inserta en un bloque de latón en una etapa de enfriamiento Peltier, aproximadamente, a 4 grados Celsius. La temperatura adecuada anestesia a las moscas en aproximadamente 30 segundos. </li><li> Ordenar las moscas en la superficie fría de la etapa de enfriamiento. A Kimwipe húmedo garantiza una refrigeración constante y absorbe la humedad de la cutícula. Seleccione la más grande de las mujeres para atar. </li><li> El uso de un pincel fino, empujar una sola mosca en la posición en el sarcófago de costumbre-construido en forma de dar cabida a un solo vuelo de tipo salvaje mujer de la fruta. <img src="/files/ftp_upload/old/1063_2008_11_26_2/1063_Figure-01_thumb.png" alt="" /> Figura 1. <a href="https://www.jove.com/files/1063_2008_10_28_1/1063_Figure-01.png">Haga clic aquí para ver una versión ampliada de la figura.</a> </li><li> Hemos puesto un alfiler minutien (multas Herramientas de la Ciencia) con una barra magnética montada sobre un micromanipulador. El pin se alinea de tal modo que el extremo romo toca el tórax dorsal justo detrás de la cabeza entre las dos alas. El PIN debe ser perfectamente vertical cuando se ve de frente hacia los ojos. Incluso una pequeña cantidad de &#39;rollo&#39; entre la marcha y el pin no se puede tolerar. El ángulo de visión lateral entre el pasador y el eje largo del cuerpo debe estar en un ángulo de la nariz hasta de 30 grados sobre el horizonte (Fig. 1). Nota: Para un funcionamiento óptimo en la sujeción del imán, los pines posible que tenga que ser ajustado para tramos más cortos. Esto dependerá enteramente de la distancia entre los imanes de suspensión. </li><li> Una vez que el pin minutien está alineado, use un alambre de acero fino para aplicar una pequeña gota de pegamento UV-activa (Electrolite Corp) con el extremo romo de la pin minutien. </li><li> Manipular los pines por lo que la gota de pegamento toca el tórax de la mosca justo por encima de la cabeza. Con el tamaño de gota adecuado, el pegamento debe recocer y el flujo de la clavija en el tórax. Tenga cuidado de no usar pegamento demasiado. </li><li> Curar la cola con dos ráfagas de 20 segundos de la luz UV. </li><li> Lugar de la mosca fija a un lado y repita. Cortar pequeños cuadrados de Kimwipe y con unas pinzas dar uno a cada volar atado a frenar el vuelo. </li><li> Después de una hora de recuperación, las moscas atadas está listo para los experimentos. </li><li> Pegado incorrecto puede dificultar la capacidad de una mosca para llevar a cabo en el campo de imán. Cada vuelo se debe comprobar la rotación suave al comienzo de cada experimento para asegurar que las moscas están bien pegados. Esto puede lograrse mediante la rotación un patrón de rayas con un período espacial lo suficientemente grande como para provocar fuertes reacciones optomotor [4]. Si la mosca se pega en el ángulo de paso incorrecto o inclinada sobre el pin con respecto al eje de balanceo, la marcha puede (i) no giran en absoluto, (ii) tienen un rango limitado de movimiento, o (iii) giran en inconsistentes velocidad. En los casos anteriores la marcha debe ser descartado y reemplazado por uno nuevo. Si esto resuelve el problema lo más probable es un caso de encolado inadecuado. (En caso de sustitución de la marcha no resuelve el problema, consulte la Parte 2.4) </li></ol> Parte 2: La alineación de los imanes La alineación de los imanes (de las tierras raras-magnets.com) correctamente es necesario para la marcha para lograr una gama suave de 360 ​​grados de movimiento. Siga estos pasos para alinear correctamente los imanes. <ol><li> Los imanes del anillo inferior se mantiene en posición horizontal por una funda de plástico y se colocan directamente en la parte superior de una cámara de vacío transparente (Fig. 1, describe con detalle en la parte 3). El imán de la barra superior se fija vertical de aproximadamente 3 / 4 &quot;por encima de los imanes más bajos. </li><li> Alrededor de alinear los imanes superiores e inferiores visual colocando el imán de la barra superior justo encima del punto central de los imanes del anillo inferior. Es útil para conectar el imán superior a un micromanipulador (Siskiyou Inc.) para el ajuste de escala fina. </li><li> Epoxi una relación V-joya (piezas pequeñas) en la superficie inferior del imán superior para minimizar la fricción de rotación y estandarizar la posición volar. </li><li> Coloque una mosca en la arena y ajustar la posición y vertical posición del imán superior hasta que la mosca puede fácilmente y de manera constante girar 360 grados en el eje de guiñada. Cada vuelo se debe comprobar la rotación suave al comienzo de cada experimento para asegurar que los imanes se alinean correctamente y que el individuo pines minutien no están dañados (ver Parte 1.11). Si los imanes no se alinean correctamente la marcha puede (i) no giran en absoluto (ii) tienen un rango limitado de movimiento (iii) giran a velocidades inconsistentes. Si el problema se resuelve mediante la sustitución de la marcha con una nueva, lo más probable debido a un problema de fijación (véase la Parte 1). Si estos problemas existen para cada vuelo, lo más probable es un problema con la alineación de los imanes y los nuevos ajustes serán necesarios. </li><li> Advertencia: al jugar con imanes de tierras raras pueden ser divertidas, son muy peligrosos. Mal uso de los resultados de los equipos dañados y / o lesiones corporales leves. Tenga mucho cuidado con estos imanes tentadora pero muy fácil subestimar. </li></ol> Parte 3: Entrega de olor <ol><li> El sistema de entrega se basa en el olor que se describe anteriormente [5] y es la parte más difícil de optimizar (Fig. 1). Se requiere de mucha paciencia y de ensayo y error. Siga estos pasos para lograr el seguimiento de olor a establo. </li><li> La puesta en marcha inicial del sistema olor requiere la ejecución de vía estrecha tubería de teflón (piezas pequeñas) de los gases de multiplexor (Sable Systems International) a la hecha a la medida del agua / olor viales. La salida de estos viales se conecta directamente a los tubos hipodérmica puerto utilizando el mismo tubo de teflón. </li><li> Una cámara de vacío de acrílico claro es colocado debajo de la arena para apoyar a los imanes. Nosotros usamos un tubo de cristal de 4 mm montado por debajo de la marcha conectada a la cámara de vacío para que el vacío de la apertura de cerca de la marcha y reducir el diámetro de la abertura. </li><li> Coloque una mosca atada a la arena con las pinzas. </li><li> A su vez en un olor atractivo (el vinagre de sidra de manzana funciona bien), y verificar visualmente que la marcha sigue el olor. Si la mosca no parece tener preferencia por el olor, siga los siguientes tres pasos. Esto puede requerir varias moscas, y varios ensayos. </li><li> Ajustar la posición del puerto hasta que el olor de la mosca que parece seguir el olor. Montamos los puertos de olor en micromanipulador (Siskiyou Inc.) y se coloca 4 mm anterior dorsal y de 3 mm para la cabeza de la mosca. </li><li> Ajustar el caudal de la masa hasta que la marcha que parece seguir el olor. Nuestros valores son 7ml/min (Sable Systems International MFC-4). </li><li> Ajustar la velocidad de flujo del vacío hasta que la marcha que parece seguir el olor. Hemos establecido el caudal de vacío en 13l/min con un regulador de flujo (Cole Parmer). Nota: Construcción de líneas de suministro de vacío puede fluctuar con el tiempo causar fluctuaciones en la estructura de la pluma. Puede ser útil para conectar una bomba de vacío externa al regulador de flujo constante de las condiciones de vacío de flujo. </li><li> Una vez que la posición del puerto y de los caudales se establecen, de forma periódica cambiar entre el vapor de agua y el vapor de la entrega de olor con un seleccionable mediante un multiplexor de gas y verificar visualmente que el volar sólo sigue el olor cuando el olor es el. De vez en cuando, el olor residual en el interior del puerto de olor puede provocar el seguimiento de olor cuando el olor está apagado. Esto puede ser eliminado sin eliminar el olor a puerto de la posición correcta de llenar los tubos hipodérmica con etanol y su recolección en el extremo de entrega con un Kimwipe. El uso de aire comprimido, sople para eliminar el etanol restante a cabo y continuar los experimentos. </li></ol> Los resultados representativos: Cuando estos pasos se siguen correctamente dos cosas deben ser observados. En primer lugar, en constante rotación estímulos visuales una mosca atada colocado en la OMT debe girar suavemente 360 ​​grados en el eje de guiñada. &quot;Espasmódicos&quot; giros (llamados movimientos sacádicos) son característicos de la conducta espontánea de inflexión en la ausencia de la constante de estímulos visuales. Si las moscas no pueden girar suavemente al parecer, pegado inadecuada (ver Parte 1) e imanes mal alineados pueden ser los culpables (véase la Parte 2). En segundo lugar, cuando el olor está encendido, la marcha deben dirigirse en la dirección del olor. Cuando el olor se apaga, la marcha debe sacada de forma espontánea, sin preferencia por ninguna posición única en la arena. Si las moscas no se activa la pista de la pluma de olor, la posición del puerto de olor, el caudal de aire y / o la tasa de flujo de vacío puede necesitar ajuste (véase la Parte 3).

<ol>
	<li>Bender, J. A., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+stimulation+of+saccades+in+magnetically+tethered+Drosophila.">Visual stimulation of saccades in magnetically tethered Drosophila.</a> J. Exp. Biol. 209, 3170-3182 (2006).</li><li>Tammero, L. F., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collision-avoidance+and+landing+responses+are+mediated+by+separate+pathways+in+the+fruit+fly,+Drosophila+melanogaster.">Collision-avoidance and landing responses are mediated by separate pathways in the fruit fly, Drosophila melanogaster.</a> J. Exp. Biol. 205, 2785-2798 (2002).</li><li>Duistermars, B. J., Frye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossmodal+visual+input+for+odor+tracking+during+fly+flight.">Crossmodal visual input for odor tracking during fly flight.</a> Curr. Biol. 18, 270-275 (2008).</li><li>Reiser, M. B., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+display+system+for+insect+behavioral+neuroscience.">A modular display system for insect behavioral neuroscience.</a> J. Neurosci. Methods. 167, 127-139 (2008).</li><li>Frye, M. A., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+output+reflects+the+linear+superposition+of+visual+and+olfactory+inputs+in+Drosophila.">Motor output reflects the linear superposition of visual and olfactory inputs in Drosophila.</a> J. Exp. Biol. 207, 123-131 (2004).</li></ol>

Atar moscas y la obtención de seguimiento olor estable en este campo es relativamente sencillo. Se requiere práctica, paciencia y ensayo y error. Muchas partes de este campo podría estar más optimizado o sustituido para producir variaciones en el potencial experimental. Por ejemplo, las nuevas versiones de la cámara de vacío son mucho más pequeños y permitir más espacio debajo de la arena. Además, la fuerza y ​​el tipo de imanes de tierras raras se puede variar un poco y puertos adicionales olor puede ser añadido para aumenta...

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a magnetic tether system to investigate visual and olfactory mediated flight control in drosophila

Ha quedado claro desde hace muchos años que los insectos usan señales visuales para estabilizar su título en una corriente de viento. Muchos animales seguimiento de los olores llevado por el viento. Por lo tanto, la estabilización visual de seguimiento directamente contra el viento ayuda en el seguimiento de olor. Pero las señales olfativas directamente influyen en el comportamiento de seguimiento visual independientemente de las señales de viento? Además, el diluvio reciente de la investigación sobre la neurofisiología y la genética neuroconductual del olfato en la mosca Drosophila ha motivado a los ensayos de comportamiento cada vez más sofisticados técnicamente y cuantitativos. Aquí, hemos modificado un sistema de sujeción magnética originalmente ideado para experimentos de visión y dotar a la arena con plumas estrechas laminar olor del flujo. Una mosca está pegado a un clavo de acero pequeñas y suspendida en un campo magnético que le permite a orientarse en libertad. De diámetro pequeño plumas olor del alimento se dirige hacia abajo sobre la cabeza de la mosca de la s, provocando el seguimiento estable por un elevado de hambre. Aquí nos centramos en los mecanismos fundamentales de la inmovilización, la alineación de los imanes, diseño de la pluma de olor, el olor y la confirmación de seguimiento estable.

Watch this Scientific Journal Video about A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila at JoVE.com

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

A continuación se describe la manera de atar una mosca en un aparato olfativo magnético de sujeción (OMT). Se describe la forma de alinear los imanes de tierras raras y los puertos de olor, y la forma de fijar las tasas de flujo de masa, tanto para la entrega de estímulos y succión al vacío para lograr el seguimiento de olor óptimo.

Un Sistema de Anclaje magnética para investigar control mediado de vuelo visual y olfativa en Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

a-magnetic-tether-system-to-investigate-visual-olfactory-mediated

Research

JoVE Journal

Biology

11.2K vistas.  University of California, Los Angeles. A continuación se describe la manera de atar una mosca en un aparato olfativo magnético de sujeción (OMT). Se describe la forma de alinear los imanes de tierras raras y los puertos de olor, y la forma de fijar las tasas de flujo de masa, tanto para la entrega de estímulos y succión al vacío para lograr el seguimiento de olor óptimo.

Video: A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Seguimiento visual mediada olor durante el vuelo en Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Investigación

Biología

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 ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Un enfoque práctico de elaboración de mapas genéticos inducible destino: una guía visual para marcar y rastrear las células En Vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Detección rápida homogénea de Ensayos Biológicos Usando el Sistema de modulación magnética biosensores

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Lípidos vesículas mediada por cromatografía de afinidad utilizando magnética clasificación de células activadas (LIMACS): un nuevo método para analizar la interacción de proteínas y lípidos

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi de detección para identificar fenotipos postembrionarios en C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

La medición cuantitativa de invadopodios mediada por proteolisis de la matriz extracelular en contextos simples y multicelulares

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

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 ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Un método mejorado para la rápida y precisa medición de rendimiento en vuelo<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

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 ...