Large-scale growth ofC. elegansand isolation of membrane protein complexes

Eric Gouaux, Sarah Clark, Hanbin Jeong, April Goehring, Yunsik Kang

Published: 2023-07-25 DOI: 10.1038/s41596-023-00852-5

C. elegans

membrane protein complexes

purification

structural studies

transgenic

AI 解读

Generation of mouse–human chimeric embryos

Genome-wide quantification of transcription factor binding at single-DNA-molecule resolution using methyl-transferase footprinting

Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning

A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues

Decoding personal biotic and abiotic airborne exposome

A clinically relevant model of focal embolic cerebral ischemia by thrombus and thrombolysis in rhesus monkeys

Bioengineering human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds

Biomimetic tail-to-head terpene cyclizations using the resorcin[4]arene capsule catalyst

Neuroscience robotics for controlled induction and real-time assessment of hallucinations

Scanorama: integrating large and diverse single-cell transcriptomic datasets

Single-cell RNA sequencing (scRNA-seq)（单细胞RNA测序）

Data integration（数据集成）

Heterogeneity（异质性）

查看全部

Sections

Figures

References

Introduction
Materials
Procedure
Troubleshooting
Timing
Anticipated results
- Reporting summary
Acknowledgements
Extended Data Fig. 1 Comparison of sonication and cryo-bead milling methods for lysis of C.

Avdesh, A. et al. Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction. J. Vis. Exp. http://doi.org/10.3791/4196 (2012).
Lishko, P. V. & Mannowetz, N. CatSper: a unique calcium channel of the sperm flagellum. Curr. Opin. Physiol. 2, 109–113 (2018).
Akopian, A. N., Chen, C. C., Ding, Y., Cesare, P. & Wood, J. N. A new member of the acid-sensing ion channel family. Neuroreport 11, 2217–2222 (2000).
Kefauver, J. M., Ward, A. B. & Patapoutian, A. Discoveries in structure and physiology of mechanically activated ion channels. Nature 587, 567–576 (2020).
Schwartz, V., Friedrich, K., Polleichtner, G. & Grunder, S. Acid-sensing ion channel (ASIC) 4 predominantly localizes to an early endosome-related organelle upon heterologous expression. Sci. Rep. 5, 18242 (2015).
Soler, D. C. et al. An uncharacterized region within the N-terminus of mouse TMC1 precludes trafficking to plasma membrane in a heterologous cell line. Sci. Rep. 9, 15263 (2019).
Kohl, K. et al. Plate-based large-scale cultivation of Caenorhabditis elegans: sample preparation for the study of metabolic alterations in diabetes. J. Vis. Exp. http://doi.org/10.3791/58117 (2018).
Heshof, R., Visscher, B., Promel, S. & Hughes, S. Large-scale cultivation of Caenorhabditis elegans in a bioreactor using a labor-friendly fed-batch approach. Biotechniques 67, 33–39 (2019).
Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).
Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).
Chatzigeorgiou, M., Bang, S., Hwang, S. W. & Schafer, W. R. tmc-1 encodes a sodium-sensitive channel required for salt chemosensation in C. elegans. Nature 494, 95–99 (2013).
Dao, J., Lee, A., Drecksel, D. K., Bittlingmaier, N. M. & Nelson, T. M. Characterization of TMC-1 in C. Elegans sodium chemotaxis and sodium conditioned aversion. BMC Genet. 21, 37 (2020).
Zhang, L. et al. TMC-1 attenuates C. elegans development and sexual behaviour in a chemically defined food environment. Nat. Commun. 6, 6345 (2015).
Wang, X., Li, G., Liu, J., Liu, J. & Xu, X. Z. TMC-1 mediates alkaline sensation in C. elegans through nociceptive neurons. Neuron 91, 146–154 (2016).
Tang, Y. Q. et al. Ankyrin is an intracellular tether for TMC mechanotransduction channels. Neuron 107, 112–125 e110 (2020).
Kaulich, E., Walker, D. S., Tang, Y. Q. & Schafer, W. R. The Caenorhabditis elegans tmc-1 is involved in egg-laying inhibition in response to harsh touch. MicroPubl Biol. 2021. http://doi.org/10.17912/micropub.biology.000439 (2021).
Wu, J. et al. GABA signaling triggered by TMC-1/Tmc delays neuronal aging by inhibiting the PKC pathway in C. elegans. Sci. Adv. 8, eadc9236 (2022).
Jeong, H. et al. Structures of the TMC-1 complex illuminate mechanosensory transduction. Nature 610, 796–803 (2022).
Dickinson, D. J. & Goldstein, B. CRISPR-based methods for Caenorhabditis elegans genome engineering. Genetics 202, 885–901 (2016).
Praitis, V., Casey, E., Collar, D. & Austin, J. Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157, 1217–1226 (2001).
Zanin, E. et al. Affinity purification of protein complexes in C. elegans. Methods Cell Biol. 106, 289–322 (2011).
Yue, X. et al. TMC proteins modulate egg laying and membrane excitability through a background leak conductance in C. elegans. Neuron 97, 571–585 e575 (2018).
Wang, C. et al. A conserved megaprotein-based molecular bridge critical for lipid trafficking and cold resilience. Nat. Commun. 13, 6805 (2022).
Caravaca, J. M. & Lei, E. P. Maintenance of a Drosophila melanogaster population cage. J. Vis. Exp. http://doi.org/10.3791/53756 (2016).
Panneels, V. & Sinning, I. Membrane protein expression in the eyes of transgenic flies. Methods Mol. Biol. 601, 135–147 (2010).
Panneels, V., Kock, I., Krijnse-Locker, J., Rezgaoui, M. & Sinning, I. Drosophila photoreceptor cells exploited for the production of eukaryotic membrane proteins: receptors, transporters and channels. PLoS One 6, e18478 (2011).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–E2586 (2012).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Clokey, G. V. & Jacobson, L. A. The autofluorescent “lipofuscin granules” in the intestinal cells of Caenorhabditis elegans are secondary lysosomes. Mech. Ageing Dev. 35, 79–94 (1986).
Zimmermann, T., Marrison, J., Hogg, K. & O'Toole, P. Clearing up the signal: spectral imaging and linear unmixing in fluorescence microscopy. Methods Mol. Biol. 1075, 129–148 (2014).
Teuscher, A. C. & Ewald, C. Y. Overcoming autofluorescence to assess GFP expression during normal physiology and aging in Caenorhabditis elegans. Bio. Protoc. http://doi.org/10.21769/BioProtoc.2940 (2018).
Lambert, T. J. FPbase: a community-editable fluorescent protein database. Nat. Methods 16, 277–278 (2019).
Stiernagle, T. Maintenance of C. elegans http://www.wormbook.org (2006).
Hibshman, J. D., Webster, A. K. & Baugh, L. R. Liquid-culture protocols for synchronous starvation, growth, dauer formation, and dietary restriction of Caenorhabditis elegans. STAR Protoc. 2, 100276 (2021).
Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A. & Ceron, J. Basic Caenorhabditis elegans methods: synchronization and observation. J. Vis. Exp http://doi.org/10.3791/4019 (2012).
Allen, P. C. New extraction method for nematode lipids. Anal. Biochem. 45, 253–259 (1972).

Large-scale growth ofC. elegansand isolation of membrane protein complexes

Extended

Extended Data Fig. 1 Comparison of sonication and cryo-bead milling methods for lysis of C. elegans .

Supplementary information

Reporting Summary

推荐阅读