This work was supported by the National Institutes of Health (1RO3AG069056-01) and the Infectious Diseases Society of America funding to DMC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank the members of the Czyz Lab for proofreading the manuscript. Cartoon figures were generated using BioRender paid license.

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The authors have declared that no conflicts of interest exist.

The described protocol outlines procedures for C. elegans culturing, imaging, and image processing that incorporates CellProfiler, an open-source image analysis software. The representative results demonstrate reproducibility, reduction of bias, and scalability. This standardized procedure will improve screening strategies employed with large bacterial, genomic, or drug libraries. While other automated C. elegans methods of object detection exists, the described technique offers a standardized, higher-throughput pipeline that integrates culturing, image acquisition, and analysis.
Several variations of worm cultivation had to be tested to optimize the protocol described herein. Initially, worms were transferred to sample bacteria immediately post age-synchronization (L1 stage). However, such an approach resulted in a population of worms with variable sizes, even among worms within the same well. C. elegans are known for pathogen avoidance19, which could have contributed to the observed variability in size and ultimately affect downstream imaging-worm detection, in particular. To eliminate such variability, the entire NGM area in each well was covered with test bacteria. Furthermore, worms were fed E. coli OP50 and allowed to fully develop into young adults for 48 h at 25 &#176;C. Allowing worms to reach adulthood on E. coli OP50 prior to transferring them onto test bacteria resulted in more consistent body size. Additionally, overcrowding and rapid food depletion by progeny were eliminated by supplementing NGM agar with FUDR. The implementation of FUDR removed progeny and enhanced automated worm identification, which was obscured by progeny mixing in with the parental population. However, it is important to be cautious and use appropriate controls when utilizing FUDR, as the compound is known to affect C. elegans proteostasis and lifespan20,21. Under the conditions described in this protocol, FUDR did not affect intestinal polyQ aggregation (Supplemental Figure 5); therefore, its utilization was suitable and beneficial to the described method.
Freezing samples prior to imaging turned out to be a critical step in the successful employment of the pipeline. The aggregate counts prior to freezing were significantly higher than manual counts (Figure 3B). Keeping worms at -20 &#176;C for 18-48 h prior to imaging reduced background fluorescence and ultimately improved aggregate detection (Figure 3A). The effects of freezing on aggregate detection have only been investigated for polyQ and should not be generalized to other models without further investigation of such effects.
Despite all the conditions being kept the same, it was observed that the average number of aggregates per worm could vary between different runs, while the ratio between the number of aggregates in animals colonized with OP50 versus MPAO1 remained consistent (Figure 6, Supplemental Figure 2, Supplemental Figure 5). Therefore, it is essential to always include E. coli OP50 control, or any additional suitable reference controls, in every run. Such variability in aggregate counts between experiments could be influenced by environmental conditions (temperature, humidity)22,23 or genetic background8. In fact, it was observed that after prolonged culture, intestinal fluorescence drastically decreased or was completely lost, which required thawing a new strain from frozen stock. The observed decrease in fluorescence could be a result of genetic changes that suppress toxic transgenes, such as those expressing polyQ. Nonetheless, the exceptional reproducibility of the results observed between different experimenters (Supplemental Figure 2), between biological replicates (Figure 5), and within the same sample (Supplemental Figure 3) emphasize on the strength of this approach.
Numerous reports have employed intestinal polyQ to study proteostasis9,11,12,13,24,25. However, a direct comparison between results cannot be made due to variability between experimental approaches and readout methods. Nonetheless, a few results from previously published data are recapitulated by the automated quantification described herein, including bacterial induction of aggregation9,13 and a comparable number of aggregates11. Collectively, the described pipeline offers a valuable tool to study proteostasis.
The method described herein has some inherent challenges. For example, it requires sufficient time to master all components of this protocol, which is especially true for section 8 of the protocol, which requires familiarity with the assay to determine if the images acquired are appropriate for pipeline analysis. Deviations from the image acquisition settings used in this protocol are possible; however, modification of the settings and worm training set will likely be required. This pipeline can distinguish aggregates of various sizes and those that are touching, which limits the &#34;blending&#34; of aggregates and ultimately increases the detection sensitivity. However, issues may arise when attempting to identify large aggregates that exceed the accepted size range, as expanding the upper size threshold may lead to errors caused by poor identification, such as the inability to differentiate aggregates that are touching. A balance between accuracy, size, and intensity must be found prior to image analysis. The efficiency of aggregate identification could be further improved by incorporating machine learning to create a neural network capable of enhancing aggregate detection. Such improvements are currently being explored and will greatly assist in addressing current issues such as the detection of aggregates that lie on different focal planes or that have abnormal shapes.
One notable weakness of the described method is the variability in automated aggregate counts, as they are not always recapitulated by manual counts in worms fed different bacterial strains. For example, based on automated counts, worms fed P. aeruginosa mutant 53 (M53) had significantly fewer aggregates compared to the wild-type strain (MPAO1) (Figure 7); however, confirmation of the hit showed no significant difference (Supplemental Figure 4). In general, high-throughput drug screens have a high rate of false-positive hit detection, and the described method is no exception26. Thus, it is a critical part of the protocol to confirm all potential hits.
While this protocol was optimized to fit a screening strategy to identify bacteria that affect host proteostasis, each step can be further modified to test the effect of genomic RNAi libraries, small molecules, or other conditions. Additional modifications can be made at each step to suit the requirements of a specific screening strategy. Furthermore, this technique provides a level of flexibility that allows for the optimization of each step to suit a specific model. For example, this approach can be extended to polyQ aggregation in other tissues or extracting other features detected in images such as monitoring gene expression using inducible fluorescent reporters (e.g., heat shock genes), assessing subcellular localization of proteins (e.g., nuclear localization of DAF-16), studying aggregation in other disease models (A&#946;1-42, &#945;-synuclein, TDP-43, etc.) or assessing physiological phenotypes, such as worm size.

Age-dependent neurodegenerative protein conformational diseases (PCDs) such as Alzheimer&#39;s, Parkinson&#39;s, and Huntington&#39;s diseases, or amyotrophic lateral sclerosis, are characterized by protein misfolding that leads to aggregation, cell death, and tissue degeneration1. While protein misfolding is recognized as the culprit, the etiology of these diseases is not clear. As such, the development of effective therapies has been hindered by the lack of knowledge regarding the factors and conditions that contribute to disease onset and progression. Recent studies suggest that changes in the microbiome influence the onset, progression, and severity of PCDs2,3,4. However, the complexity of the human, or even murine, microbiome makes it difficult to conduct studies that would reveal the exact influence of microbes on their host. Therefore, simpler organisms, such as Caenorhabditis elegans, are often used as a discovery tool5,6,7,8. Recent studies have employed C. elegans to investigate the effect of bacteria on host proteostasis and disease pathogenesis9,10. Bacterial colonization, hormesis, and genomic changes are among exemplar conditions that affect the aggregation of polyglutamine (polyQ) tracts9,11,12. Additionally, these misfolded protein clusters exhibit polyQ length- and age-dependent accumulation within the host and are associated with impaired motility9,13. The relatively simple approach of quantifying fluorescently labeled puncta can generate important data on conditions, factors, or drugs that affect protein folding and aggregation.
Though quantification of fluorescent puncta has proven to be a reliable and relatively simple procedure, the challenge remains to develop a protocol that would facilitate large-scale screening of compounds, bacteria, or conditions that affect protein aggregation. The concept of automated C. elegans image processing and puncta quantification is not entirely novel, as a number of practical support tools have been developed14,15. However, the integration of culturing, image acquisition, and a processing pipeline are essential in eliminating variability in results and allowing for higher-throughput screens.
As such, the intent of this manuscript is to standardize the procedure used to quantify polyQ aggregation in C. elegans as a proxy to detect changes in proteostasis. This task was accomplished by employing CellProfiler, an open-source image analysis software16 capable of automated worm and aggregate identification, and is integrated into a larger protocol for culturing worms, acquiring images, and processing data.

<table><tbody><tr><td>1.7 mL Microtubes</td><td>Olympus Plastics</td><td>Cat#24-282</td><td>Microcentrifuge tubes</td></tr><tr><td>10 mL Serological Pipettes</td><td>GenClone</td><td>Cat#12-104</td><td>Plastic pipettes</td></tr><tr><td>15 mL Centrifuge Tubes, Racked</td><td>Olympus Plastics</td><td>Cat#28-101</td><td>Conical tubes</td></tr><tr><td>5-Fluoro-2&amp;prime;-deoxyuridine</td><td>Fisher Scientific</td><td>D2235100MG</td><td>FUDR</td></tr><tr><td>Accu-jet Pro Pipette Controller</td><td>Genesee Scientific</td><td>91-600RB</td><td>Pipette gun</td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>Cat#BP1423-2</td><td>Granulated agar</td></tr><tr><td>BioRender</td><td>BioRender</td><td></td><td>Graphical figure generator</td></tr><tr><td>Bleach (Regular)</td><td>Clorox</td><td>Bar# 044600324111, Splash-less Bleach</td><td>4.5% sodium hypochlorite</td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt;: AM140: &lt;em&gt;rmIs132&lt;/em&gt; [&lt;em&gt;unc-54p::q35::yfp&lt;/em&gt;]</td><td>Morimoto Lab (Northwestern University)</td><td>AM140, Q35::YFP</td><td>Muscle polyQ</td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt;: AM738: &lt;em&gt;rmIs297&lt;/em&gt;[&lt;em&gt;vha-6&lt;/em&gt;p::&lt;em&gt;q44&lt;/em&gt;::&lt;em&gt;yfp&lt;/em&gt;; &lt;em&gt;rol-6&lt;/em&gt;(&lt;em&gt;su1006&lt;/em&gt;)]</td><td>Morimoto Lab (Northwestern University)</td><td>AM738, Q44::YFP</td><td>Intestinal polyQ</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;sub&gt;&amp;middot;&lt;/sub&gt;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>Cat#C79-500</td><td>Calcium chloride dihydrate</td></tr><tr><td>CellProfiler</td><td>Broad Institute</td><td></td><td>Image analysis software</td></tr><tr><td>Cholesterol</td><td>Fisher Scientific</td><td>Cat#ICN10138201</td><td>Cholesterol</td></tr><tr><td>Circulating Water Bath Head</td><td>Lauda</td><td>26LE</td><td>Lauda E 100</td></tr><tr><td>CoolLED pE300lite 365 dir mount STEREO</td><td>CoolLED</td><td>8114931</td><td>Fluorescent light control and emitter</td></tr><tr><td>16 mL Culture Tubes</td><td>Olympus Plastics</td><td>21-129</td><td>Culture tubes, 17 mm x 100 mm</td></tr><tr><td>Escherichia coli OP50</td><td>Caenorhabditis Genetics Center</td><td>OP50</td><td>&lt;em&gt;E. coli &lt;/em&gt;control strain</td></tr><tr><td>Ethanol, 200 Proof</td><td>Decon Labs, Inc.</td><td>2701</td><td></td></tr><tr><td>Eyepiece 10x/23B, adjustable, 3d gen</td><td>Leica Microsystems</td><td>10450910</td><td>Eyepiece set</td></tr><tr><td>Filter Set ET GFP - MZ10F</td><td>Leica Microsystems</td><td>10450588</td><td>Filter cube</td></tr><tr><td>GraphPad Prism v9.2.0</td><td>GraphPad Software, Inc.</td><td></td><td>Statistical analysis tool</td></tr><tr><td>Heratherm Incubator IMP180</td><td>Thermo</td><td>51031562</td><td>Refrigerated incubator</td></tr><tr><td>Innova 4000</td><td>New Brunswick Scientific</td><td>M1192-0000</td><td>Shaking incubator</td></tr><tr><td>K5 Camera</td><td>Leica Microsystems</td><td>11547112</td><td>Stereomicroscope camera</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;P0&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>Cat#P285-3</td><td>Potassium phosphate monobasic</td></tr><tr><td>LAS X Imaging Software</td><td>Leica Microsystems</td><td></td><td>Microscope imaging software</td></tr><tr><td>Leica MZ10 F Optics Carrier</td><td>Leica Microsystems</td><td>10450103</td><td>Stereomicroscope</td></tr><tr><td>Levamisole</td><td>Fisher Scientific</td><td>Cat#0215522805</td><td>Levamisole hydrochloride</td></tr><tr><td>Luria Broth (Lennox)</td><td>Apex Bioresearch Products</td><td>Cat#11-125</td><td>LB</td></tr><tr><td>Magnetic Stir Plate</td><td>Fisher Scientific</td><td>11-100-49S</td><td>Stir plate</td></tr><tr><td>MgSO&lt;sub&gt;4&amp;middot;&lt;/sub&gt;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Alfa Aesar</td><td>A14491</td><td>Magnesium sulfate heptahydrate</td></tr><tr><td>Microscope Slides</td><td>Premiere</td><td>8205</td><td>Single frosted microscope slides</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&amp;middot;&lt;/sub&gt;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>Cat#S373-500</td><td>Sodium phosphate dibasic heptahydrate</td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>Cat#S671-500</td><td>Sodium chloride</td></tr><tr><td>NaOH</td><td>Fisher Scientific</td><td>Cat#S318-3</td><td>Sodium hydroxide pellets</td></tr><tr><td>Objective Achromat, f = 100 mm</td><td>Leica Microsystems</td><td>10411597</td><td>Objective microscope lens</td></tr><tr><td>Petri Dishes</td><td>Genesee Scientific</td><td>Cat#32-107G</td><td>100 mm x 15 mm</td></tr><tr><td>&lt;em&gt;Pseudomonas aeruginosa&lt;/em&gt; Mutant Library</td><td>Manoil Lab (University of Washington)</td><td></td><td>&lt;em&gt;P. aeruginosa&lt;/em&gt; mutant library</td></tr><tr><td>Suspension Culture Plate 24-Well, Flat Bottom</td><td>Olympus Plastics</td><td>25-102</td><td>Used for worm growth and imaging</td></tr><tr><td>Trinocular Tube 100% M-series</td><td>Leica Microsystems</td><td>10450043</td><td></td></tr><tr><td>Trypticase Peptone</td><td>ThermoFisher, Difco</td><td>Cat#211921</td><td></td></tr><tr><td>TX-400 Rotor</td><td>Thermo Scientific</td><td>Cat#75003181</td><td>Swing bucket rotor</td></tr><tr><td>Vacuum Driven Filter System</td><td>GenClone</td><td>Cat#25-227</td><td>500 mL, PES Membrane, .22 &amp;micro;m</td></tr><tr><td>Video Objective with C-Mount</td><td>Leica Microsystems</td><td>10447367</td><td>0.63x camera adapter tube</td></tr></tbody></table>

automating aggregate quantification in caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

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Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

automating-aggregate-quantification-in-caenorhabditis-elegans

Research

JoVE Journal

Biology

2.7K Views.  University of Florida. Een eenvoudige aanpak werd gekozen om veranderingen in proteostase te monitoren door polyglutamineaggregatie te beoordelen en een raamwerk te bieden dat kan worden gebruikt voor toepassingen met een hoge doorvoer. Het scoren van een fenotype zoals het aantal aggregaten door ik kan subjectief worden. Het automatiseren van aggregaattellingen stelt ons in staat om dergelijke vertekeningen te elimineren, de doorvoer en reproduceerbaarheid te verhogen.Deze methode helpt bij het identificere...

Video: Automating Aggregate Quantification in Caenorhabditis elegans

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

Genetics

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Bioengineering

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Neuroscience

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

Behavior

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Biochemistry

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Immunology and Infection

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble proteins over 40% of the time results in an extension of the lifespan in C. elegans, suggesting that many of these proteins are key determinants of the aging process. Isolation and quantitative identification of these insoluble proteins are crucial to understand key biological processes that occur during aging. Here, we present a modified and improved protocol that details how to extract and isolate the SDS-insoluble proteins (insolublome) from C. elegans more efficiently to streamline mass spectrometric workflows via a novel label-free quantitative proteomics analysis. This improved protocol utilizes a highly efficient sonicator for worm lysis that greatly increases efficiency for protein extraction and allows us to use significantly less starting material (approximately 3,000 worms) than in previous protocols (typically using at least 40,000 worms). Subsequent quantitative proteomic analysis of the insolublome was performed using data-dependent acquisition (DDA) for protein discovery and identification and data-independent acquisition (DIA) for comprehensive and more accurate protein quantification. Bioinformatic analysis of quantified proteins provides potential candidates that can be easily followed up with other molecular methods in C. elegans. With this workflow, we routinely identify more than 1000 proteins and quantify more than 500 proteins. This new protocol enables efficient compound screening with C. elegans. Here, we validated and applied this improved protocol to wild-type C. elegans N2-Bristol strain and confirmed that aged day-10 N2 worms showed greater accumulation of the insolublome than day-2 young worms.

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

This novel workflow efficiently extracts and isolates SDS-insoluble proteins (insolublome) from Caenorhabditis elegans with minimal starting material for quantitative differential proteomic analysis. The protocol uses a comprehensive data-independent acquisition mass spectrometry analysis to quantify the insolublome and bioinformatic analysis to gain biological insights into aging mechanisms and pathologies.

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble ...

Automating Aggregate Quantification in Caenorhabditis elegans

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