Experimental considerations for study of C. elegans lysosomal proteins.
Experimental considerations for study of C. elegans lysosomal proteins.
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DOI：
10.1093/g3journal/jkad032
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发表时间：
2023-04-11
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影响因子：
--
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中科院分区：
文献类型：
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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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影响因子：
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作者：
Mok DZ;Sternberg PW;Inoue T
通讯作者：
Inoue T
影响因子：
3.3
作者：
Cohen, Jennifer D.;Flatt, Kristen M.;Sundaram, Meera, V
通讯作者：
Sundaram, Meera, V
影响因子：
9.9
作者：
Meeuse, Milou W. M.;Hauser, Yannick P.;Grosshans, Helge
通讯作者：
Grosshans, Helge
DOI：
10.1083/jcb.201602090
发表时间：
2016-10-24
期刊：
The Journal of cell biology
影响因子：
--
作者：
Li Y;Chen B;Zou W;Wang X;Wu Y;Zhao D;Sun Y;Liu Y;Chen L;Miao L;Yang C;Wang X
通讯作者：
Wang X
影响因子：
3.3
作者：
Ghanta KS;Mello CC
通讯作者：
Mello CC