Abstract
Caenorhabditis elegans is a premier model genetic system for discovering new information about the assembly and maintenance of striated muscle. The localization of a protein within a nematode muscle cell can reveal important clues to its function. In C. elegans, proteins can be localized by two different methods at the light microscopy level: GFP tagged proteins and indirect immunofluorescence. Although there are advantages and disadvantages of each method, antibodies can be used to localize proteins expressed at endogenous levels and without tags that might interfere with function. Immunolocalization requires efficient and effective methods of fixation. Here, we describe in detail two different methods for fixation of adult worms, the Nonet method and the Constant Spring method. We also discuss the advantages and the disadvantages of each, and how to choose between them. These methods are also useful for localizing proteins expressed in other cell types.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Xiong G, Qadota H, Mercer KB, McGaha LA, Oberhauser AF, Benian GM (2009) A LIM-9 (FHL)/SCPL-1 (SCP) complex interacts with the C-terminal protein kinase regions of UNC-89 (obscurin) in Caenorhabditis elegans muscle. J Mol Biol 386:976–988
Miller RK, Qadota H, Stark TJ, Mercer KB, Wortham TS, Anyanful A, Benian, GM (2009) CSN-5, a component of the COP9 signalosome complex, regulates the levels of UNC-96 and UNC-98, two components of M-lines in Caenorhabditis elegans muscle. Mol Biol Cell 20:3608–3616
Qadota H, McGaha LA, Mercer KB, Stark TJ, Ferrara TM, Benian, GM (2008) A novel protein phosphatase is a binding partner for the protein kinase domains of UNC-89 (Obscurin) in Caenorhabditis elegans. Mol Biol Cell 19:2424–2432
Stevenson TO, Mercer KB, Cox EA, Szewczyk NJ, Conley CA, Hardin JD, Benian, GM (2007) unc-94 encodes a tropomodulin in Caenorhabditis elegans. J Mol Biol 374:936–950
Qadota H, Mercer KB, Miller RK, Kaibuchi K, Benian GM (2007) Two LIM domain proteins and UNC-96 link UNC-97/pinch to myosin thick filaments in Caenorhabditis elegans muscle. Mol Biol Cell 18:4317–4326
Miller RK, Qadota H, Landsverk ML, Mercer KB, Epstein HF, Benian, GM (2006) UNC-98 links an integrin-associated complex to thick filaments in Caenorhabditis elegans muscle. J Cell Biol 175:853–859
Mercer KB, Miller RK, Tinley TL, Sheth S, Qadota H, Benian, GM (2006) Caenorhabditis elegans UNC-96 is a new component of M-lines that interacts with UNC-98 and paramyosin and is required in adult muscle for assembly and/or maintenance of thick filaments. Mol Biol Cell 17:3832–3847
Small TM, Gernert KM, Flaherty DB, Mercer KB, Borodovsky M, Benian GM (2004) Three new isoforms of Caenorhabditis elegans UNC-89 containing MLCK-like protein kinase domains. J Mol Biol 342:91–108
Mercer KB, Flaherty DB, Miller RK, Qadota H, Tinley TL, Moerman DG, Benian, GM (2003) Caenorhabditis elegans UNC-98, a C2H2 Zn finger protein, is a novel partner of UNC-97/PINCH in muscle adhesion complexes. Mol Biol Cell 14:2492–2507
Miller DM, Shakes DC (1995) ImmunoÂfluorescence microscopy. Methods Cell Biol 48:365–394
Nonet ML, Grundahl K, Meyer BJ, Rand, JB (1993) Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin. Cell 73:1291–1305
Benian GM, Tinley TL, Tang X, Borodovsky M (1996) The Caenorhabditis elegans gene Âunc-89, required fpr muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains. J Cell Biol 132:835–848
Finney M, Ruvkun G (1990) The unc-86 gene product couples cell lineage and cell identity in C. elegans. Cell 63:895–905
Takeda K, Watanabe C, Qadota H, Hanazawa M, Sugimoto A (2008) Efficient production of monoclonal antibodies recognizing specific structures in Caenorhabditis elegans embryos using an antigen subtraction method. Genes Cells 13:653–665
Acknowledgments
We thank the NIH for research support (grant AR052133), and for a Fellowship in Research and Science Teaching (FIRST) postdoctoral fellowship (K12GM000680), also from the NIH, for supporting K.J.W.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Wilson, K.J., Qadota, H., Benian, G.M. (2012). Immunofluorescent Localization of Proteins in Caenorhabditis elegans Muscle. In: DiMario, J. (eds) Myogenesis. Methods in Molecular Biology, vol 798. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-343-1_10
Download citation
DOI: https://doi.org/10.1007/978-1-61779-343-1_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-342-4
Online ISBN: 978-1-61779-343-1
eBook Packages: Springer Protocols