Immunofluorescent localization of proteins in Caenorhabditis elegans muscle

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.

Figure 1. Comparison of immunolocalization after Nonet or Constant Spring fixation

Figure modified from (4). (A) Nonet method fixation of wild type worms with anti-UNC-94 (TMD-1) and either anti-UNC-89 (obscurin) or anti-α-actinin. The Nonet method shows clearly that UNC-94 (TMD-1) localizes to two closely spaced parallel lines flanking the M-line. (B) Constant-spring method fixation of wild type worms with anti-UNC-94 (TMD-1) shows broad I-band localization with no hint of the lines revealed by the Nonet method. The localization obtained with the Nonet method is consistent with likely localization to the pointed ends of the thin filaments and the known biochemical activity of other tropomodulins.