PMC

Dependence of CLASP2 deletion mutants on CLIP-170 for their localization to the MT tips and EB1 recruitment to the MTs. (A and B) COS-7 cells were transfected with GFP-CLASP2-ΔM and stained for CLIP-170. (C) Schematic representation the structure of CLIP-170 and the dominant negative construct, used in this study. (D–F) COS-7 cells were cotransfected with GFP-CLASP2-ΔM and the dominant negative CLIP-170, and stained for EB1 and the endogenous CLIP-170, using the antibody against its NH₂ terminus. The cells, expressing the dominant negative CLIP-170 construct, can be recognized by the diffuse pattern of endogenous CLIP-170 staining. (G–I) COS-7 cells were cotransfected with GFP-CLASP2-M and the dominant negative CLIP-170, and stained for EB1, the endogenous CLIP-170 (using the antibody against its NH₂ terminus) and the dominant negative CLIP-170 (using the antibody against its COOH terminus). (J–L) COS-7 cells were transfected with GFP-CLASP2γ (J) and stained for CLIP-170 (K) and EB1 (L). (M–O) COS-7 cells were transfected with GFP-CLASP2-ΔC (M) and stained for CLIP-170 (N) and EB1 (O). Enlarged portions of the boxed areas are shown in the insets. Bars, 10 μm.

Characterization of CLASP-specific antibodies and RNAi tools. (A) Lysates of HeLa cells, transfected with GFP-CLASP1α (lane 1), GFP-CLASP2α (lane 2), or mock transfected (lane 3), were analyzed by Western blotting with the indicated antibodies. (B) Lysates of HeLa cells, transfected with the indicated siRNAs, were prepared 24, 48, or 72 h after transfection and analyzed by Western blotting with the indicated antibodies. (C and D) Lysates of HeLa cells, transfected with the indicated siRNAs, were prepared 72 h after transfection and analyzed by Western blotting with the indicated antibodies. (E) HeLa cells, transfected either with the control siRNA, CLASP1+2#A or #B siRNAs, were stained with the mixture of antibodies #402 or #2358 72 h after transfection and analyzed by FACS. (F) HeLa cells were stained with a mixture of antibodies #402 (CLASP1) and #2358 (CLASP2) and either α-tubulin or the Golgi marker GM130. Bars, 10 μm. (G) HeLa cells, transfected either with the control siRNA or the CLASP1+2#B siRNAs, were stained with antibodies #402 or #2358 72 h after transfection. Bars, 10 μm.

The COOH-terminal domain of CLASP2 is responsible for association with the cell cortex and Golgi complex. (A–F) TIRF microscopy images of live HeLa cells, expressing GFP-CLASP1α (A), GFP-CLASP2γ (B), GFP-α-tubulin (C and D), or EB3-GFP (E and F). Cells were either not treated with siRNAs (A and B), or treated for 72 h with control (C and E) or CLASP1+2#B siRNAs (D and F). The contrast is inverted. Bars, 10 μm. (G) Schematic representation of CLASP2γ and the relevant deletion mutants. (H) HeLa cells were transfected with GFP-CLASP2-C and stained for the Golgi marker GM130. Bar, 10 μm. (I) HeLa cells were transfected with GFP-CLASP2 or GFP-CLASP2-ΔC and were either fixed directly or treated with 10 μM nocodazole for 1 h before fixation and stained for α-tubulin. Bars, 10 μm.

Effect of CLASP knockdown on MT dynamics. (A) Time-lapse images of HeLa cells, stably expressing GFP-α-tubulin, 72 h after transfection with the control or CLASP1+2#A siRNAs. For rescue, CLASP1+2#A siRNA-treated cells were transfected with mRFP-CLASP2α 48 h after the siRNA transfection and observed 24 h later. In the “rescue” panel mRFP-CLASP2α is shown in red and GFP-α-tubulin is shown in green. Bars, 5 μm. (B) Time of staying of MT ends within 1 μm distance from the cell edge, determined from the time-lapse image series as shown in A (cells were imaged with 2-s interval). The difference between the control and CLASP knockdown is statistically significant (P < 0.001), the difference between control and rescue is not significant. (C) Time-lapse images of HeLa cells, stably expressing EB1-GFP, 72 h after transfection with the control or CLASP1+2#A siRNAs. Rescue was performed as described in A. In the “rescue” panel mRFP-CLASP2α is shown in red and EB1-GFP is shown in green. Cell margin is indicated by an arrow. Bars, 5 μm. (D) Length of the shortening excursions from the cell edge, measured from the same dataset as in A and B. Error bars represent the SD.

Identification of the MT plus-end binding and EB1-binding domains in CLASP1 and 2. (A–G) GFP fusions of CLASP2α, CLASP2γ, different CLASP2γ deletion mutants, CLASP1α and its deletion mutants were transfected in COS-1 or COS-7 cells, fixed and counterstained for EB1, and the MT plus-end accumulation was assessed as: ++, strong; +, clearly visible; +/−, weakly detectable, −, undetectable. B, C, and F, GFP signal; D, E, and G, EB1 staining. Bars, 10 μm. (H) Alignment of the repetitive part of the CLASP2-M fragment with the corresponding regions of CLASP1 and D. melanogaster Orbit/MAST. Repeats of different types are indicated by different arrows. (I) Coomassie-stained gel, showing purified HIS-tagged GFP-CLASP2-M and GFP. (J) MT pelleting assay with purified GFP-CLASP2-M and GFP. Coomassie-stained gel is shown for tubulin and Western blots with anti-GFP antibodies for the GFP fusions. S, supernatant; P, pellet. (K) Immunoprecipitation with anti-GFP antibodies from COS-1 cells, transfected with the indicated GFP-CLASP1 and 2 deletion mutants. (L) Schematic representation of EB1 structure and the deletion mutants used in this study. CH, calponin homology domain; CC, coiled coil; Ac, acidic tail domain. (M) GST pull-down assays with the indicated GST fusions. Purified GFP-CLASP2-M and GFP or extracts of COS-1 cells, overexpressing GFP-CLASP1α, GFP-CLASP2, and GFP-CLASP2-ΔM were used. Coomassie-stained gel is shown for the GST fusions and Western blots with anti-GFP antibodies for the GFP fusions. 10% of the input and 25% of the material, bound to the beads, were loaded on gel. (N) Immunoprecipitation with anti-GFP antibodies from COS-1 cells, transfected with the indicated GFP-EB1 fusions. (O) Immunoprecipitation with a rabbit polyclonal antibody against EB1 or a control rabbit IgG from untransfected COS-1 cells.

Rescue of CLASP knockdown with CLASP2 deletion mutants and a model for CLASP action at the cell cortex. (A–F) HeLa cells, depleted for CLASPs as described in A, were transfected with different rescue constructs (indicated on the left), fixed with methanol and stained for α-tubulin (A–D) or EB1 (E and F). Cell images were collected with the confocal microscope (1-μm-thick optical sections). GFP (green) and tubulin/EB1 (red) signals are superimposed on the right. Enlarged portions of the boxed areas are shown in the insets. Bars, 10 μm. (G) Average of pixel intensity of α-tubulin staining (∼10 cells per construct). (H) Ratio of the average pixel intensity of MT fluorescence (I_MT) to the integrated intensity of GFP fluorescence (I_GFP; ∼10 cells per construct). (I) Proposed model of CLASP rescue activity at the cell cortex. CLASPs can interact with the plus end of a growing MT directly and/or through the association with EB1 and the same time make contact with the cell cortex. After the MT undergoes a catastrophe, most of the EB1 proteins are lost from the tip, but CLASPs remain associated with the cortex and the peripheral stretches of the depolymerizing MT. By enhancing the affinity of EB1–MT interaction at these sites, CLASPs help to retain and/or recruit EB1 to such MTs, leading to their rescue. The oscillations of EB1 signals at the cortical MT tips in control cells support this idea (with strong EB1 accumulation corresponding to growth episodes, and weak EB1 accumulation observed during pausing/depolymerization events). Error bars represent the SD.

Effect of CLASP knockdown on the interphase MT network. (A and B) HeLa cells 72 h after transfection with the control siRNA (A) or CLASP1+2#B siRNAs (B), fixed with methanol, and stained for α-tubulin. Bars, 10 μm. (C) Plots of integrated intensity of fluorescence of α-tubulin staining, which was performed as in A and B, in HeLa cells 72 h after transfection with the indicated siRNAs. The integrated intensity was measured within a 5-μm² box at 5 μm distance from the cell edge, with subtracting the background. Measurements were performed in ∼30 cells per siRNA in three different cell regions. Values significantly different from the control (P < 0.001) are indicated by asterisks. (D) Number of MT ends in a trapezoid part of the cell sector (with a base b and side h) in HeLa cells 72 h after transfection with the control siRNA (30 cells, n = 587) or CLASP1+2#B siRNAs (30 cells, n = 401). (E) Integrated intensity within a 5-μm² box was measured along the cell radius in the same cells as described in D. MTOC, the MT organizing center, was determined as the site of radial gathering of MTs. (F) HeLa cells, transfected either with the control siRNA or the CLASP1+2#B siRNAs, were methanol fixed and stained for CLASPs (using with the mixture of antibodies #402 or #2358) and for tubulin 72 h after transfection. Representative cells were selected to include examples with both high and low levels of CLASP staining. Integrated intensity of CLASP staining was plotted versus integrated intensity of tubulin staining. Each dot represents an average of five independent measurements in different areas at the periphery of one cell. The regression line is shown in red and the 95% confidence interval is indicated by gray lines. (G) Distribution of distances from MT ends to the cell edge within the trapezoid part of the cell sector shown in D. Analyzed images were the same as in D. (H) Distribution of angles of distal segments of MTs to the cell radius. Analyzed images were the same as in D. (I) Confocal microscope images (1-μm optical sections) of HeLa cells, fixed with formaldehyde 72 h after transfection with the control or CLASP1+2#B siRNAs and stained for α-tubulin. Control cells were also incubated for 1 h at 4°C before fixation, to depolymerize MTs (“cold treated”). Bar, 10 μm. (J) Plots of the ratio of the intensity of soluble tubulin staining I_S (after subtracting background) to the intensity of polymer staining I_P (after subtracting background and I_S), obtained from the images as shown in H. Measurements were performed in 20 control cells (n = 158), 35 CLASP-knockdown cells (n = 225), and 18 cold-treated cells (n = 130). Error bars represent the SD.