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Experimental considerations for study of C. elegans lysosomal proteins. | LitMetric

Experimental considerations for study of C. elegans lysosomal proteins.

G3 (Bethesda)

Department of Molecular, Cell, and Developmental Biology, University of California-Santa Cruz, Santa Cruz, CA 95064, USA.

Published: April 2023

AI Article Synopsis

Article 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.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10085801PMC
http://dx.doi.org/10.1093/g3journal/jkad032DOI Listing

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