TTLL10 recruitment to microtubules decreases with increased polyglycylation. (A) TTLL10-SNAP(647) (cyan) association with monoglycylated microtubules (unlabeled, circled in yellow) and polygylcylated microtubules (HiLyte 488-labeled, magenta). The weighted mean of the number of glycines denoted as in Figure 1. For LC-MS of microtubules used in this assay see Figure S6. Assays performed at 500 nM TTLL10. Scale bar, 5 μm. (B). Quantification of TTLL10-SNAP(Alexa647) recruitment of differentially polyglycylated microtubules normalized to levels on monoglycylated microtubules; n = 59, 20, 28, and 33 microtubules from 10, 3, 3, and 4 independent experiments for the β+2.8G, β+4.3G, β+5.4G, and β+7.7G conditions, respectively. Statistical significance determined by Welsh’s t test, with p < 0.0001 (****) or p < 0.05 (*). (C) Representative images showing TTLL10-SNAP(647) association with monoglycylated and unmodified human microtubules as a function of time, in the presence of 1mM ATP with 1mM glycine (top two panels) and without 1mM glycine (bottom two panels). Scale bar, 5 μm. (D) Time courses of TTLL10-SNAP(Alexa647) recruitment to microtubules monoglycylated by TTLL8 (<nG>α ∼ 1.1, <nG>β ∼ 2.8) in the presence of 1 mM ATP with 1mM glycine (blue), 1 mM ATP without 1mM glycine (yellow), and unmodified microtubules in the presence of 1 mM ATP with 1mM glycine (black), and without 1mM glycine (green); n = 12 monoglycylated and 7 unmodified microtubules for the 1mM glycine time courses, and 8 monoglycylated and 8 unmodified microtubules for the glycine-free time courses. The same effect was observed across 3 independent experiments.