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. 2015 Sep 15;128(18):3420–3434. doi: 10.1242/jcs.169318

Fig. 2.

Fig. 2.

Impact of GFP-IPAM90-268 on MT organization, centrosome integrity and MT assembly. (A) HeLa cells expressing GFP-IPAM90-268 (green) were fixed and stained for γ-tubulin or pericentrin (both red). Fluorescence panels show maximum projection of three confocal z-sections spanning the centrosome. Scale bars: 5 µm. (B) 3D reconstructions from structural illumination microscopy (SIM). Scale bars: 2 µm. (C) Representative images of HeLa cells expressing high levels of GFP-IPAM90-268 (green) that had been fixed and stained for γ-tubulin or pericentrin (both red). Scale bars: 5 µm. (D) Following confocal imaging, the features indicated by γ-tubulin (top panels) or pericentrin (bottom panels) labeling were categorized (as shown in supplementary material Fig. S2B) using cells expressing GFP, GFP-IPAM90-268 low or GFP-IPAM90-268 high. *P<0.05, **P<0.01, ***P<0.005. (E) HeLa cells expressing low or high levels of GFP-IPAM90-268 (green) were fixed and stained for α/β-tubulin (red). Panels show maximum projection of the entire z-stack. Scale bars: 5 µm. (F) HeLa cells were transfected with GFP-IPAM90-268 or GFP (green) and labeled for DNA (blue) and for α/β-tubulin (red). Panels show cells in division. Scale bars: 5 µm. (G) MTs were depolymerized in HeLa cells expressing GFP-IPAM90-268 (green) and allowed to regrow for 5 min prior to fixation and staining for α/β-tubulin (red). Panels show maximum projection of four confocal z-sections. Cell periphery is outlined in white, N indicates the nucleus. Scale bars: 5 µm. (H) MTs were depolymerized in HeLa cells expressing GFP, GFP-IPAM90-268 low or GFP-IPAM90-268 high (green). Panels show SIM reconstructions of MTs allowed to regrow for 5 min prior to fixation and staining for α/β-tubulin (red). The control GFP signal is absent because diffuse signals cannot be visualized by using SIM. Scale bars: 2 µm. (I) Following confocal imaging, MT architecture in individual cells expressing GFP or GFP-IPAM90-268 was categorized (as shown in supplementary material Fig. S2C) after MT depolymerization and 5 min of regrowth. ***P<0.005.