We thank Cindy Voisine and Yoko Shibata for helpful discussion and critical comments on the manuscript. We acknowledge the High Throughput Analysis Laboratory (HTAL) and the Biological Imaging Facility (BIF) at Northwestern University for their assistance. This work was funded by grants from the National Institutes of Health (NIGMS, NIA, NINDS), the Ellison Medical Foundation, and the Daniel F. and Ada L. Rice Foundation (to R.I.M.). C.I.N.-K. was supported by the Deutsche Forschungsgemeinschaft (KR 3726/1-1).

1. Monitoring Transcellular Spreading of Prion-like Proteins By In Vivo Time-lapse Imaging
NOTE: Grow C. elegans wild-type (WT) (N2) and transgenic lines according to standard methods and carefully control the cultivation temperature47.
<ol>
	<li>Generate transgenic lines of C. elegans expressing the prion-like protein, tagged with monomeric red fluorescent protein (mRFP). Watch this video that demonstrates how to use microinjection48. For further details and methods describing how to integrate these extrachromosomal lines, see49.</li>
	<li>Prepare synchronized populations by egg laying or bleaching according to standard methods50.
	<ol>
		<li>Synchronization by egg laying
		<ol>
			<li>Transfer 10 - 20 gravid adults on a plate and let them lay eggs for 1 - 2 hr. Remove adults from the plate and let the progeny grow until the desired age.</li>
		</ol>
		</li>
		<li>Synchronization by bleaching
		<ol>
			<li>Collect an unsynchronized population of gravid adults and bleach them with alkaline hypochlorite solution (250 mM NaOH and 1:4 (v/v) dilution of commercial bleach in H2O). Wash the eggs twice (218 x g for 1 min) with M9 buffer47 (21 mMNa2HPO4&#183;7H2O, 22 mMKH2PO4, 86 mMNaCl, 1 mMMgSO4&#183;7H2O, add dH2O up to 1 L).</li>
			<li>Allow them to hatch in M9 buffer with gentle agitation O/N at 20 &#176;C. Worm development will arrest at the L1 stage in the absence of a food source, leaving a synchronized population. Transfer L1s onto fresh Nematode Growth Media (NGM) plates seeded with OP50 E. coli bacteria and let the progeny develop until the desired age47.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare 2% agarose pads (in H2O) on a microscope slide as described50.
	<ol>
		<li>Prepare two microscope slides with labeling tape placed over their entire length to be used as spacers. Place a third microscope slide between them.</li>
		<li>Dissolve 2% agarose in H2O and pipette one drop onto the clean slide.</li>
		<li>Place a fourth slide perpendicular to the three other slides on top of the agar drop. Gently press it down to flatten the pad to the same thickness as the labeling tape.</li>
		<li>Let it dry for 1 min before removing the spacers and gently pulling the slides apart. The agar pad will stick to one of them.</li>
	</ol>
	</li>
	<li>Pipette ~10 &#181;l anesthetic (2 mM levamisole in M9 buffer) to the pad and transfer ~10 animals using a platinum wire pick. Cover with a cover slip (~22 x 22 mm) and take images within 1 hr.</li>
	<li>Alternatively, to acquire movies over a longer period of time or to further reduce the possibility of any movement of the animals, use a combination of anesthetic and bead immobilization51.
	<ol>
		<li>Prepare 10% agarose pads (in M9 buffer) as described51 and add worms to 3 &#181;l nanosphere size standards solution (polystyrene beads, 100 nm) plus 3 &#181;l anesthetic (4 mM levamisole in M9 buffer). Cover gently with a cover slip. To avoid desiccation, seal the cover slip with VALAP (mixture of equal amounts of Vaseline, lanolin, and paraffin wax).</li>
	</ol>
	</li>
	<li>Image immobilized worms using a confocal microscope. 
	NOTE: Results are obtained using a Spinning Disc AF Confocal Microscope equipped with an EM-CCD camera and a Microscopy Automation &#38; Image Analysis Software such as MetaMorph and outline the specifics below, but other comparable confocal imaging systems can also be used.
	<ol>
		<li>Use the 63X or 100X/1.4NA oil objective and place the microscope slide containing worms into the microscope slide holder.</li>
		<li>Open the software. Adjust laser power and filters for mRFP imaging. Use the 561 nm laser at 10% power and emission filter &#62;600 nm.</li>
		<li>Open &#8220;MultiDimensional Acquisition&#8221;. Under the &#8220;Main&#8221; tab, check the boxes for &#8220;Timelapse&#8221; and &#8220;Run Journals&#8221; (Hardware Auto Focus: off).</li>
		<li>Under the &#8220;Saving&#8221; tab select or create the directory folder where the files should be saved. Assign a name to the file.</li>
		<li>Under the &#8220;Wave Length&#8221; tab select the appropriate illumination and adjust exposure and gain. Use &#8220;YokoQuad Red&#8221; (or an equivalent illumination setting for mRFP imaging) with an exposure between 100 and 300 msec and a camera gain between 100 and 300, depending on the individual sample (assessed by using the live image).</li>
		<li>Under the &#8220;Timelapse&#8221; tab select &#8220;Number of time points&#8221; = 301, &#8220;Duration&#8221; = 5 min and &#8220;Time/Interval&#8221; = 1 sec.</li>
		<li>Under the &#8220;Journals&#8221; tab select Journal: &#8220;AFC SET Z HOLD&#8221; and &#8220;AFC Return to Z HOLD&#8221;, Type: &#8220;Special&#8221; (twice), Initial Point: &#8220;Start of time point&#8221; and &#8220;End of time point&#8221;. This autofocus option is important to image the same section over a longer period of time.</li>
		<li>When the setup is done, press &#8220;Acquire&#8221;. The timelapse video is safed as separate TIFF files.</li>
		<li>Open all .tiff files of a given time series in ImageJ. Go to &#8220;Image&#8221; &#8594;&#8220;Stacks&#8221;&#8594;&#8220;Images to Stacks&#8221;. Optionally, adjust brightness and contrast, add size bar, etc. Export the movie under &#8220;File&#8221;&#8594;&#8220;Save as&#8221;&#8594;&#8220;AVI&#8230;&#8221; (for an example, see Video 1 and 2 corresponding to Figure 1).</li>
	</ol>
	</li>
</ol>
2. Using Folding Sensors and Stress Reporters to Investigate Cell Autonomous and Non cell Autonomous Affects on Proteostasis and Toxicity
<ol>
	<li>Generate transgenic lines that coexpress a folding sensor or stress reporter together with the prion-like protein. For methods on how to establish crosses or generate transgenic lines, see48,49,52. See Table 1 for a list of C. elegans strains that can be used.</li>
	<li>Prepare synchronized populations of transgenic animals and grow them until the desired age as described above (section 1.2)50.</li>
	<li>Using a stereomicroscope, examine the respective phenotype of the folding sensor or stress reporter.
	<ol>
		<li>For the folding sensor, determine the number of animals harboring aggregates on each day after synchronization (for an example, see Figure 2E and F).</li>
		<li>For the stress reporter, test if the coexpression of the prion-like protein results in an increased fluorescence of the reporter (for an example, see Figure 3).</li>
	</ol>
	</li>
</ol>
3. Genome-wide Screen for Suppression of Prion-induced Toxicity in C. elegans
<img alt="Figure 4" src="/files/ftp_upload/52321/52321fig4highres.jpg" /> 
Figure 4.&#160;Schematic representation of the RNAi screening protocol. See protocol section 3 for a detailed description of the individual steps.
<ol>
	<li>Synchronization of C. elegans worms and duplication of RNAi library
	<ol>
		<li>For the RNAi screen, use the Ahringer RNAi library (or the Vidal RNAi library)53,54. Rearray the RNAi library from the original 384 well format into 96 well plates by filling the plates with 100 &#181;l LB-amp media (50 &#956;g/ml Ampicillin in LB) supplemented with 10% glycerol and inoculate using a 96-pin replicator. Grow at 37 &#176;C O/N with agitation and store at -80 &#176;C.</li>
		<li>Maintain C. elegans WT and transgenic lines at 20 &#176;C on NGM plates seeded with OP50 E. coli bacteria according to standard methods47.</li>
		<li>Synchronize prion domain transgenic and WT (control) nematodes by bleaching (see section 1.2).</li>
		<li>On the same day that the worms are bleached, prepare LB media supplemented with 50 &#956;g/ml ampicillin. Use an automated reagent dispenser to dispense (or pipette manually) 65 &#956;l of the LB-amp media into each well of a 96 well plate.</li>
		<li>Remove 96 well Ahringer RNAi library plates from -80 &#176;C and bring them to a sterile hood lined with paper towels. Immediately remove the seal-tape by holding the plate upside down while plates are still frozen, being careful to avoid contamination from ice on the outside of the plates. Reinvert the plates and let them thaw for about 30 min.</li>
		<li>Dip one sterile 96 pin replicator into one RNAi library plate, and then into one fresh LB-amp plate. Use a fresh sterile replicator for each plate. Seal all plates with adhesive foil tape and return the library plates to -80 &#176;C. The replicators can be soaked in bleach, rinsed, and autoclaved to be used again.</li>
		<li>Allow duplicated RNAi plates to grow O/N in an incubator set to 37 &#176;C and 300 rpm. To use HT115 E. coli bacteria harboring the empty vector (L4440) as a control, grow it separately in a culture tube and then pipette 65 &#956;l of the culture with a multichannel pipette into one quarter of two 96 well plates.</li>
	</ol>
	</li>
	<li>Induction of bacterial dsRNA production and addition of worms
	<ol>
		<li>Dilute Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) to a 5 mM concentration in ddH2O. Use an automated workstation with a multichannel head to dispense (or pipet manually) 10 &#956;l of diluted IPTG into each well of the RNAi bacteria and the control plates. Reseal and place the plates back into the incubator. Let them shake for 3 hr at 37 &#176;C.</li>
		<li>While bacteria are shaking, prepare the worms. Mix the M9/worm suspension well, and pipette a small sample (~5 - 10 &#181;l) to a glass microscope slide. Count the number of worms under a stereomicroscope and calculate the number of worms per &#956;l.</li>
		<li>Using sterile technique, prepare a solution of supplemented M9 (M9+) with the following final concentrations: 0.20 mg/ml IPTG, 8.0 &#956;g/ml Cholesterol, 50 &#956;g/ml Ampicillin, 9.6 &#956;g/ml Tetracycline, 0.0835 &#956;g/ml Fungizone, and 15 worms per 50 &#956;l.
		<ol>
			<li>Make separate solutions for prion domain transgenic and WT worms. The final volume of M9+ worm solution needed will depend on the number of RNAi plates copied (see below), plus ~30 ml of dead volume that will remain in the reservoir, if an automated dispenser is used.</li>
		</ol>
		</li>
		<li>After the 3 hr induction of bacterial dsRNA production, take the plates out of the incubator and let them cool to RT (~30 min) to avoid heat stressing the animals.</li>
		<li>Dispense 50 &#956;l M9+ prion domain transgenic worm solution to each well of the RNAi plates and one of the empty vector control plates. Make sure to mix the worm solution before each step as the animals tend to sediment to the bottom of the reservoir.</li>
		<li>Dispense WT worms into the second control plate. Leave the plates unsealed to allow aeration. To keep the liquid culture from evaporating, stack 4 - 5 plates together and wrap with a damp paper towel and aluminum foil. Incubate at 200 rpm at 20 &#176;C for 4 days.</li>
	</ol>
	</li>
	<li>Scoring 
	NOTE: After 4 days in the incubator, the worms will be at the second day of adulthood and are ready to screen. Let the animals adjust to non shaking conditions for 30 min before screening to ensure undisturbed thrashing.
	<ol>
		<li>Using a Falcon 4M60 camera connected to a computer with a monitor, screen visually for increased thrashing as compared with control treated prion domain transgenic animals.</li>
		<li>Compile a list of preliminary hits to confirm using wrMTrck (see next section).</li>
	</ol>
	</li>
</ol>
4. Confirmation of Preliminary Screen Hits
<ol>
	<li>Motility assay on solid plates
	<ol>
		<li>Prepare plates with NGM supplemented with 100 &#181;g/ml ampicillin, 12.5 &#181;g/ml tetracycline and 1 mM IPTG, according to standard methods47. If possible, use a plate-pouring machine to assure all plates have the same height of media, which will allow for a more streamlined video acquisition process.</li>
		<li>Grow the different RNAi clones in ~1 ml LB + 50 &#181;g/ml ampicillin, O/N at 37 &#176;C and 250 rpm.</li>
		<li>The next day, induce the expression of the dsRNA with 1 mM IPTG for 3 hr. Seed the plates with 150 &#181;l of each RNAi bacterial clone, spread into a thin layer. Let the bacteria dry for 2 days at RT in the dark. Prepare 3 plates per RNAi clone.</li>
		<li>Synchronize the worm population by bleaching according to standard methods50 (see section 1.2), and let the eggs hatch O/N&#160;in M9 media.</li>
		<li>Take a sample of the worm suspension and determine the amount of nematodes per &#181;l at the stereomicroscope. Then, pipette the proper volume of M9 plus worms into each experimental plate so that it contains 25 - 30 L1 worms. Grow the nematodes at 20 &#176;C for 4 days until the worms reach day 2 of adulthood.</li>
	</ol>
	</li>
	<li>Quantitative analysis of worm motility with the wrMTrck plugin for ImageJ 
	NOTE: The videos were recorded using a stereomicroscope at 10X magnification with a Hamamatsu Orca-R2 digital camera C10600-10B and the Hamamatsu Simple PCI imaging software.
	<ol>
		<li>Turn on the camera and the Simple PCI imaging software. Click on &#8220;Live&#8221; to allow for adjustment of the imaging conditions.</li>
		<li>Set up the video conditions as follows: Gain = 0; Light Mode = High; Speed Index = 1; Binning = 2. Click &#8220;Auto Expose&#8221; and then adjust the lighting conditions, moving the microscope mirrors and the brightness and contrast dials. 
		NOTE: The video needs to have a high contrast without being overexposed, so that the animals appear as black shapes on a bright background.</li>
		<li>Click on &#8220;Time Scan&#8221; and choose a folder and a file name. Set &#8220;Field Delay&#8221; to 20 msec and &#8220;Stop at Time&#8221; to 30 sec. Press &#8220;Live Review&#8221; in order to choose the area of the plate to record (where most worms are). Tap the plate 3 or 4 times on the stage, quickly confirm its position in the field of view and press &#8220;Start&#8221;.</li>
		<li>After the movie finishes recording, right click on the image and choose &#8220;Export Montage Sequence&#8221; to export the movie from a .cxd format to an .avi.</li>
	</ol>
	</li>
	<li>Analyzing the motility videos
	<ol>
		<li>Open the ImageJ software, go to the &#8220;Plugins&#8221; tab, then &#8220;wrmtrck&#8221; and select &#8220;wrMTrck Batch&#8221;. Select the directory containing all the files to be analyzed.</li>
		<li>In the main input window of wrMTrck_Batch, load the input values as detailed in Figure 5C. Explanation for each of the parameters can be found in the instructions that accompany the plugin. Click &#8220;OK&#8221; and let the movement analysis run.</li>
		<li>To curate the results and confirm that all detected tracks are from actual C. elegans and eliminate artifacts, open each of the .txt files created for each movie and copy the information to a data analysis software file. Open the &#8220;*_labels.zip&#8221; files created and run the resulting &#8220;*_labels.tif&#8221; to manually check and eliminate false worm tracks.</li>
	</ol>
	</li>
</ol>

The authors declare no competing financial interests.

The methods described here help to illustrate spreading and the complex cell autonomous and non cell autonomous toxicity of prion-like proteins. We recently discovered that an aggregation-prone cytosolic prion domain is taken up into membrane-bound vesicles in an autophagy related process. A specific subset of these vesicles transports the prion domain within and between cells and tissues40. The key to monitor their movement in the living animal is that the protein has to be tagged with mRFP, because only mRFP...

Many neurodegenerative diseases, including Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), and transmissible spongiform encephalopathies (TSEs), are associated with aggregation-prone proteins and are hence collectively known as protein misfolding disorders (PMDs). TSEs or prion diseases constitute a unique class of PMDs in that they can be infectious in both humans and animals1. At the molecular level, prions replicate by recruiting and converting monomeric &#945;-helix-rich host-encoded cellular PrP (PrPC) into the pathological &#946;-sheet-rich PrPSc conformation2,3. Self-propagating protein aggregates have been also identified in fungi, which share important characteristics with mammalian prions4,5. Additionally, mammalian prions are capable of moving from cell-to-cell and infect na&#239;ve cells6,7.
While PMDs other than TSEs are not infectious, they share a common pathogenic principle with prion diseases8,9. Although the proteins linked to each of the PMDs are not related in structure or function, they all form aggregates via a crystallization-like process called nucleated or seeded polymerization; moreover proteinaceous seeds grow by recruiting their soluble isoforms2,10,11. The efficiency to self-propagate varies in vivo, depending on the intrinsic properties of the protein, which together with additional cellular factors such as molecular chaperones ultimately determine rates of aggregate nucleation, seeding, fragmentation and spreading12-15. Hence, there must exist a fine balance among these factors that allows efficient propagation of protein aggregation. This might also explain why only some amyloidogenic aggregates harbor the characteristics of a prion, and thus not all PMDs are infectious. Prions seem to represent &#8216;top-performers&#8217; of a wide spectrum of self-replicating proteinaceous aggregates, which makes them a powerful tool to study PMDs8,13.
Intriguingly, the toxicity associated with disease-related aggregates often has a non cell autonomous component16,17. This means that they affect neighboring cells that do not express the corresponding gene, in contrast to a strictly cell autonomous effect, which implies that only the cells expressing the gene exhibit the specific phenotype. This was compellingly demonstrated by tissue-specific expression or knock down of the respective proteins in numerous models of neurodegenerative diseases18-26. Various mechanisms have been suggested as a basis for this non-cell autonomous toxicity in PMDs, including diminished nutrient supply, imbalance in neuronal signaling, glutamate excitotoxicity, and neuroinflammation16,27,28. In addition, a prion-like movement of disease-linked aggregates between cells might contribute to this aspect29,30. Increasing evidence suggests that protein inclusions other than prions can transmit from cell-to-cell, which may explain the characteristic spreading of pathology observed in many PMDs30-36. However, it has yet to be determined whether there is a clear causal link between intercellular movement of disease proteins and the toxic effect on neighboring cells. Therefore, a better understanding of the cellular pathways that underlie cell-to-cell transmission and non cell autonomous toxicity is necessary and essential for the development of novel therapeutics. However, many aspects of prion-like spreading and cellular factors that influence cell-to-cell transmission of misfolded proteins in metazoans are not well understood, in particular at the organismal level.
The nematode Caenorhabditis elegans has several advantages that provide the potential to discover new facets of prion-like spreading in metazoans17. It is transparent, allowing for in vivo tracking of fluorescently tagged proteins in the living organism. Furthermore, many cellular and physiological processes affected by disease are conserved from worms to human, and C. elegans is also amenable to a wide variety of genetic manipulations and molecular and biochemical analyses37-39. Exactly 959 somatic cells make up the adult hermaphrodite with a simple body plan that still has several distinct tissue types, including muscle, neurons and intestine.
To establish a new prion model in C. elegans, we chose to exogenously express the well characterized glutamine/asparagine (Q/N)-rich prion domain NM of the cytosolic yeast prion protein Sup35, since there are no known endogenous prion proteins in worms4,40. Yeast prions have been invaluable in elucidating basic mechanisms of prion replication41-44. Furthermore, NM is the first cytosolic prion-like protein that has been shown to recapitulate the full life cycle of a prion in mammalian cell culture45,46. Likewise, when expressed in C. elegans, the Sup35 prion domain adopted remarkably well to the different requirements for propagation in metazoan cells compared to yeast cells and exhibited key features of prion biology40. NM aggregation was associated with a profound toxic phenotype, including the disruption of mitochondrial integrity and appearance of various autophagy related vesicles on the cellular level, as well as embryonic and larval arrest, developmental delay, and a widespread disturbance of the protein folding environment on the organismal level. Strikingly, the prion domain exhibits cell autonomous and non cell autonomous toxicity, affecting neighboring tissues in which the transgene was not expressed. Furthermore, the vesicular transport of the prion domain within and between cells is monitored real time in vivo40.
Here we describe how to examine prion-like dissemination in C. elegans. We will explain how to monitor the intra- and intercellular transport of vesicles containing the prion domain using time-lapse fluorescence microscopy. We will emphasize the use of tissue-specific folding sensors and ubiquitously expressed stress reporters to evaluate cell autonomous and non cell autonomous effects on cellular fitness. Finally, we will describe the procedure of a recently performed genome wide RNA interference (RNAi) screen to identify new modifiers of prion-induced toxicity. In combination, these methods can help to tease apart genetic pathways involved in the intercellular movement of proteins and their non cell autonomous toxicity.

<table border="0" cellpadding="0" cellspacing="0" width="665">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Reagent</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Nanosphere size standards 100 nm</td>
			<td style="width:107px;">ThermoScientific</td>
			<td style="width:92px;">3100A</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Levamisole</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:92px;">L-9756</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">IPTG</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:92px;">15502-10G</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">Ahringer RNAi library</td>
			<td style="width:107px;">Source BioScience LifeSciences&#160;</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.lifesciences.sourcebioscience 
			.com/clone-products/non-mammalian/c-elegans/c-elegans-rnai-library/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Equipment</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">Sorvall Legend XTR Refrigerated Centrifuge, 120VAC</td>
			<td style="width:107px;">ThermoScientific</td>
			<td style="width:92px;">75004521</td>
			<td style="width:255px;">http://www.coleparmer.com/Product/Thermo_Scientific_Sorvall_Legend_ 
			XTR_Refrigerated_Centrifuge_120 
			VAC/EW-17707-60</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">96 pin replicator&#160;</td>
			<td style="width:107px;">Scionomix&#160;&#160;</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.scinomix.com/all-products/96-pin-replicator/</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">HiGro high-capacity, incubating shaker&#160;</td>
			<td style="width:107px;">Digilab</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.digilabglobal.com/higro</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">Multidrop Combi Reagent Dispenser&#160;</td>
			<td style="width:107px;">Titertrek</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://groups.molbiosci.northwestern.edu/hta/titertek.htm</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">Biomek FX AP96 Automated Workstation&#160;</td>
			<td style="width:107px;">Beckman Coulter</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://groups.molbiosci.northwestern.edu/hta/biomek_multi.htm</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:211px;">Innova44 shaker</td>
			<td style="width:107px;">New Brunswick</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.eppendorf.com/int///index.php?sitemap=2.3&#38;pb=d78efbc05310ec 
			04&#38;action=products&#38;contentid=1&#38; 
			catalognode=83389</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:211px;">M205 FA&#160;</td>
			<td style="width:107px;">Leica</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.leica-microsystems.com/de/produkte/stereomikroskope-makroskope/fluoreszenz/details/product/leica-m205-fa/</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">ORCA-R2 C10600-10BDigital CCD camera</td>
			<td style="width:107px;">Hamamatsu</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.hamamatsu.com/jp/en/community/life_science_camera/product/search/C10600-10B/index.html</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:211px;">Spinning Disc AF Confocal Microscope&#160;</td>
			<td style="width:107px;">Leica</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.leica-microsystems.com/products/light-microscopes/life-science-research/fluorescence-microscopes/details/product/leica-sd-af/</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">Falcon 4M60 camera&#160;</td>
			<td style="width:107px;">Teledyne Dalsa&#160;</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.teledynedalsa.com/imaging/products/cameras/area-scan/falcon/PT-41-04M60/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Software</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">MetaMorph Microscopy Automation &#38; Image Analysis Software</td>
			<td style="width:107px;">Molecular Devices</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.moleculardevices.com/products/software/meta-imaging-series/metamorph.html</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">Hamamatsu SimplePCI Image Analysis Software</td>
			<td style="width:107px;">Meyer Instruments</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://meyerinst.com/imaging-software/hamamatsu/index.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">ImageJ</td>
			<td style="width:107px;">NIH</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://rsbweb.nih.gov/ij/download.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">wrMTrck plugin for ImageJ</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.phage.dk/plugins/wrmtrck.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">C. elegans strains</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">N2 (WT)</td>
			<td style="width:107px;">Caenorhabditis Genetics Center (CGC)</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.cgc.cbs.umn.edu/strain.php?id=10570</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">AM815&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; rmIs323[myo-3p::sup35(r2e2)::rfp]</td>
			<td style="width:107px;">Morimoto lab</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">available from our laboratory&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">See table 1 for a source for folding sensor and stress reporter strains</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

investigating the spreading and toxicity of prion-like proteins using the metazoan model organism c. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Monitoring intercellular spreading of prion-like proteins by in vivo time-lapse imaging
Transgenic C. elegans lines expressing the prion domain are particularly well suited for the analysis of certain aspects of prion-like proteins, e.g., cell-to-cell transmission and non cell autonomous toxicity. The transparency of the animals enables tracking of fluorescently tagged proteins from within the living organism at ever...

Watch this Scientific Journal Video about Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans at JoVE.com

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

investigating-spreading-toxicity-prion-like-proteins-using-metazoan

Research

JoVE Journal

Biology

15.8K Views.  Northwestern University. Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Video: Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.