automated-analysis-of-c-elegans-fluorescence-images-using-segelegans

Automated Analysis of <em>C. elegans</em> Fluorescence Images using SegElegans

Microscopy, particularly of the fluorescent kind, is a frequently used tool in C. elegans research. The analysis of data from microscopy experiments can, ...

University of Crete

Foundation for Research and Technology-Hellas

Mitochondria constitute the main energy-producing centers of eukaryotic cells. In addition, they are involved in several crucial cellular processes, such as lipid metabolism, calcium buffering, and apoptosis. As such, their malfunction can be detrimental for proper cellular physiology and homeostasis. Mitophagy is a mechanism that protects and maintains cellular function by sequestering harmful or dysfunctional mitochondria to lysosomes for degradation. In this report, we present experimental procedures for quantitative, in vivo monitoring of mitophagy events in the nematode Caenorhabditis elegans.

Monitoring Mitophagy During Aging in Caenorhabditis elegans.

Mitochondria are critical to tissues and organs characterized by high-energy demands, such as the nervous system. They provide essential energy and metabolites, and maintain Ca2+ balance, which is imperative for proper neuronal function and development. Emerging findings further underline the role of mitochondria in neurons. Technical advances in the last decades made it possible to investigate key mechanisms in neuronal development and the contribution of mitochondria therein. In this article, we discuss the latest findings relevant to the involvement of mitochondria in neuronal development, placing emphasis on mitochondrial metabolism and dynamics. In addition, we survey the role of mitochondrial energy metabolism and Ca2+ homeostasis in proper neuronal function, and the involvement of mitochondria in axon myelination.

Mitochondrial contributions to neuronal development and function.

The cytoskeleton consists of filamentous protein polymers that form organized structures, contributing to a multitude of cell life aspects. It includes three types of polymers: the actin microfilaments, the microtubules and the intermediate filaments. Decades of research have implicated the cytoskeleton in processes that regulate cellular and organismal aging, as well as neurodegeneration associated with injury or neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, or Charcot Marie Tooth disease. Here, we provide a brief overview of cytoskeletal structure and function, and discuss experimental evidence linking cytoskeletal function and dynamics with aging and neurodegeneration.

The Cytoskeleton as a Modulator of Aging and Neurodegeneration.

The term lipophagy is used to describe the autophagic degradation of lipid droplets, the main lipid storage organelles of eukaryotic cells. Ever since its discovery in 2009, lipophagy has emerged as a significant component of lipid metabolism with important implications for organismal health. This review aims to provide a brief summary of our current knowledge on the mechanisms that are responsible for regulating lipophagy and the impact the process has under physiological and pathological conditions.

Emerging Roles of Lipophagy in Health and Disease.

The in-vivo elucidation of the molecular mechanisms underlying muscles dysfunction due to aging via non-invasive label free imaging techniques is an important issue with high biological significance. In this study, polarization-dependent second-harmonic generation (PSHG) was used to evaluate structural alterations in the striated muscles during Caenorhabditis elegans lifespan. Young and old wild-type animals were irradiated. The obtained results showed that it was not feasible to detect differences in the structure of myosin that could be correlated with the aging of the worms, via the implementation of the classical, widely used, cylindrical symmetry model and the calculation of the SHG anisotropy values. A trigonal symmetry model improved the extracted results; however, the best outcome was originated from the application of a general model. Myosin structural modifications were monitored via the estimation of the difference in spectral phases derived from discrete Fourier transform analysis. Age classification of the nematodes was achieved by employing both models, proving the usefulness of the usage of PSHG microscopy as a potential diagnostic tool for the investigation of muscle diseases.

Monitoring aging-associated structural alterations in Caenorhabditis elegans striated muscles via polarization-dependent second-harmonic generation measurements.

The nematode Caenorhabditis elegans is a powerful experimental platform for cell biology studies. The molecular mechanisms that mediate cell death and neurodegeneration have been characterized extensively in the nematode. In addition, the availability of a wide arsenal of genetic and molecular tools and methodologies renders C. elegans an organism of choice for modeling human neurodegenerative diseases. Indeed, neuronal necrosis can readily be observed and examined in vivo, in the worm. In this chapter, we describe the two main approaches that are routinely used for monitoring and quantifying neuronal cell death in C. elegans. The first is based on direct visualization of dying cells via Nomarski differential interference contrast (DiC) microscopy, and the second on the assessment of neuronal survival by fluorescence microscopy.

Assessment of Neuronal Cell Death in Caenorhabditis elegans.

SNARE proteins reside between opposing membranes and facilitate vesicle fusion, a physiological process ubiquitously required for secretion, endocytosis and autophagy. With age, neurosecretory SNARE activity drops and is pertinent to age-associated neurological disorders. Despite the importance of SNARE complex assembly and disassembly in membrane fusion, their diverse localization hinders the complete understanding of their function. Here, we revealed a subset of SNARE proteins, the syntaxin SYX-17, the synaptobrevins VAMP-7, SNB-6 and the tethering factor USO-1, to be either localized or in close proximity to mitochondria, in vivo. We term them mitoSNAREs and show that animals deficient in mitoSNAREs exhibit increased mitochondria mass and accumulation of autophagosomes. The SNARE disassembly factor NSF-1 seems to be required for the effects of mitoSNARE depletion. Moreover, we find mitoSNAREs to be indispensable for normal aging in both neuronal and non-neuronal tissues. Overall, we uncover a previously unrecognized subset of SNAREs that localize to mitochondria and propose a role of mitoSNARE assembly and disassembly factors in basal autophagy regulation and aging.

MitoSNARE Assembly and Disassembly Factors Regulate Basal Autophagy and Aging in .

Caenorhabditis elegans is a great model for exploring organismal, cellular, and subcellular biology through optical and fluorescence microscopy, with its research applications steadily expanding. However, manual processing of numerous microscopic images is prone to errors and demands significant labor due to worms tendency to touch or cluster with each other. Here, we present a new system for segmenting whole-body instances of Caenorhabditis elegans in microscopic images (referred to as SegElegans), employing a combination of neural network architecture and conventional image processing techniques. Our method effectively overcomes previous challenges and resolves many instances of contact and overlap between worms in highly populated images in a timely manner. The results obtained show an average Intersection over Union value of 96.3% per worm and an average improvement of 6% over other existing methods for automated analysis of worm images. SegElegns is a user-friendly application for Caenorhabditis elegans segmentation that will benefit whole-worm phenotypic screenings essential for studying development, behavior, aging, and disease.

Article	Year
Monitoring Mitophagy During Aging in Caenorhabditis elegans. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28324491	2017
Mitochondrial contributions to neuronal development and function. Biological chemistry\| PubMed ID: 29476663	2018
The Cytoskeleton as a Modulator of Aging and Neurodegeneration. Advances in experimental medicine and biology\| PubMed ID: 31493230	2019
Emerging Roles of Lipophagy in Health and Disease. Frontiers in cell and developmental biology\| PubMed ID: 31552248	2019
Monitoring aging-associated structural alterations in Caenorhabditis elegans striated muscles via polarization-dependent second-harmonic generation measurements. Journal of biophotonics\| PubMed ID: 34405541	2021
Assessment of Neuronal Cell Death in Caenorhabditis elegans. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 35776360	2022
MitoSNARE Assembly and Disassembly Factors Regulate Basal Autophagy and Aging in . International journal of molecular sciences\| PubMed ID: 36835643	2023
SegElegans: Instance segmentation using dual convolutional recurrent neural network decoder in Caenorhabditis elegans microscopic images. Computers in biology and medicine\| PubMed ID: 40120179	2025

Konstantinos Kounakis

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