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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Growth and maintenance of C. elegans strains

1. Seed Nematode Growth Medium (NGM, see Table of Materials) agar plates with 300 µL of the slow-growing E. coli strain OP50 in a laminar flow cabinet.
2. Leave the NGM agar plates in the laminar flow cabinet to dry.
   NOTE: In the absence of laminar flow cabinet, plates can be left to dry on the bench but they are more prone to contamination.
3. Transfer at least 20 animals to each of two OP50-seeded NGM a.......

Discussion

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 und.......

Materials

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