Funding provided by the National Science Foundation (M.F.).

Introduction
The OMT is an adaptation of a magnetic tether system [1] designed to incorporate a &#8220;virtual plume simulator&#8221;. The following protocols will explain how to properly tether flies (Part 1) and provide strategies for setting up the magnets (Part 2) and mass-flow regulated odor delivery system (Part3). The information described here is optimized for this particular system and may vary for components with other technical specifications.
Part 1: Tethering flies
The steps for tethering described here are similar to many previous studies [2] but adapted for use in an OMT [3]. Proper tethering is crucial to ensure robust and repeatable experimental trials. The importance of careful tethering can not be overstated.
<ol>
	<li>Collect flies 4-6 days post eclosion and starve them for 4-6 hours. Place a kimwipe moistened with water in the foodless vial to prevent desiccation.</li>
	<li>Transfer a batch of flies from a bottle to a small vial and insert it into a brass block on a Peltier cooling stage set at approximately 4 degrees Celsius. The proper temperature anesthetizes the flies within approximately 30 seconds.</li>
	<li>Sort the flies on the cold surface of the cooling stage. A damp Kimwipe ensures constant cooling and moisture wicking from the cuticle. Select the largest females for tethering.</li>
	<li>Using a fine brush, push a single fly into position in the custom-built sarcophagus shaped to accommodate a single wild type female fruit fly. 
	<img alt="" src="/files/ftp_upload/old/1063_2008_11_26_2/1063_Figure-01_thumb.png" /> 
	Figure 1. 
	<a href="https://www.jove.com/files/ftp_upload/old/1063_2008_10_28_1/1063_Figure-01.png">Please click here to see a larger version of the the figure.</a></li>
	<li>We position a minutien pin (Fines Science Tools) with a magnetic rod mounted on a micromanipulator. The pin is to be aligned such that the blunt end touches the dorsal thorax just behind the head between the two wings. The pin should be perfectly vertical when viewed head-on toward the eyes. Even a small amount of &#8216;roll&#8217; between the fly and pin can not be tolerated. The side-view angle between the pin and the long axis of the body should be at a nose-up angle of 30 degrees relative to the horizon (Fig. 1). Note: For optimal operation in the magnet tether, the pins may need to be trimmed to shorter lengths. This will depend entirely on the distance between the suspension magnets.</li>
	<li>Once the minutien pin is aligned, use a thin steel wire to apply a small drop of UV-activated glue (ElectroLite Corp) to the blunt end of the minutien pin.</li>
	<li>Manipulate the pin so the glue droplet touches the fly&#8217;s thorax just above the head. With the proper drop size, the glue should anneal and flow from the pin onto the thorax. Be careful not to use too much glue.</li>
	<li>Cure the glue with two 20 second bursts of UV light.</li>
	<li>Place the fixed fly aside and repeat. Slice small squares of Kimwipe and with forceps give one to each tethered fly to restrain flight.</li>
	<li>After one hour of recovery, the tethered flies are ready for experiments.</li>
	<li>Improper gluing may hinder a fly&#8217;s ability to perform in the magnet arena. Every fly should be checked for smooth rotation at the start of every experiment to ensure the flies are properly glued. This can be accomplished by rotating a striped pattern with a sufficiently large spatial period to elicit strong optomotor responses [4]. If the fly is glued at the wrong pitch angle or tilted on the pin with respect to the roll axis, the fly may (i) not spin at all, (ii) have a restricted range of motion, or (iii) rotate at inconsistent velocity. In the above cases the fly should be discarded and replaced with a new one. If this solves the problem it was most likely a case of improper gluing. (if replacing the fly does not solve the problem, see Part 2.4)</li>
</ol>
Part 2: Aligning the magnets
Aligning the magnets (Rare-earth-magnets.com) properly is necessary for the fly to achieve a smooth 360 degree range of motion. Follow these steps to properly align the magnets.
<ol>
	<li>The lower ring magnets are held horizontally by a plastic sleeve and placed directly on top of a clear vacuum chamber (Fig. 1; described further in part 3). The upper rod magnet is fixed vertically approximately 3/4&#34; above the lower magnets.</li>
	<li>Roughly align the upper and lower magnets visually by placing the upper rod magnet directly above the center point of the lower ring magnets. It is useful to attach the upper magnet to a micromanipulator (Siskiyou Inc.) for fine scale adjustment.</li>
	<li>Epoxy a V-jewel bearing (Small Parts) on the bottom surface of the upper magnet to minimize rotational friction and standardize fly positioning.</li>
	<li>Place a fly in the arena and adjust the horizontal and vertical position of the upper magnet until the fly can smoothly and steadily rotate 360 degrees in the yaw axis. Every fly should be checked for smooth rotation at the start of every experiment to ensure the magnets are properly aligned and that individual minutien pins are not damaged (see Part 1.11). If the magnets are not properly aligned the fly may (i) not spin at all (ii) have a restricted range of motion (iii) rotate at inconsistent velocities. If the problem is solved by replacing the fly with a new one, it was most likely due to a gluing problem (see Part 1). If these problems exist for every fly, it is likely an issue with the alignment of the magnets and further adjustment will be required.</li>
	<li>Warning: while playing with rare earth magnets can be fun, they are very dangerous. Misuse results in damaged equipment and/or minor bodily harm. Exercise extreme caution with these tempting yet easily underestimated magnets.</li>
</ol>
Part 3: Odor Delivery
<ol>
	<li>The odor delivery system is based on one described previously [5] and is the toughest part to optimize (Fig. 1). It requires a lot of patience and trial-and-error. Follow these steps to achieve stable odor tracking.</li>
	<li>The initial set-up of the odor system requires running narrow gauge Teflon tubing (Small Parts) from the gas multiplexer (Sable Systems International) to the custom built water/odor vials. The outlet of these vials connects directly to the hypodermic port tubes using the same Teflon tubing.</li>
	<li>A clear acrylic vacuum chamber is positioned under the arena to support the magnets. We use a 4mm glass tube mounted below the fly connected to the vacuum chamber to bring the vacuum opening closer to the fly and reduce the diameter of the opening.</li>
	<li>Place a tethered fly into the arena using forceps.</li>
	<li>Turn on an attractive odor (apple cider vinegar works well), and visually verify that the fly tracks the odor. If the fly appears to have no preference for the odor, follow the next three steps. This may require several flies, and several trials.</li>
	<li>Adjust the position of the odor port until the fly appears to track the odor. We mounted the odor ports on micromanipulators (Siskiyou Inc.) and positioned 4mm dorsal and 3mm anterior to the fly&#8217;s head.</li>
	<li>Adjust the mass flow rate until the fly appears to track the odor. Our settings are 7ml/min (Sable Systems International MFC-4).</li>
	<li>Adjust the flow rate of the vacuum until the fly appears to track the odor. We set the vacuum flow rate at 13l/min using a flow regulator (Cole Parmer). Note: Building vacuum supply lines may fluctuate over time causing fluctuations in plume structure. It may be useful to connect an external vacuum pump to the flow regulator for constant vacuum flow conditions.</li>
	<li>Once the position of the port and the flow rates are set, periodically switch between delivering water vapor and odor vapor using a switchable using a gas multiplexer and visually verify that the fly only tracks the odor when the odor is on. Occasionally, residual odor inside the odor port can elicit odor tracking when the odor is turned off. This can be eliminated without removing the properly positioned odor port by filling the hypodermic tubes with ethanol and collecting it on the delivery end with a Kimwipe. Using compressed air, blow any remaining ethanol out and resume experiments.</li>
</ol>
Representative Results:
When these steps are followed correctly two things should be observed. First, under constant rotating visual stimuli a tethered fly placed in the OMT should rotate smoothly 360 degrees in the yaw axis. &#8220;Jerky&#8221; turns (called saccades) are characteristic of spontaneous turning behavior in the absence of constant visual stimuli. If flies can apparently not rotate smoothly, improper gluing (see Part 1) and misaligned magnets may be to blame (see Part 2). Second, when the odor is turned on, the fly should head in the direction of the odor. When the odor is turned off, the fly should saccade spontaneously with no preference for any single position in the arena. If flies do not actively track the odor plume, the position of the odor port, the air flow rate and/or the vacuum flow rate may need adjustment (see Part 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>

Tethering flies and eliciting stable odor tracking in this arena is relatively straightforward. It requires practice, patience and trial and error. Many parts of this arena could be further optimized or substituted to yield variations in experimental potential. For example, newer versions of the vacuum chamber are much smaller and allow more space below the arena. Also, the strength and type of rare-earth magnets can be varied slightly and additional odor ports could be added to increase the number of odor types and/or c...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cooling Stage</td>
<td>&nbsp;</td>
<td>Teca</td>
<td>LHP-300CP</td>
<td>&nbsp;</td>
<tr>
<td>Brass Cooling Block</td>
<td>&nbsp;</td>
<td>Custom Machined</td>
<td>&nbsp;</td>
<td>~4 x 8 x 2cm</td>
</tr>
<tr>
<td>Sarcophagus</td>
<td>&nbsp;</td>
<td>Custom Machined</td>
<td>&nbsp;</td>
<td>Contact us for details</td>
</tr>
<tr>
<td>UV-Activated Glue</td>
<td>&nbsp;</td>
<td>Kemxert corp</td>
<td>KOA300</td>
<td>1oz bottle lasts years</td>
</tr>
<tr>
<td>Panel System</td>
<td>&nbsp;</td>
<td>Caltech</td>
<td>http://www.dickinson.caltech.edu/panels</td>
<td>[4]</td>
</tr>
<tr>
<td>Minutien Pins</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>&nbsp;</td>
<tr>
<td>Lower Ring Magnets</td>
<td>&nbsp;</td>
<td>Rare-earth-magnets.com</td>
<td>NSN0615</td>
<td>5 - 0.75"o.d. x 0.375" i.d. x 0.125"</td>
</tr>
<tr>
<td>Upper Rod Magnet</td>
<td>&nbsp;</td>
<td>Rare-earth-magnets.com</td>
<td>NSN0750</td>
<td>0.125" x 1"</td>
</tr>
<tr>
<td>V-Jewel Bearing</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>VJ-0469-01</td>
<td>&nbsp;</td>
<tr>
<td>MFC-4 Flow Regulators</td>
<td>&nbsp;</td>
<td>Sable Systems International</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gas Multiplexer</td>
<td>&nbsp;</td>
<td>Sable Systems International</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Teflon Tubing</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>STT-20-C</td>
<td>&nbsp;</td>
<tr>
<td>20 Gauge Hypodermic Tubes</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>HTX-20R-06</td>
<td>&nbsp;</td>
<tr>
<td>Glass Tubing</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>4mm o.d.</td>
</tr>
<tr>
<td>Vacuum Flow Regulator</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>EW-32464-52</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a magnetic tether system to investigate visual and olfactory mediated flight control in 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. 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? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

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

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

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 ビュー.  University of California, Los Angeles. ここでは、どのようにテザー嗅覚磁気テザー（OMT）装置でフライをして説明します。我々は、希土類磁石と臭気のポート、およびどのように刺激の配信と最適な臭気の追跡を実現するために真空吸引の両方の質量流量を設定することを整列する方法について説明します。

ビデオ: 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.

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 ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

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.

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.

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.

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.

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.

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

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.

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.

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.

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