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Experimental considerations for study of C. elegans lysosomal proteins

Lysosomes are an important organelle required for the degradation of a range of cellular components. Lysosome function is critical for development and homeostasis as dysfunction can lead to inherited genetic disorders, cancer, and neurodegenerative and metabolic diseases. The acidic and protease-ric...

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Autores principales: Clancy, John C, Vo, An A, Myles, Krista M, Levenson, Max T, Ragle, James Matthew, Ward, Jordan D
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10085801/
https://www.ncbi.nlm.nih.gov/pubmed/36748711
http://dx.doi.org/10.1093/g3journal/jkad032
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author Clancy, John C
Vo, An A
Myles, Krista M
Levenson, Max T
Ragle, James Matthew
Ward, Jordan D
author_facet Clancy, John C
Vo, An A
Myles, Krista M
Levenson, Max T
Ragle, James Matthew
Ward, Jordan D
author_sort Clancy, John C
collection PubMed
description Lysosomes are an important organelle required for the degradation of a range of cellular components. Lysosome function is critical for development and homeostasis as dysfunction can lead to inherited genetic disorders, cancer, and neurodegenerative and metabolic diseases. The acidic and protease-rich environment of lysosomes poses experimental challenges. Many fluorescent proteins are quenched or degraded, while specific red fluorescent proteins can be cleaved from translational fusion partners and accumulate. While studying MLT-11, a Caenorhabditis elegans molting factor that localizes to lysosomes and the cuticle, we sought to optimize several experimental parameters. We found that, in contrast to mNeonGreen fusions, mScarlet fusions to MLT-11 missed cuticular and rectal epithelial localization. Rapid sample lysis and denaturation were critical for preventing MLT-11 fragmentation while preparing lysates for western blots. Using a model lysosomal substrate (NUC-1), we found that rigid polyproline linkers and truncated mCherry constructs do not prevent cleavage of mCherry from NUC-1. We provide evidence that extended localization in lysosomal environments prevents the detection of FLAG epitopes in western blots. Finally, we optimize an acid-tolerant green fluorescent protein (Gamillus) for use in C. elegans. These experiments provide important experimental considerations and new reagents for the study of C. elegans lysosomal proteins.
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spelling pubmed-100858012023-04-12 Experimental considerations for study of C. elegans lysosomal proteins Clancy, John C Vo, An A Myles, Krista M Levenson, Max T Ragle, James Matthew Ward, Jordan D G3 (Bethesda) Investigation Lysosomes are an important organelle required for the degradation of a range of cellular components. Lysosome function is critical for development and homeostasis as dysfunction can lead to inherited genetic disorders, cancer, and neurodegenerative and metabolic diseases. The acidic and protease-rich environment of lysosomes poses experimental challenges. Many fluorescent proteins are quenched or degraded, while specific red fluorescent proteins can be cleaved from translational fusion partners and accumulate. While studying MLT-11, a Caenorhabditis elegans molting factor that localizes to lysosomes and the cuticle, we sought to optimize several experimental parameters. We found that, in contrast to mNeonGreen fusions, mScarlet fusions to MLT-11 missed cuticular and rectal epithelial localization. Rapid sample lysis and denaturation were critical for preventing MLT-11 fragmentation while preparing lysates for western blots. Using a model lysosomal substrate (NUC-1), we found that rigid polyproline linkers and truncated mCherry constructs do not prevent cleavage of mCherry from NUC-1. We provide evidence that extended localization in lysosomal environments prevents the detection of FLAG epitopes in western blots. Finally, we optimize an acid-tolerant green fluorescent protein (Gamillus) for use in C. elegans. These experiments provide important experimental considerations and new reagents for the study of C. elegans lysosomal proteins. Oxford University Press 2023-02-07 /pmc/articles/PMC10085801/ /pubmed/36748711 http://dx.doi.org/10.1093/g3journal/jkad032 Text en © The Author(s) 2023. Published by Oxford University Press on behalf of the Genetics Society of America. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Investigation
Clancy, John C
Vo, An A
Myles, Krista M
Levenson, Max T
Ragle, James Matthew
Ward, Jordan D
Experimental considerations for study of C. elegans lysosomal proteins
title Experimental considerations for study of C. elegans lysosomal proteins
title_full Experimental considerations for study of C. elegans lysosomal proteins
title_fullStr Experimental considerations for study of C. elegans lysosomal proteins
title_full_unstemmed Experimental considerations for study of C. elegans lysosomal proteins
title_short Experimental considerations for study of C. elegans lysosomal proteins
title_sort experimental considerations for study of c. elegans lysosomal proteins
topic Investigation
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10085801/
https://www.ncbi.nlm.nih.gov/pubmed/36748711
http://dx.doi.org/10.1093/g3journal/jkad032
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