Name: using single-worm data to quantify heterogeneity in caenorhabditis elegans-bacterial interactions
Uploaded: 2022-07-22T14:20:00
Description: Watch this Scientific Journal Video about Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions at JoVE.com

The authors would like to acknowledge H. Schulenberg and C. LaRock for their generous sharing of bacterial strains used in these experiments. This work was supported by funding from Emory University and NSF (PHY2014173).

Worms used in these experiments were obtained from the Caenorhabditis Genetic Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Bristol N2 is the wild-type. DAF-2/IGF mutants daf-16(mu86) I (CGC CF1038) and daf-2(e1370) III (CGC CB1370)&#160;are used to illustrate differences in intestinal bacterial load.
HT115(DE3) E. coli carrying the pos-1 RNAi vector is from the Ahringer library21. ...

<ol>
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Here data are presented on the advantages of single-worm quantification of bacterial load in C. elegans, along with a 96-well disruption protocol to allow the rapid and consistent acquisition of large data sets of this type. As compared with existing methods33, these protocols allow higher-throughput measurement of intestinal microbial communities in the worm.
This approach has plating as a rate-limiting step and is not truly &#34;high-throughput&#34;. Large-ob...

Heterogeneity in host-microbe associations is observed ubiquitously, and variation between individuals is increasingly recognized as a contributing factor in population-level processes from competition and coexistence1 to disease transmission2,3,4. In C. elegans, &#34;hidden heterogeneity&#34; within isogenic populations has been observed repeatedly, with sub-populations of individuals showing distinct phenotypes in heat shock response5,6, ageing, and lifespan7,8,9,10,11, and many other aspects of physiology and development12. Most analyses that attempt to identify sub-population structure provide evidence for two sub-populations in experimental populations of isogenic, synchronized worms5,7,8, though other data suggest the possibility of within-population distributions of traits rather than distinct groups7,12,13. Of relevance here, substantial heterogeneity in intestinal populations is observed even within isogenic populations of worms colonized from a shared source of microbes13,14,15,16, and this heterogeneity can be concealed by the batch digest measurements that are widely used17,18,19,20 for bacterial quantification in the worm.
This work presents data suggesting a need for greater reliance on single-worm measurements in host-microbe association, as well as protocols for increasing accuracy and throughput in single-worm disruption. These protocols are designed to facilitate mechanical disruption of large numbers of individual C. elegans for quantification of viable bacterial load, while providing better repeatability and lower effort per sample than pestle-based disruption of individual worms. A recommended gut-purging step, where worms are permitted to feed on heat-killed E. coli prior to the preparation for disruption, is included to minimize contributions from recently ingested and other transient (non-adhered) bacteria. These protocols include a cold-paralysis method for cleaning the cuticle with a low-concentration surface bleach treatment; surface bleaching can be used as a preparatory step in single-worm disruption or as a method for preparing live, externally germ-free worms. This surface-bleaching method is sufficient to remove a wide range of external microbes, and cold treatment provides an alternative to conventional levamisole-based paralysis; while levamisole will be preferred for cold-sensitive experiments, cold paralysis minimizes contributions to hazardous waste streams and allows rapid resumption of normal activity. While the full protocol describes a laboratory experiment where worms are colonized with known bacteria, the procedures for cleaning worms and single-worm disruption can readily be applied to worms isolated from wild samples or colonized in microcosm experiments. The protocols described here produces live bacteria extracted from the worm intestine, suitable for plating and quantification of colony forming units (CFUs) in individual worms; for sequencing-based intestinal community analysis, subsequent cell lysis and nucleic acid extraction steps should be added to these protocols.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well flat-bottom polypropylene plates, 300 uL</td>
			<td>Evergreen Labware</td>
			<td>290-8350-03F</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate sealing mat, silicon, square wells (AxyMat)</td>
			<td>Axygen</td>
			<td>AM-2ML-SQ</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates, 2 mL, square wells</td>
			<td>Axygen</td>
			<td>P-2ML-SQ-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well polypropylene plate lids</td>
			<td>Evergreen Labware</td>
			<td>290-8020-03L</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>443570050</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead mill adapter set for 96-well plates</td>
			<td>QIAGEN</td>
			<td>119900</td>
			<td>Adapter plates for use with two 96-well plates on the TissueLyser II</td>
		</tr>
		<tr>
			<td>Bead mill tissue homogenizer (TissueLyser II)</td>
			<td>QIAGEN</td>
			<td>85300</td>
			<td>Mechanical homogenizer for medium to high-throughput sample disruption</td>
		</tr>
		<tr>
			<td>BioSorter</td>
			<td>Union Biometrica</td>
			<td>By quotation</td>
			<td>Large object sorter equipped with a 250 micron focus for C. elegans</td>
		</tr>
		<tr>
			<td>Bleach, commercial, 8.25% sodium hypochlorite</td>
			<td>Clorox</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Breathe-Easy 96-well gas permeable sealing membrane</td>
			<td>Diversified Biotech</td>
			<td>BEM-1</td>
			<td>Multiwell plate gas permeable polyurethane membranes. Thin sealing film is permeable to O2, CO2, and water vapors and is UV transparent down to 300 nm. Sterile, 100/box.</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Fisher Scientific</td>
			<td>AC423525000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>VWR</td>
			<td>AAA11470-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid monohydrate</td>
			<td>Fisher Scientific</td>
			<td>AC124910010</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper (II) sulfate pentahydrate</td>
			<td>Fisher Scientific</td>
			<td>AC197722500</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 6765 LSE Mini Microcentrifuge</td>
			<td>Corning</td>
			<td>&#160;COR-6765</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium EDTA</td>
			<td>Fisher Scientific</td>
			<td>409971000</td>
			<td></td>
		</tr>
		<tr>
			<td>DL 1,4 Dithiothreitol, 99+%, for mol biology, DNAse, RNAse and Protease free, ACROS Organics</td>
			<td>Fisher Scientific</td>
			<td>327190010</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 1.5 mL microcentrifuge tubes, natural</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424R microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5406000640</td>
			<td>24-place refrigerated benchtop microcentrifuge</td>
		</tr>
		<tr>
			<td>Eppendorf 5810R centrifuge with rotor S-4-104</td>
			<td>Eppendorf</td>
			<td>22627040</td>
			<td>3L benchtop centrifuge with adaptors for 15-50 mL tubes and plates</td>
		</tr>
		<tr>
			<td>Eppendorf plate bucket (x2), for Rotor S-4-104</td>
			<td>Eppendorf</td>
			<td>22638930</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads, 2.7 mm</td>
			<td>Life Science Products</td>
			<td>LS-79127</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads, acid-washed, 425-600 &#181;m</td>
			<td>Sigma</td>
			<td>G877-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass plating beads</td>
			<td>VWR</td>
			<td>76005-124</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>VWR</td>
			<td>BDH7204-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (II) sulfate heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>423731000</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimble Kontes pellet pestle motor</td>
			<td>DWK Life Sciences</td>
			<td>749540-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimble Kontes polypropylene pellet pestles and microtubes, 0.5 mL</td>
			<td>DWK Life Sciences</td>
			<td>749520-0590</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMi8 motorized inverted microscope with motorized stage</td>
			<td>Leica</td>
			<td>11889113</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica LAS X Premium software</td>
			<td>Leica</td>
			<td>11640687</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>AC124900010</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese(II) chloride tetrahydrate</td>
			<td>VWR</td>
			<td>470301-706</td>
			<td></td>
		</tr>
		<tr>
			<td>PARAFILM M flexible laboratory sealing film</td>
			<td>Amcor</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Fisher Scientific</td>
			<td>BP1420-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, round, 10 cm</td>
			<td>VWR</td>
			<td>25384-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, round, 6 cm</td>
			<td>VWR</td>
			<td>25384-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, square, 10 x 10 cm</td>
			<td>VWR</td>
			<td>10799-140</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-buffered saline (1X PBS)</td>
			<td>Gold Bio</td>
			<td>P-271-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene autoclave tray, shallow</td>
			<td>Fisher Scientific</td>
			<td>13-361-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>AC134062500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP363-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Fisher Scientific</td>
			<td>BP362-1</td>
			<td></td>
		</tr>
		<tr>
			<td>R 4.1.3/RStudio 2022.02.0 build 443</td>
			<td>R Foundation</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Scoop-type laboratory spatula, metal</td>
			<td>VWR</td>
			<td>470149-438</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon carbide 36 grit</td>
			<td>MJR Tumblers</td>
			<td>n/a</td>
			<td>Black extra coarse silicon carbide grit. Available in 0.5-5 lb sizes from this vendor.</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP166-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>VWR</td>
			<td>BDH7247-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic anhydrous</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodum chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>AC419760010</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-potassium citrate monohydrate</td>
			<td>Fisher Scientific</td>
			<td>AC611755000</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc sulfate heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>AC205982500</td>
			<td></td>
		</tr>
	</tbody>
</table>

using single-worm data to quantify heterogeneity in caenorhabditis elegans-bacterial interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Bleach sterilization of live worms 
Surface-bleached worms are effectively free of external bacteria until motility returns and excretion resumes. Under the conditions used here, rapid extinction of bacteria in buffer is observed (Figure 1A-C, Supplementary Figure 2, Video 1) without disturbing the gut-associated bacteria in cold-paralyzed worms (Figure 1D-F, Video 2). Thes...

Watch this Scientific Journal Video about Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions at JoVE.com

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

This protocol demonstrates mechanical disruption of sea elegans. In a 96 well format allowing medium throughput quantification of bacterial load in individual worms. This technique increases the speed and uniformity of mechanical disruption as compared to Pasel based methods allowing researchers to easily produce larger data sets of high quality single worm measurements.This technique involves many moving parts and it's easy to lose your place. Make sure that you have all materials, labeled tubes, buffers and plates set up before beginning. Demonstrating the procedure will be Megan Taylor a research specialist lead from my laboratory.Begin by re suspending the worm sample in one milliliter of M nine TX zero one in a micro centrifuge tube. Collect the adult worms by centrifugation and discard the supernatant. Using the same centrifugation parameters.Rinse the worms twice with one milliliter of M9TX01 and once with one milliliter of M9 worm buffer, to minimize the external bacteria. Then, re suspend each sample in one milliliter of S medium plus two x heat killed OP50 in a culture tube. Incubate the tubes at 25 degrees Celsius for 20 to 30 minutes to pass the non-ad adhered bacteria from the gut.After incubation, rinse the purged worms twice with one milliliter of cold M9TX01 and discard the super natant. Put the tubes on ice for 10 minutes to paralyze the worms. Then add one milliliter of ice cold M nine worm buffer with unscented bleach to each tube.Incubate the tubes on ice for a minimum of 10 minutes to kill external bacteria. Discard the bleach buffer and return the tubes to the ice to prevent the worms from pumping. Next, add one milliliter of cold M9TX01 to each tube and centrifuge for five seconds.In a mini centrifuge. Return the tubes to ice and remove the supernatant. Repeat this step once.For chemical permeabilization of worm cuticle. Transfer the tubes containing the worms in 20 microliters of buffer to a room temperature tube rack. Then add 100 microliters of SDS/DTT solution to each warm sample.Incubate the tubes for up to eight minutes on the bench to partially break down the cuticle of the adult worms. At this point, the worms will die and settle at the bottom of the tube. After incubation, carefully discard the SDS/DTT supernatant.Then, add one milliliter of M9TX01 to each tube and centrifuge briefly to pellet the worms. Discard the supernatant and re suspend the worms in one milliliter M nine worm buffer with 0.1%Triton x-100. To prepare for mechanical disruption of worms, obtain a sterile two milliliter deep well 96 well plate and a silicone plate cover.Use a sterile scoop spatula to add a small amount of sterile 36 grid silicone carbide to each well such that the grid barely covers the bottom of the well. Add 180 microliters of M nine worm buffer to each well. Label the columns and rows and then cover the plate loosely with the silicone plate cover.Next, transfer the permeabilized worms to a small Petri dish with M9TX01 filled up to a depth of one centimeter. Using a dissecting microscope pipette out individual worms in 20 microliter volumes and transfer them to individual wells of the 96 well plate to eject any worms stuck to the pipette, aspirate 20 microliters of M9TX01 from a clear area of the Petri dish and release it back into the dish. Once all worms are transferred cover the 96 well plate with a sheet of flexible ceiling film with the paper backed side facing down onto the sample wells.Place the silicone ceiling mat lightly on top of the flexible ceiling film without pressing the cover down into the wells. Place the plate at four degrees Celsius for 30 to 60 minutes to prevent overheating during disruption. After chilling the plate press the silicone ceiling mat down firmly into the wells to seal them.Then secure plates in the tissue disruptor. Shake the plates for one minute at 30 hertz. Then rotate the plates by 180 degrees and repeat shaking for one minute.Tap the plates firmly on the bench two or three times to dislodge any grit from the flexible ceiling film. Centrifuge the plate set 2, 400 times G for two minutes to collect the samples at the bottom of the wells. Then remove the silicone lid and pull off the ceiling film.To prepare tenfold serial dilution of the worm digests, fill 180 microliters of one X PBS in rows B to D of a 96 well plate. Using a multi-well pipetter set to 200 microliters, slowly mix the worm digest by pipetting. Transfer the maximum amount of the liquid to row A of the 96 well plate containing PBS.Using a multichannel pipette, remove 20 microliters from the top row and dispense into row B.Followed by mixing. Repeat this step for row B to C and then row C to D to get a 1000 fold dilution For bacterial quantification of mono colonized worms. Plate 10 to 20 microliters of each dilution on solid agar plates.For multi-species colonization, plate 100 microliters of each dilution on 10 centimeter agar plates. Using this protocol, effective external sanitization was observed after surface bleaching of cold paralyzed worms as seen by the decline of external bacteria in buffer. Without affecting the gut associated bacteria.Manual disruption of worms resulted in more heterogeneity. Silicone carbide grit was ideal for disruption with or without Tri Andex. In the 96 well method, large glass beads were not suitable for the 96 well technique.Small glass beads produced consistent results but clogged the pipe at tip. Worms colonized on the same pool of bacteria showed high variation in intestinal load when observed for individual bacteria and multi-species bacterial communities. Worms colonized with bacteria expressing GFP, showed heterogeneity in individual worm measurements.For this GFP expressing bacterial strain comparing the colonization of wild type Bristol Len two worms. To DAF two IGF mutants showed that the DAF 16 mutant supports larger populations of bacteria, while DAF two is resistant to colonization. This heterogeneity was characteristic and consistent over different runs of the same experiment.Re sampling of data to simulate batch digests was found to alter the distribution as compared to individual worm data. The batch extrapolated CFU per worm was centered around the arithmetic mean of the individual data leading to loss of biological variation. Instead of proceeding to digest live surface bleach and rinse worms can be used for further experiments.Movement will resume quickly once worms are removed from the cold.

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Research

JoVE Journal

Biology

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

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 ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful in cases of attenuated, oligo- or mono-symptomatic forms of CF usually diagnosed later in life, and of CF-related disorders such as congenital bilateral absence of vas deferens, idiopathic chronic pancreatitis, allergic bronchopulmonary aspergillosis, and bronchiectasis. In both clinical and preclinical settings, the test has been used as a biomarker to quantify responses to targeted therapeutic strategies for CF. Adapting the test to a mouse is challenging and can entail an associated mortality. This paper describes the adequate depth of anesthesia required to maintain a nasal catheter in situ for continuous perfusion. It lists measures to avoid broncho-aspiration of solutions perfused in the nose. It also describes the animal care at the end of the test, including administration of a combination of antidotes of the anesthetic drugs, leading to rapidly reversing the anesthesia with full recovery of the animals. Representative data obtained from a CF and a wild-type mouse show that the test discriminates between CF and non-CF. Altogether, the protocol described here allows reliable measurements of the functional status of trans-epithelial chloride and sodium transporters in spontaneously breathing mice, as well as multiple tests in the same animal while reducing test-related mortality.

Here, we present a protocol to measure nasal potential difference in mice. The test quantifies the function of transmembrane ion transporters such as the cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel. It is valuable to evaluate the efficacy of novel therapies for cystic fibrosis.

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

Collecting Hemolymph from Small Arthropods

A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods

Arthropods are known to transmit a variety of viruses of medical and agricultural importance through their hemolymph, which is essential for virus transmission. Hemolymph collection is the basic technology for studying virus-vector interactions. Here, we describe a novel and simple method for the quantitative collection of hemolymph from small arthropods using Laodelphax striatellus (the small brown planthopper, SBPH) as a research model, as this arthropod is the main vector of rice stripe virus (RSV). In this protocol, the process begins by gently pinching off one leg of the frozen arthropod with fine-tipped tweezers and pressing the hemolymph out of the wound. Then, a simple micropipette consisting of a capillary and a pipette bulb is used to collect the transudative hemolymph from the wound according to the principle of capillary forces. Finally, the collected hemolymph can be dissolved into a specific buffer for further study. This new method for collecting hemolymph from small arthropods is a useful and efficient tool for further research on arboviruses and vector-virus interactions.

Author Spotlight: A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods  

We describe a method to collect quantifiable hemolymph efficiently from small arthropods for subsequent analysis.