We thank members of the Neumann lab for valuable discussions and input. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The authors thank WormBase for its wealth of information on C. elegans, and acknowledge Monash Micro Imaging, Monash University, for the provision of instrumentation, training and technical support. This work was supported by CMTAA research grants (2015 and 2018), and NHMRC Project Grants 1101974 and 1099690 awarded to B.N.

<ol>
	<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. , (1974).</li><li>Markaki, M., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+human+diseases+in+Caenorhabditis+elegans.">Modeling human diseases in Caenorhabditis elegans.</a> Biotechnology Journal. 5 (12), 1261-1276 (2010).</li><li>Laranjeiro, R., Harinath, G., Burke, D., Braeckman, B. P., Driscoll, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+swim+sessions+in+C.+elegans+induce+key+features+of+mammalian+exercise.">Single swim sessions in C. elegans induce key features of mammalian exercise.</a> BMC Biology. , (2017).</li><li>Alexander, A. G., Marfil, V., Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+C.+elegans+as+a+model+to+study+Alzheimer's+disease+and+other+neurodegenerative+diseases.">Use of C. elegans as a model to study Alzheimer's disease and other neurodegenerative diseases.</a> Frontiers in Genetics. , (2014).</li><li>Zheng, C., Jin, F. Q., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hox+Proteins+Act+as+Transcriptional+Guarantors+to+Ensure+Terminal+Differentiation.">Hox Proteins Act as Transcriptional Guarantors to Ensure Terminal Differentiation.</a> Cell Reports. 13 (7), 1343-1352 (2015).</li><li>Koushika, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+Caenorhabditis+elegans+cytoplasmic+dynein+components+reveal+specificity+of+neuronal+retrograde+cargo.">Mutations in Caenorhabditis elegans cytoplasmic dynein components reveal specificity of neuronal retrograde cargo.</a> Journal of Neuroscience. 24 (16), 3907-3916 (2004).</li><li>Byrne, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+mitochondrial+dynamics+affects+behaviour+and+lifespan+in+Caenorhabditis+elegans.">Disruption of mitochondrial dynamics affects behaviour and lifespan in Caenorhabditis elegans.</a> Cellular and Molecular Life Sciences. , (2019).</li><li>Campagnola, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+High-Resolution+Second-Harmonic+Generation+Imaging+of+Endogenous+Structural+Proteins+in+Biological+Tissues.">Three-Dimensional High-Resolution Second-Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues.</a> Biophysical Journal. 82 (1), 493-508 (2002).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Sommer, C., Straehle, C., Kothe, U., Hamprecht, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ilastik:+Interactive+learning+and+segmentation+toolkit.">Ilastik: Interactive learning and segmentation toolkit.</a> 2011 IEEE International Symposium on Biomedical Imaging: From Nano to Macro. , (2011).</li><li>Mondal, S., Ahlawat, S., Koushika, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+microfluidic+devices+for+in+vivo+imaging+of+C.+elegans,+Drosophila+and+zebrafish.">Simple microfluidic devices for in vivo imaging of C. elegans, Drosophila and zebrafish.</a> Journal of Visualized Experiments. , (2012).</li><li>Paul, G., Cardinale, J., Sbalzarini, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+image+restoration+and+segmentation:+A+generalized+linear+model/bregman+perspective.">Coupling image restoration and segmentation: A generalized linear model/bregman perspective.</a> International Journal of Computer Vision. , (2013).</li><li>Rizk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmentation+and+quantification+of+subcellular+structures+in+fluorescence+microscopy+images+using+Squassh.">Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh.</a> Nature Protocols. 9, 586-586 (2014).</li><li>Akella, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEC-17+is+an+alpha-tubulin+acetyltransferase.">MEC-17 is an alpha-tubulin acetyltransferase.</a> Nature. 467 (7312), 218-222 (2010).</li><li>Neumann, B., Hilliard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+MEC-17+leads+to+microtubule+instability+and+axonal+degeneration.">Loss of MEC-17 leads to microtubule instability and axonal degeneration.</a> Cell Reports. 6 (1), 93-103 (2014).</li><li>Shida, T., Cueva, J. G., Xu, Z., Goodman, M. B., Nachury, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+alpha-tubulin+K40+acetyltransferase+alphaTAT1+promotes+rapid+ciliogenesis+and+efficient+mechanosensation.">The major alpha-tubulin K40 acetyltransferase alphaTAT1 promotes rapid ciliogenesis and efficient mechanosensation.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (50), 21517-21522 (2010).</li><li>Teoh, J. -. S., Wang, W., Chandhok, G., Pocock, R., Neumann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+maintenance+of+synaptic+structure+is+mediated+by+the+alpha-tubulin+acetyltransferase+MEC-17/&#945;TAT1.">Development and maintenance of synaptic structure is mediated by the alpha-tubulin acetyltransferase MEC-17/&#945;TAT1.</a> bioRxiv. 84, 522904-522904 (2019).</li><li>Bird, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Charcot-Marie-Tooth Neuropathy Type 2. , (1998).</li><li>Chandhok, G., Lazarou, M., Structure Neumann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function,+and+regulation+of+mitofusin-2+in+health+and+disease.">Structure, function, and regulation of mitofusin-2 in health and disease.</a> Biological Reviews. 93 (2), 933-949 (2018).</li><li>Soh, M. S., Cheng, X., Liu, J., Neumann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+genes+associated+with+Charcot-Marie-Tooth+type+2+lead+to+common+behavioural,+cellular+and+molecular+defects+in+Caenorhabditis+elegans.">Disruption of genes associated with Charcot-Marie-Tooth type 2 lead to common behavioural, cellular and molecular defects in Caenorhabditis elegans.</a> bioRxiv. , 605584 (2019).</li><li>Kamerkar, S. C., Kraus, F., Sharpe, A. J., Pucadyil, T. J., Ryan, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin-related+protein+1+has+membrane+constricting+and+severing+abilities+sufficient+for+mitochondrial+and+peroxisomal+fission.">Dynamin-related protein 1 has membrane constricting and severing abilities sufficient for mitochondrial and peroxisomal fission.</a> Nature Communications. , (2018).</li><li>Zhu, P. -. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-+and+Intermolecular+Domain+Interactions+of+the+C-terminal+GTPase+Effector+Domain+of+the+Multimeric+Dynamin-like+GTPase+Drp1.">Intra- and Intermolecular Domain Interactions of the C-terminal GTPase Effector Domain of the Multimeric Dynamin-like GTPase Drp1.</a> Journal of Biological Chemistry. 279 (34), 35967-35974 (2004).</li><li>Osellame, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+and+independent+roles+of+the+Drp1+adaptors+Mff,+MiD49+and+MiD51+in+mitochondrial+fission.">Cooperative and independent roles of the Drp1 adaptors Mff, MiD49 and MiD51 in mitochondrial fission.</a> Journal of Cell Science. , (2016).</li><li>Dima, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+segmentation+algorithms+for+fluorescence+microscopy+images+of+cells.">Comparison of segmentation algorithms for fluorescence microscopy images of cells.</a> Cytometry Part A. 79 (7), 545-559 (2011).</li><li>Trevisan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+Mitochondria+Dynamics+Reveals+Separate+Roles+for+Form+and+Function+in+Mitochondria+Distribution.">Manipulation of Mitochondria Dynamics Reveals Separate Roles for Form and Function in Mitochondria Distribution.</a> Cell Reports. , (2018).</li></ol>

The authors declare that they have no competing interests.

Morphological variations have frequently been assessed via manual counting of noticeable differences or using arbitrary thresholds to determine defects in comparison to a wild-type phenotype. More recently, however, quantitative methods have been used for comparative studies of morphology to accurately measure and describe changes on a cellular and subcellular level in an unbiased fashion. The ability to identify subtle yet biologically relevant differences between phenotypes is a powerful means for understanding the underlying molecular mechanisms that control changes in morphology caused by environmental factors or genetic diseases. The continued increases in computation power and microscopy imaging resolution, coupled with the ease of growth and the versatility of C. elegans genetics has provided the perfect platform for the combination of sophisticated yet free imaging software with fine detailed confocal images. Here, we have demonstrated methods to evaluate and quantify synaptic size and protein localization, body wall muscle area and fiber length, and mitochondrial morphology in a range of genetic backgrounds.
While these analyses have demonstrated an ability to quantify subtle morphological differences, there are limitations. Although each of these analyses make use of open-source and readily available software, they rely on high quality, sharp images to accurately resolve cellular structures and subcellular organelles. This can often be a limiting step, as sensitive high-end equipment, such as confocal microscopes with advanced detectors, are required to capture images with satisfactory resolution. These are not always accessible to laboratories, and analysis with sub-par technology can lead to inaccurate and unrepeatable results. In addition, while each technique aims to reduce experimenter bias by using computer algorithms, there are still steps that can involve an element of human error. The manual definition of the synapse by hand, the outlining of the muscle cell area, the parameter optimizations of mitochondrial segmentation by eye are all areas that can be improved in this respect. To reduce the chance for human error, studies could be conducted in a blinded fashion or alternatively image analysis software could be used to define the region of interest itself. Using cell masks, in which the desired area for analysis is defined by the fluorescence at a different wavelength, may help this. This is used to limit the analysis to a section of an image displaying fluorescence above a set threshold, such as a cell&#8217;s nucleus or a transfected cell13,24. Furthermore, the use of single time point images limits the questions these techniques can address. The processes that govern organelle morphology, synapse formation and muscle structure are most likely dynamic, and differences can be seen within the same cell depending on the developmental or biological status. Analysis of these structures at a specific end-point can therefore be limiting and time-lapse imaging would be beneficial for monitoring structural changes over time. Finally, while morphological changes can be a good indicator of disruption in cellular processes, correlation is not always implicit. For example, recent findings in different species have shown that mitochondrial function and form can indeed be dissociated7,25. Therefore, additional assays must be taken into account when inferring functional changes as the result of morphological differences.
We have been able to demonstrate the use of these quantitative techniques in a particular set of tissues and genetic backgrounds; however, their usage can be applied more broadly. Fluorescence quantification via imaging software can be used to analyze a range of different subcellular organelles such as nuclei and endosomes, or different structures such as neurons and epithelial cells, all within the context of different mutant or disease backgrounds.

The nematode Caenorhabditis elegans (C. elegans) is increasingly utilized as a model system to uncover the biological and molecular processes involved in human disease. An adult nematode has a body length of just over 1 mm, and can produce a large brood of up to 300 eggs1. After hatching, C. elegans only require 3-4 days to reach adulthood, and live for around 2 to 3 weeks2. Due to its ease of culturing, C. elegans is currently one of the most sought-after in vivo animal models for conducting cost-effective, rapid drug screening to identify therapeutics for human diseases. Additionally, its genetic conservation, well defined behavioral paradigms, transparent body for fluorescence or light microscopy, and ease of genetic manipulation make the study of cellular and molecular consequences of genetic mutations readily achieveable3. The C. elegans genome shares approximately 60-80% orthology with human genes, and about 40% of those genes are known to be disease-related. Some of human diseases that have been modelled and studied in C. elegans include neurodegenerative disorders (Alzheimer&#8217;s disease, Parkinson&#8217;s disease, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease), muscle-associated diseases (Duchenne muscular dystrophy), and metabolic diseases (hyperglycemia)2,4. In most human disorders, disease-induced cellular and organelle localization and morphological changes occur, which can readily be evaluated in the nematode model.
Fluorescent markers have been widely used to label tissues and organelles for dynamic visualization under the microscope. However, in C. elegans, conventional methods that assess morphological irregularities due to genetic mutations have largely relied on visual descriptions. While qualitative assessments can cover wider ranges of phenotypic descriptions (synaptic morphology, GFP clumping, specific axonal shape, muscle fiber thickness, etc.) and provide a bird&#8217;s eye view of the morphological changes, they are less well suited for comparing small variations across different groups. Furthermore, qualitative assessments are based on visual, subjective evaluation, which may lead to over- or under-estimations of morphological abnormalities. Finally, qualitative observations can also vary greatly between individuals, creating difficulties with data replication.&#160;
In recent years, a number of user-friendly, readily available computational algorithms that can quantitatively analyse images have been developed. However, the utilization of such image analysis software for some morphological studies, especially in relation to body wall muscles and mitochondria, in C. elegans research has lagged behind. To improve underlying structural analysis in C. elegans, some of the readily available, open-source image analysis software were trialed to quantitatively compare the effects of genetic mutations on muscle mitochondria, body wall muscle and synaptic morphology. These experimental procedures outline in detail how these programs (Fiji, ilastik, CellProfiler, SQUASSH) can be used to evaluate changes in synaptic size and synaptic protein localization, body wall muscle area and fiber length, and mitochondrial size and circularity as a result of genetic mutations in the nematode.&#160;

<table>
	<tbody>
		<tr>
			<td>Agar-agar</td>
			<td>Merck</td>
			<td>1.01614.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Invitrogen</td>
			<td>16500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>TCS SP8</td>
			<td>Inverted platform</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Carl Zeiss AG</td>
			<td>Zeiss Axio Imager M2</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips #1</td>
			<td>Thermo scientifique</td>
			<td>MENCS22221GP</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips #1.5</td>
			<td>Zeiss</td>
			<td>474030-9000-000</td>
			<td>Made by SCHOTT</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Thermo scientifique</td>
			<td>MENS41104A/40</td>
			<td></td>
		</tr>
		<tr>
			<td>Light LED</td>
			<td>Schott</td>
			<td>KL 300 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ51</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone (Peptone from casein)</td>
			<td>Merck</td>
			<td>107213</td>
			<td>Ingredients for Lysogeny Broth (LB) medium</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Merck</td>
			<td>103753</td>
			<td>Ingredients for Lysogeny Broth (LB) medium</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>106404</td>
			<td>Ingredients for Lysogeny Broth (LB) medium</td>
		</tr>
		<tr>
			<td>Peptone (Peptone from meat)</td>
			<td>Merck</td>
			<td>107214</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma</td>
			<td>A1296</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>106404</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8667-25G</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Merck</td>
			<td>102382</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Merck</td>
			<td>105886</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Dipotassium phosphate</td>
			<td>Merck</td>
			<td>105101</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Merck</td>
			<td>104873</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Disodium phosphate</td>
			<td>Merck</td>
			<td>106586</td>
			<td>Ingredients for M9 buffer</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>106404</td>
			<td>Ingredients for M9 buffer</td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Merck</td>
			<td>104873</td>
			<td>Ingredients for M9 buffer</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Merck</td>
			<td>105886</td>
			<td>Ingredients for M9 buffer</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Corning</td>
			<td>CLS7095D5X-200EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Corning</td>
			<td>CLS430589-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum wire</td>
			<td>Sigma</td>
			<td>267201-2G</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Met-app</td>
			<td>2616</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramisole hydrochloride</td>
			<td>Sigma</td>
			<td>L9756-5G</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans at JoVE.com

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.

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 ...

quantitative-approaches-for-studying-cellular-structures-organelle

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

Cells, Genomes, and Evolution

SCIENTISTS IN ACTION

9.6K Views.  Monash University, Melbourne VIC 3800, Australia.. 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 C. elegans to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

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

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

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 ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

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

Summary

Explore More Videos

Chapters in this video

Measuring Caenorhabditis elegans Life Span on Solid Media

A high-throughput method to globally study the organelle morphology in S. cerevisiae

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Analyzing and Building Nucleic Acid Structures with 3DNA

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans