Brightness and contrast were optimized individually for display

Brightness and contrast were optimized individually for display. Analysis of microtubule nucleation in egg extracts Number of MTs and their growth speed in extracts was measured using a combination of custom-built and existing MATLAB software. essential for MT nucleation in egg extracts. The concentration of XMAP215 determines the extent of MT nucleation. Even though XMAP215 and -tubulin ring complex (-TuRC) possess minimal nucleation activity individually, together these factors synergistically stimulate MT nucleation from high concentrations of -tubulin dimers1. In contrast, spontaneous MT formation rarely occurs and egg extracts to resolve individual nucleation events, we show that XMAP215 determines the extent of MT nucleation in the cytoplasm. XMAP215 and -TuRC operate synergistically to generate MTs egg extracts induced by the dominant active form of the small GTPase Ran (RanQ69L)36. We visualized MT nucleation from pre-existing MTs using time-lapse total internal reflection microscopy with Cy5-labeled MTs and mCherry-labeled end-binding protein 1 (EB1) SIRT-IN-1 to spotlight the growing MT plus-ends (Supplementary Fig. 1A). To assess XMAP215s role, we first added recombinant XMAP215 to egg extracts, which contains approximately 120 nM endogenous XMAP21526. We expected the branched structures to appear larger due to longer and faster growing MTs, but less dense due to unaltered level of MT nucleation26. We quantified the number of MTs by SIRT-IN-1 detecting each plus-tip as the readout for MT nucleation. Surprisingly, XMAP215 increased the number of MTs in a concentration-dependent manner (Supplementary Fig. 1ACB, Supplementary Video 1). Addition of 480 nM XMAP215 increased MT numbers up to 30-fold, while the growth velocity of MTs remained nearly constant (Supplementary Fig. 1BCC). These results show that XMAP215 has a dramatic effect on MT generation. XMAP215 increases nucleation events, but is it required to generate MTs? To assess this, we immunodepleted XMAP215 from extracts (Fig. 1A). Remarkably, MT nucleation was completely abolished in the absence of XMAP215 (Fig. 1B). This was confirmed by observing the reaction up to an hour, throughout the entire sample (2000 fields of view), and across tens of extract preparations. This phenotype has previously been observed only by immunodepletion of -tubulin, the universal MT nucleator36. To verify the specificity of this effect, we added back purified XMAP215, which rescued the branched structures with comparable MT count to the control-depleted extracts (Fig. 1ACB, Supplementary Fig. 1D). Proteins such as TACC3, -tubulin, augmin, TPX2 and Rabbit Polyclonal to MAP9 -TuRC were not depleted (Supplementary Fig. 1E). In summary, XMAP215 is required for generating MTs in egg extracts. Open in a separate window Physique 1 XMAP215 stimulates microtubule nucleation in egg extract(ACB) Western blot SIRT-IN-1 analysis of XMAP215 or IgG immunodepletion from extracts and add-back of wild-type XMAP215-GFP, probed using antibody against TOG12 domain name. Branching MT nucleation in extracts in the presence of 5.5M RanQ69L. EB1-mCherry (pseudo-colored as green) and Cy5-labeled tubulin (red) was added. XMAP215-GFP was added back at 85nM (equivalent to 120nM in IgG-depleted extract). Representative images are displayed at 20 minutes of the reaction. Scale bar, 10m. The experiments were repeated at least three times with impartial extract preparations. (C) Increasing concentration of XMAP215-GFP added back to immunodepleted extract with 5.5M RanQ69L. Representative images are displayed at 480 seconds. Scale bar, 10m. See Supplementary Video 2. The experiment was repeated four occasions with impartial extract preparations. (D) EB1 comets in the entire field of view were detected, counted and plotted with time. The analyses in (DCF) were repeated at least thrice with impartial extract preparations. (E) Growth velocity of MTs was obtained by tracking all EB1 comets observed over during the experiment. No MTs nucleated below 30nM XMAP215, and growth speed was not SIRT-IN-1 measured. At 30nM, growth velocity was measured manually as 2.30.8 m/min (mean s.d.; n=25). For 60nM and above, growth velocity was computed via image analysis: 60nMC8.01.9 (n=1470), 85nMC8.51.8 (n=18190), 120nMC9.32.0 (n=45090), 240nMC9.62.2 (n=59297), 480nMC10.52.4 (n=79381) and 720nMC11.12.7 m/min (n=147008). n.