Fig. 2. MgcRacGAP tracks with EB3 at growing microtubule plus ends. (A) Amino acid sequence alignment surrounding the SxIP motif (yellow box) of five known EB1-binding proteins and the SxIP motif in the C-terminus of X. laevis Mgc. Basic arginine (R) and lysine (K) residues are shown in blue. Stop codons are indicated by *. (B) Mgc domain diagram. Endogenous Mgc is knocked down with MOs that target the 5′UTR; MgcWT and MgcSxNN constructs do not contain the endogenous 5′ UTR sequence, so they are not targeted by the MOs. The location of the putative SxIP motif in Mgc (red circle) and the SxNN mutation (blue square) are indicated. (C) TIRF microscopy image of a Cos-7 cell transfected with EB3–mChe (red) and MgcWT–mNeon (green). Scale bar: 20 µm. (C′) An enlarged view of the boxed region in C showing that EB3–mChe and MgcWT–mNeon accumulate on the plus ends of microtubules (yellow arrowheads). Scale bar: 5 µm. (D) Representative kymographs following an EB3–mChe comet in cells co-expressing EB3–mChe and MgcWT–mNeon. Scale bar: 4.3 µm (distance), 1 sec (time). (E) TIRF microscopy image of a Cos-7 cell expressing EB3–mChe (red) and MgcSxNN–mNeon (green). Scale bar: 20 µm. (E′) An enlarged view of the boxed region in E showing EB3–mChe comets (yellow arrowheads) and the strongly reduced corresponding signal in the MgcSxNN–mNeon channel. Scale bar: 5 µm. (F) Representative kymographs following an EB3–mChe comet in cells co-expressing EB3–mChe and MgcSxNN–mNeon. The total time represented is 6.5 s; scale bar: 4.3 µm (distance). (G–I) Quantification of (G) Mgc–mNeon intensity at EB3 comets. MgcWT=26 kymographs, 9 cells; MgcSxNN=25 kymographs, 8 cells. (H) EB3–mChe run duration on MT plus ends. Control=35 EB3 comets, 7 cells; MgcWT=50 EB3 comets, 10 cells; MgcSxNN=50 EB3 comets, 10 cells. (I) Velocity of EB3–mChe comets. Control=15 EB3 comets, 7 cells; MgcWT=17 EB3 comets, 9 cells; MgcSxNN=17 EB3 comets, 9 cells. Mean±s.e.m. (unpaired Student’s t-test). All n values represent cumulative values from three independent experiments. ns, not significant.
Image published in: Breznau EB et al. (2017)
Copyright © 2017. Image reproduced with permission of the Publisher, The Company of Biologists Ltd.
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