Based on our success using a green fluorescent protein (GFP) fused to an actin binding protein, we constructed a second generation fusion protein. It consists of humanized (for codon bias) GFP containing the S65T mutation (CLONTECH Laboratories), which speeds protein folding and increases the quantum efficiency of the GFP, fused to the same fragment of moesin that includes the extended helical region and the actin binding sequences (Edwards et al. 1997). Our original construct (called hsGFPmoe) used a heat shock driven promoter because we feared that constitutive expression of the moesin fusion construct might have deleterious effects on fly development. Based on our observations that flies harboring the hsGFPmoe transgene could be heat-shocked daily and still survive as a viable stock, we used a promoter/enhancer construct from the ubiquitously expressed spaghetti squash gene, which encodes the single, nonmuscle myosin II regulatory light chain (Karess et al. 1991; Wheatley et al. 1995; Edwards and Kiehart 1996; Jordan and Karess 1997). This construct, called sGMCA, was used to establish stable transgenic fly lines through P element–based germ line transformation. The construct appears to be expressed ubiquitously and does not appear to be deleterious to any aspect of fly development or behavior, although the stocks are not as robust as our healthiest stocks (e.g., wild-type Oregon R or w1118). The line that we use most is called sGMCA-3.1 and has the transgene construct inserted on the third chromosome, but other insertions behave in an indistinguishable fashion. Fly stocks can be established in which the sGMCA-3.1 chromosome is homozygous, demonstrating that the transgenic construct is inserted in a nonessential part of the genome. The complete sequence of sGMCA in its P element vector is provided as Supplemental Figure 1 (sGMCA sequence and annotation), which is available at http://www. jcb.org/cgi/content/full/149/2/471/DC1.