SLAIN2 and ch-TOG promote MT growth. (A) HeLa cells stably expressing GFP– or mCherry–α-tubulin were transfected with the indicated siRNAs. FRAP assay was performed 72 h later in an internal part of the lamella indicated by a stippled line. Video frames at 33 s after FRAP are shown. Newly polymerized MTs are indicated by arrows. (B) Share of freshly polymerized MT segments longer than 2 µm 30 s after FRAP. Approximately 230–300 growth episodes were analyzed in 15–20 cells for each condition. (C and D) Mean MT elongation rate was measured in internal cytoplasm over periods of 10–50 s from the moment of appearance of the growing MT end until the end of the video or until a catastrophe leading to a processive MT-shortening episode with the length of >1 µm. Analysis was performed in HeLa cells stably expressing EB3-GFP that were transfected with the indicated siRNAs (C) or in HeLa cells stably expressing mCherry–α-tubulin transfected with GFP or GFP–SLAIN1/2-N1 (D). Approximately 70–100 growth episodes in 10–20 cells were analyzed for each condition. (E) Kymographs illustrating MT growth using mCherry–α-tubulin or EB3-GFP after different siRNA treatments or in cells expressing GFP alone (control) or GFP–SLAIN1/2-N1. (F) CHO cells were transiently transfected with EB3-GFP and the indicated shRNAs. Live images were collected with a 0.5-s time interval. Single frames (top) and maximum intensity projections of 100 frames (bottom) are shown. (G) Length of EB3-GFP comets in control, SLAIN2, or ch-TOG–depleted cells determined from live imaging experiments shown in F. Approximately 300 MT tips were analyzed in ∼15 cells per condition. (H and I) Proportion of MT tracks originating from the centrosome with the length exceeding 7.5 µm (H) and MT nucleation frequency from the centrosome in CHO cells transiently transfected with EB3-GFP and the indicated shRNAs (I). In H, ∼100 MT growth episodes were analyzed in ∼15 cells per condition. In I, ∼10 cells were analyzed per condition. (J and K) MT recovery after nocodazole washout. HeLa cells expressing GFP or GFP–SLAIN1/2-N1 for 1 d were treated with 10 µm nocodazole for 2 h. The drug was washed out with fresh medium, and cells were fixed and stained 10 min later (J). The number of MTs per 100 µm² was counted in 20 cells for each condition (K). (L and M) MT organization in HeLa cells transiently expressing GFP or GFP–SLAIN1/2-N1 1 d after transfection. Cells were stained for β-tubulin (L), and the angles of MT segments in relation to the long axis of the lamella were measured. Angle distributions measured in 10 cells and their Gaussian fits are shown for each condition (M). Transfected cells are indicated by asterisks in J and L. In C, D, G–I, and K, the values significantly different from controls are indicated with asterisks (**, P < 0.01; ***, P < 0.001).