Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3.

The regulation of microtubule dynamics is critical to ensure essential cell functions. End binding protein 1 (EB1) is a master regulator of microtubule dynamics that autonomously binds an extended GTP/GDP-Pi structure at growing microtubule ends and recruits regulatory proteins at this location. However, negative regulation of EB1 association with growing microtubule ends remains poorly understood. We show here that microtubule-associated tumor suppressor ATIP3 interacts with EB1 through direct binding of a non-canonical proline-rich motif. Results indicate that ATIP3 does not localize at growing microtubule ends and that in situ ATIP3-EB1 molecular complexes are mostly detected in the cytosol. We present evidence that a minimal EB1-interacting sequence of ATIP3 is both necessary and sufficient to prevent EB1 accumulation at growing microtubule ends in living cells and that EB1-interaction is involved in reducing cell polarity. By fluorescence recovery of EB1-GFP after photobleaching, we show that ATIP3 silencing accelerates EB1 turnover at microtubule ends with no modification of EB1 diffusion in the cytosol. We propose a novel mechanism by which ATIP3-EB1 interaction indirectly reduces the kinetics of EB1 exchange on its recognition site, thereby accounting for negative regulation of microtubule dynamic instability. Our findings provide a unique example of decreased EB1 turnover at growing microtubule ends by cytosolic interaction with a tumor suppressor.

(model INUG2E-ZILCS, Tokai Hit) set at 37°C, a Photometrics Evolve 512 EMCCD camera and controlled with MetaMorph 7.7 software. For excitation of GFP we used a Cobolt Calypso 491 nm (100 mW) laser and green fluorescent light was collected via a ET-GFP filter set (Chroma). Inset montages were denoised using the ImageJ Safir Filter plugin [3]. Four iterations and a patch size of 1 were used as parameters of the denoising process.

Fluorescence recovery after photobleaching (FRAP) analyses
FRAP was performed on an inverted Ti-E (Nikon) Nikon spinning disk microscope. EB1 comets were bleached using a FRAP-on-fly-spiral option (Roper Scientific France/PICT-IBiSA), which allows bleaching of a selected circular region of 0.5 µm in diameter. We used 100% (7-15 kW/cm 2 ) of 491nm laser power for photobleaching. Time-lapse images were acquired for at least 3s before bleaching and for 10s after bleaching. Image acquisition was done at 150 ms of exposure.
For analysis, Safir software (Roper Scientific France) was used to denoise time-lapse images.
Fluorescence recovery was measured in a region of 2 x 2 pixels that comprised part of the fluorescent MT end. Bleached MT ends and non-bleached cytoplasmic regions (background) were analyzed. To derive exponential decay curves for EB1-GFP, cytoplasmic background values were subtracted, and curves were normalized to 1. The average decay of fluorescence peaks was fitted as a first-order exponential decay with a robust fitting routine: y = (y 0plateau) * exp (-kdecay*x) + plateau, where y is normalized intensity, y 0 is the initial fluorescence value, plateau is the y value at infinite times, (y 0plateau) is the span of the reaction, k decay is the reaction constant, and x is the time. Quantification was performed on bleached comets of 17 cells (siCtrl) and 16 cells (siATIP3) from 2 independent experiments.
The same experimental procedure and analysis were used to measure fluorescence recovery of cytosolic EB1-GFP signals, except that bleaching was performed in selected regions (2 x 2 pixels) of the cytosol.  Number of EB1-GFP comets analyzed is under brackets.
(B). Quantification of EB1 decoration time (s) calculated as the ratio of comet length to microtubule growth rate. Time-lapse TIRF videomicroscopy analysis of HeLa cells co-transfected with siRNAs (control (siCtrl) or ATIP3-specific (siATIP3)) and EB1-GFP is described in Molina et al. [17]. Quantification was performed on 10 cells (siCtrl) and 7 cells (siATIP3) from 2 independent experiments. Number of EB1-GFP comets analyzed is under brackets.
(D). Quantification of k recovery (s-1 ), association time Tau in seconds (s) and half-time of recovery in seconds (s), of EB1-GFP fluorescence recovery in the cytosol of control and ATIP3-depleted cells.
For each condition, 4 bleached areas were analyzed from 4 single cells in 2 independent experiments.