Impact of EB1 and/or ch-TOG depletion on microtubule organisation in interphase HeLa cells.
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(A) Parental HeLa cells and mEB1-expressing HeLa cells were treated with EB1 siRNA, fixed and stained for tubulin, and observed using a confocal microscope. Note that the microtubule density was reduced at the cell periphery in the EB1-depleted cells, while the expression of mEB1 rescued the phenotype. Scale bar, 20 µm. (B) The HeLa/RFP-α-tubulin clone (1A2) was treated with the indicated siRNAs and observed by TIRF microscopy. Many microtubules are visible at the cell periphery in cells expressing EB1 (arrowheads), but not in EB1-depleted cells (arrows). For rescue experiments, mEB1 or EB3 were stably introduced into the HeLa/RFP-α-tubulin clone (1A2) and treated with siRNAs against endogenous EB1. In both mEB1- and EB3-expressing cells, the microtubule density at the cell periphery was restored. Scale bar, 20 µm. (C) The microtubule numbers visualised by TIRF were analysed and plotted. Values significantly different from control are indicated with asterisks (***, P < 0.001; n = 146 in 41 cells for mock, n = 143 in 33 cells for EB1 siRNA, n = 106 in 39 cells for, n = 126 in 33 cells for EB1 + ch-TOG siRNA, n = 112 in 32 cells for EB1 siRNA + mEB1, n = 102 in 32 cells for EB1 siRNA + EB3). (D) A schema depicting EB1 function in the microtubule-anchoring process. EB1 binds to cortical +TIPs, including CLASPs, to mediate microtubule tethering. The linkage between EB1 and the cortex should still allow for ch-TOG-mediated tubulin addition at the very ends of the microtubules.