Lanine) (Fig. S3A). Taking into consideration that in total five serines and four threonines were identified to be phosphorylated, it was conceivable that the general phosphorylation degree of EB1 was not significantly altered by single-site or single-motif mutations. By contrast, the phosphorylation of Y71 and Y217 contributed significantly for the all round tyrosine phosphorylation of EB1, as both of the phospho-deficient mutants showed dramatically reduced signals in the immunoblot, with antibodies against phosphorylated tyrosine (Fig. S3B). We then overexpressed wild-type or mutant EB1 to investigate whether the phosphorylation at certain internet sites affects EB1 interaction with microtubules/ tubulin. It turned out that none with the EB1 mutants changed its interaction with -tubulin within the GST pulldown assays (Fig. S3C ). Immunofluorescence microscopy further revealed that all the mutants of EB1 have been located in the plus finish of microtubules within a pattern equivalent to wildtype EB1 (Fig. S3F). To analyze the effect of EB1 phosphorylation on microtubule dynamics, we overexpressed GFP-EB1 wild-type and mutants and took serial photos by time-lapse microscopy. We then utilized the PlusTipTracker software program to analyze the dynamics of microtubules (Matov et al., 2010). As outlined by the imply development speed (15 m/min) and mean development time (9 s) of wild-type EB1, we divided the microtubule population into four groups (Fig. 1A and 1B). We considered cells using a high percentage of fast-growth and long-lived microtubules highly dynamic. Each T33A and T33D decreased the percentage of fast-growth and long-lived microtubules, and also the development speed and development length decreased significantly as in comparison with wild-type EB1 (Fig. 1C ), indicating the value of T33 for EB1 to regulate microtubule dynamics. As for Y71, TSSS, and T206, we found that all the phospho-deficient mutants decreased the dynamics, development speed, and development length of microtubules, whereas the phospho-mimic mutants either maintained or promoted the dynamics, growth speed, and development length of microtubules (Fig. 1C ). As for S27 and ST, despite the fact that the phospho-deficient mutants didn’t significantly lower the dynamics, growth speed, or growth length?The Author(s) 2014. This short article is published with open access at Springerlink and journal.1379812-12-0 custom synthesis hep.Vanadium(IV)bis(acetylacetonato)oxide Chemical name cnProtein CellLETTERJie Chen et al.PMID:23710097 AFast-growth, long-lived ( )15 /min 9s15 /min 9sC40 30 20 ten 0 ** *** *** **** ** ****D Growth speed ( /min)20 15 10 five * *** *** *** * *** **B WT S27A S27D T33AProtein CellT33D Y71F Y71D TSSSAAAA TSSSDDDD STAA STDD T206A T206D F WT Y217FE Development length ( )3.0 2.five two.0 1.five 1.0 * ** ** *** * *** *** **Y217DComet/BackgroundEnlargementFigure 1. EB1 phosphorylation at diverse websites modulates microtubule dynamics to diverse extents. (A ) HeLa cells had been transfected with GFP-EB1 or many mutants, and time-lapse images of GFP-EB1 were taken by confocal microscopy at 2-second intervals. The pictures had been analyzed with the PlusTipTracker software and also the Quadrant Scatter Plot tool. Microtubules have been classified into 4 subpopulations based on the mean growth speed (15 m/min) and imply growth time (9 s) of GFP-EB1, and representative images had been shown in (A). The partitioning in the 4 subpopulations of microtubules was shown in (B), and the percentage of fast-growth and long-lived microtubules was shown in (C). (D) Imply growth speed of microtubules. (E) Imply development length of microtubules. (F) Cells had been transfected with GFP-EB1 wild-type or the Y.
