Experimental considerations for study of C. elegans lysosomal proteins.
G3 (Bethesda)
; 13(4)2023 04 11.
Article
em En
| MEDLINE
| ID: mdl-36748711
ABSTRACT
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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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Caenorhabditis elegans
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
G3 (Bethesda)
Ano de publicação:
2023
Tipo de documento:
Article