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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 temperature.
<ol>
	<li>Generate transgenic lines of C. elegans expressing the prion-like protein, tagged with a monomeric red fluorescent protein (mRFP).</li>
	<li>Prepare synchronized populations by egg laying or bleaching according to standard methods.
	<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 an alkaline hypochlorite solution (250 mM sodium hydroxide (NaOH) and 1:4 (v/v) dilution of commercial bleach in water (H2O)). Wash the eggs twice (218 x g for 1 min) with M9 buffer (21 mM Na2HPO4&#183;7H2O (di-sodium hydrogen phosphate heptahydrate), 22 mM (potassium dihydrogen phosphate) KH2PO4, 86 mM sodium chloride (NaCl), 1 mM MgSO4&#183;7H2O (magnesium sulfate heptahydrate), add dH2O up to 1 L).</li>
			<li>Allow them to hatch in M9 buffer with gentle agitation overnight (O/N) at 20 &#176;C. Worm development will be arrested 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 age.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare 2% agarose pads (in H2O) on a microscope slide as described.
	<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 minute 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 immobilization.
	<ol>
		<li>Prepare 10% agarose pads (in M9 buffer) as described 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 coverslip with VALAP (a mixture of equal amounts of Vaseline, lanolin, and paraffin wax).</li>
	</ol>
	</li>
	<li>Image immobilized worms using a confocal microscope. 
	&#8203;NOTE: Results are obtained using a Spinning Disc AF Confocal Microscope equipped with an EM-CCD camera and Microscopy Automation &#38; Image Analysis Software such as MetaMorph 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 &#34;MultiDimensional Acquisition&#34;. Under the &#34;Main&#34; tab, check the boxes for &#34;Timelapse&#34; and &#34;Run Journals&#34; (Hardware Auto Focus: off).</li>
		<li>Under the &#34;Saving&#34; tab, select or create the directory folder where the files should be saved. Assign a name to the file.</li>
		<li>Under the &#34;Wave Length&#34; tab, select the appropriate illumination and adjust exposure and gain. Use &#34;YokoQuad Red&#34; (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 &#34;Timelapse&#34; tab, select &#34;Number of time points&#34; = 301, &#34;Duration&#34; = 5 min, and &#34;Time/Interval&#34; = 1 sec.</li>
		<li>Under the &#34;Journals&#34; tab, select Journal: &#34;AFC SET Z HOLD&#34; and &#34;AFC Return to Z HOLD&#34;, Type: &#34;Special&#34; (twice), Initial Point: &#34;Start of time point&#34; and &#34;End of time point&#34;. This autofocus option is important to image the same section over a longer period of time.</li>
		<li>When the setup is complete, press &#34;Acquire.&#34; The timelapse video is saved as separate TIFF files.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanosphere size standards 100 nm</td>
			<td>ThermoScientific</td>
			<td>3100A</td>
			<td></td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>Sigma</td>
			<td>L-9756</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend XTR Refrigerated Centrifuge, 120VAC</td>
			<td>ThermoScientific</td>
			<td>75004521</td>
			<td>http://www.coleparmer.com/Product/Thermo_Scientific_Sorvall_Legend_ 
			XTR_Refrigerated_Centrifuge_120 
			VAC/EW-17707-60</td>
		</tr>
		<tr>
			<td>96 pin replicator</td>
			<td>Scionomix</td>
			<td></td>
			<td>http://www.scinomix.com/all-products/96-pin-replicator/</td>
		</tr>
		<tr>
			<td>HiGro high-capacity, incubating shaker</td>
			<td>Digilab</td>
			<td></td>
			<td>http://www.digilabglobal.com/higro</td>
		</tr>
		<tr>
			<td>Multidrop Combi Reagent Dispenser</td>
			<td>Titertrek</td>
			<td></td>
			<td>http://groups.molbiosci.northwestern.edu/hta/titertek.htm</td>
		</tr>
		<tr>
			<td>Biomek FX AP96 Automated Workstation</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>http://groups.molbiosci.northwestern.edu/hta/biomek_multi.htm</td>
		</tr>
		<tr>
			<td>Innova44 shaker</td>
			<td>New Brunswick</td>
			<td></td>
			<td>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>
			<td>M205 FA</td>
			<td>Leica</td>
			<td></td>
			<td>http://www.leica-microsystems.com/de/produkte/stereomikroskope-makroskope/fluoreszenz/details/product/leica-m205-fa/</td>
		</tr>
		<tr>
			<td>ORCA-R2 C10600-10BDigital CCD camera</td>
			<td>Hamamatsu</td>
			<td></td>
			<td>http://www.hamamatsu.com/jp/en/community/life_science_camera/product/search/C10600-10B/index.html</td>
		</tr>
		<tr>
			<td>Spinning Disc AF Confocal Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>http://www.leica-microsystems.com/products/light-microscopes/life-science-research/fluorescence-microscopes/details/product/leica-sd-af/</td>
		</tr>
		<tr>
			<td>Falcon 4M60 camera</td>
			<td>Teledyne Dalsa</td>
			<td></td>
			<td>http://www.teledynedalsa.com/imaging/products/cameras/area-scan/falcon/PT-41-04M60/</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph Microscopy Automation &#38; Image Analysis Software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>http://www.moleculardevices.com/products/software/meta-imaging-series/metamorph.html</td>
		</tr>
		<tr>
			<td>Hamamatsu SimplePCI Image Analysis Software</td>
			<td>Meyer Instruments</td>
			<td></td>
			<td>http://meyerinst.com/imaging-software/hamamatsu/index.htm</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>http://rsbweb.nih.gov/ij/download.html</td>
		</tr>
		<tr>
			<td>wrMTrck plugin for ImageJ</td>
			<td></td>
			<td></td>
			<td>http://www.phage.dk/plugins/wrmtrck.html</td>
		</tr>
		<tr>
			<td>C. elegans strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2 (WT)</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td>http://www.cgc.cbs.umn.edu/strain.php?id=10570</td>
		</tr>
	</tbody>
</table>

time-lapse imaging to monitor the transcellular spreading of prion-like proteins in transgenic nematodes

Source: Nussbaum-Krammer, C. I., et al. <a href="https://app.jove.com/v/52321">Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans.</a> J. Vis. Exp. (2015)
This video demonstrates the procedure for in vivo imaging of C. elegans expressing prion-like proteins tagged with red fluorescent protein. The protocol involves immobilizing nematodes on an agarose pad for time-lapse confocal microscopy, revealing protein aggregation and transport within and between cells. This technique is crucial for studying protein aggregation related to neurodegenerative diseases.

Time-Lapse Imaging to Monitor the Transcellular Spreading of Prion-Like Proteins in Transgenic Nematodes

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 ...

Take transgenic nematodes that are maintained at the same developmental stage.
The body wall muscle cells of these nematodes express prion-like proteins, which are tagged with a red fluorescent protein and localized within acidic vesicles.
Prion-like proteins are misfolded, aggregating proteins that are associated with neurodegenerative diseases.
Next, transfer the nematodes onto an agarose pad containing polymeric nanoparticles and an anesthetic solution.
The combined effects of the nanoparticles and the anesthetic solution immobilize the nematodes on the agarose pad.
Place a coverslip over the agarose pad and seal it.
Now, position the agarose pad assembly with the nematodes under a confocal microscope and set the appropriate imaging parameters.&#160;
Acquire time-lapse images of the fluorescent-tagged proteins within the nematodes to monitor the vesicular transport of the prion-like proteins within and between the body wall muscle cells.

time-lapse-imaging-to-monitor-transcellular-spreading-prion-like

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

39 Views. מקור: Nussbaum-Krammer, C. I., et al. חקירת ההתפשטות והרעילות של חלבונים דמויי פריונים באמצעות אורגניזם המודל Metazoan C. elegans. J. Vis. Exp. (2015) סרטון זה מדגים את ההליך להדמיית in vivo של C. elegans המבטאים חלבונים דמויי פריונים המתויגים בחלבון פלואורסצנטי אדום. הפרוטוקול כולל השתקת נמטודות על משטח אגרוז ל...

Video: הדמיה בהילוך מהיר לניטור ההתפשטות הטרנסתאית של חלבונים דמויי פריונים בנמטודות טרנסגניות - Concept

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

בשנת הדמיה Vivo סידן בג. אלגיה שרירי הגוף

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

JoVE Journal

Biochemistry

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

ניטור-לתא שידור של אגרגטים חלבון פריאון-כמו בדרוזופילה Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations of bacteria move over large distances in characteristic tendril-shaped communities over the course of hours. Swarming is sensitive to several factors including medium moisture, humidity, and nutrient content. In addition, the collective stress response, which is observed in P. aeruginosa that are stressed by antibiotics or bacteriophage (phage), repels swarms from approaching the area containing the stress. The methods described here address how to control the critical factors that affect swarming. We introduce a simple method to monitor swarming dynamics and the collective stress response with high temporal resolution using a flatbed document scanner, and describe how to compile and perform a quantitative analysis of swarms. This simple and cost-effective method provides precise and well-controlled quantification of swarming and may be extended to other types of plate-based growth assays and bacterial species.

We detail a simple method to produce high-resolution time-lapse movies of Pseudomonas aeruginosa swarms that respond to bacteriophage (phage) and antibiotic stress using a flatbed document scanner. This procedure is a fast and simple method for monitoring swarming dynamics and may be adapted to study the motility and growth of other bacterial species.

זמן הדמיה של נחילים חיידקיים ואת תגובת הלחץ הקולקטיבי

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations ...

Time-Lapse Imaging to Monitor the Transcellular Spreading of Prion-Like Proteins in Transgenic Nematodes

Transcript

Time-Lapse Imaging to Monitor the Transcellular Spreading of Prion-Like Proteins in Transgenic Nematodes