1. Volume 11, Issue 1, Pages 21-32 (July 2006)
CLASPs Attach Microtubule Plus Ends to the Cell Cortex through a Complex with LL5β 
Gideon Lansbergen, Ilya Grigoriev, Yuko Mimori-Kiyosue, Toshihisa Ohtsuka, Susumu Higa, Isao Kitajima, Jeroen Demmers, Niels Galjart, Adriaan B. Houtsmuller, Frank Grosveld, Anna Akhmanova 
Developmental Cell 
Volume 11, Issue 1, Pages (July 2006)
DOI: /j.devcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Identification of LL5β and ELKS as CLASP Binding Partners
(A–C) Streptavidin pull-down assays were performed with lysates of HeLa cells coexpressing bio-GFP-CLASP2α together with BirA, bio-GFP-ELKSα together with BirA, bio-GFP-LL5β together with BirA, or BirA alone. Proteins bound to streptavidin beads were analyzed on a Coomassie-stained gel (A) or by Western blotting with the indicated antibodies (B and C). In (A), only the fraction bound to the beads was loaded. In (B) and (C), the extract before the pull down (lanes marked “E”) and proteins bound to the beads (lanes marked “B”) are shown. Lanes marked “E” correspond to 25% of the extract used to obtain lanes marked “B.”
(D) Protein extracts of HEK293 cells overexpressing GFP-CLASP1α, HA-LL5β, or Myc-ELKSɛ were mixed in the indicated combinations, followed by IP with the anti-GFP antibody. IPs were analyzed by Western blotting with the indicated antibodies.
(E) IPs from extracts of untransfected HeLa cells with the indicated antibodies. Lane marked “Ext” shows 5% of the input. In the experiments shown in this figure, rabbit antibodies against LL5β and ELKS were used.
Developmental Cell  , 21-32DOI: ( /j.devcel )
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3. Figure 2
Colocalization of CLASPs, LL5β, and ELKS at Peripheral Cortical Sites of HeLa Cells
(A–C) HeLa cells were fixed with methanol and stained with a mixture of antibodies against CLASP1 and CLASP2 ([A and B], green in the overlay), mouse anti-LL5β antibody ([A], red in the overlay), rabbit anti-LL5β antibody ([C], green in the overlay) and mouse anti-ELKS antibody ([B and C], red in the overlay).
(D) HeLa cells were transfected with RFP-LL5β (red in the overlay) and GFP-ELKSɛ (green in the overlay), fixed with PFA, and imaged directly. Note that the perinuclear signal observed with the rabbit anti-LL5β antibody in (C) is probably background because it is not present in the staining with the mouse anti-LL5β antibody or in the RFP-LL5β pattern.
(E) Epifluorescence (EPI, green in the overlay) and TIRF microscopy (red in the overlay) images of live HeLa cells expressing GFP-LL5β.
(F) HeLa cells were transfected with GFP-LL5β (green in the overlay), fixed with PFA, and stained with antibodies against a FA marker phosphotyrosine (red in the overlay). White rectangles indicate the portion of the figure shown enlarged in the left corner of each panel. Bars, 10 μm.
Developmental Cell  , 21-32DOI: ( /j.devcel )
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4. Figure 3
LL5β and ELKS Are Required for Efficient Cortical Accumulation of CLASPs in HeLa Cells
(A) Western blot analysis of the extracts of HeLa cells cultured for 72 hr after transfection with the indicated siRNAs.
(B–I) HeLa cells were cultured for 72 hr after transfection with the indicated siRNAs. Cells were fixed with methanol (B–E and G–I) and stained with a mixture of antibodies against CLASP1 and CLASP2 (B, C, D, G, and I), mouse anti-LL5β antibodies (B, D, and I), rabbit anti-LL5β antibodies (E and H), and mouse anti-ELKS antibodies (C, E, G, and H). In (I), the part of the image indicated by the white square is enlarged and shown as an overlay, with CLASP staining in green and LL5β staining in red. In (F), cells were transfected with GFP-ELKSɛ 48 hr after siRNA transfection, fixed with PFA 24 hr later, and imaged directly. Bar, 10 μm.
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5. Figure 4 LL5β Binds to CLASPs and ELKS through Different Domains
(A and B) Hela cells were cotransfected with GFP-LL5β together with either RFP-CLASP2 or RFP-CLASP2-ΔC. Twenty-four hours later, cells were treated with 10 μM nocodazole for 1 hr, PFA fixed, and imaged directly. Bar, 10 μm.
(C–E) Schematic representation of the structure of CLASP1, CLASP2, LL5β, ELKS, and their deletion mutants used in this study. CC, region with heptad repeats (potential coiled coil); PH, pleckstrin homology domain.
(F) A scheme of the identified interactions between CLASP, LL5β, and ELKS.
(G) GST pull-down assays with the indicated GST fusions. Extracts of COS-1 cells overexpressing GFP-CLASP1α or GFP-CLASP2α or purified HIS-CLASP1-C and HIS-CLIP-170-N were used for binding. Coomassie-stained gel is shown for the GST fusions and Western blots with anti-GFP or anti-HIS antibodies for the bound proteins. Ten percent of the input and 25% of the material, bound to the beads, were loaded on gel.
(H) IPs from the extracts of COS-1 cells transiently expressing the indicated GFP-LL5β fusions. The precipitates were analyzed on Western blots with the indicated antibodies. Extr., 5% of the input.
(I and J) GST pull-down assays with the indicated GST fusions. Extracts of HEK293 cells overexpressing full-length (FL) myc-ELKSα, ELKSɛ, or different ELKS myc-tagged deletion mutants were used for binding. Western blots with anti-myc antibodies are shown. Input lanes contain 6% of the extract used. In (J), the inputs are shown on the left, while the material bound to GST-LL5β-M1-containing beads is shown on the right.
(K) GST pull-down assays with the indicated GST-LL5β proteins and bacterially purified MBP (arrow) or MBP-ELKS-M1 fusion (arrowhead). Western blot with anti-MBP antibodies is shown. Input lanes contain 3% of the protein sample used for binding.
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6. Figure 5
Relationship between LL5β-ELKS Complexes and MTs in HeLa Cells
(A and B) HeLa cells fixed with cold methanol were stained with antibodies against α-tubulin and LL5β. In (A), an enlargement of the rightmost cell margin is shown on the right.
(B) Correlation between the LL5β fluorescence intensity and the number of MTs in a 5 × 1 μm box at the cell edge. For this plot, cells showing strong variability of the peripheral LL5β labeling were chosen. The regression line is shown in red; Pearson's correlation coefficient r is indicated.
(C and D) HeLa cells were cultured for 72 hr after transfection with the indicated siRNAs, fixed with methanol, and stained for α-tubulin. (C) Images of individual HeLa cells treated with the indicated siRNAs. (D) Plots show MT numbers in a 5 × 1 μm box located at the indicated distance from the cell edge. Measurements were performed in 20 cells per siRNA. Whiskers indicate SD; numbers above the plots indicate the size of each bar in % compared to the adjacent control bar; values significantly different from control are indicated by asterisks (asterisk, p < 0.05; double asterisk, p < 0.001).
(E and F) HeLa cells stably expressing GFP-α-tubulin were imaged by TIRF microscopy 72 hr after transfection with the indicated siRNAs. In (E), the contrast is inverted.
(F) Quantification of the number of MTs visible by TIRF in a 5 × 1 μm box at the cell edge (∼60 regions in approximately ten cells per siRNA). Plots are constructed as in Figure 5D.
(G and H) HeLa cells were treated with 10 μM nocodazole for 10 min, fixed with methanol, and stained for tubulin. (H) MT numbers were quantified and plotted as in Figure 5D with the same color codes.
(I–L) Analysis of MT dynamics by time-lapse imaging of HeLa cells stably expressing GFP-α-tubulin 72 hr after transfection with the control or LL5β siRNAs. Cells were imaged with a 2 s interval. For details, see Movie 1 and Table S2. (I) Life-history plots of individual MTs at the periphery of control and LL5β-depleted HeLa cells expressing GFP-α-tubulin (see also Movie 1). Plots for three different MTs are shown by red, green, and blue lines. The vertical axis indicates the distance from the cell edge (the position of which is indicated by a horizontal orange line). (J) Frequency of MT entry into lamella. Plots are constructed as in Figure 5D. (K and L) Percentage of time spent by MT plus ends in different phases of dynamic instability (K) or in the 1 μm region at the cell edge (L). Bars, 10 μm.
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7. Figure 6
Inhibition of PI3 Kinase Activity Affects the Recruitment and Maintenance of Cortical LL5β Clusters
(A–C) HeLa cells transiently expressing GFP-LL5β-ΔPH (A) or GFP-LL5β-PH (B and C) were fixed with PFA and imaged directly. In (C), cells were preincubated for 1 hr with 100 nM wortmannin.
(D) Confocal images of the basal plasma membrane of HeLa cells transiently expressing YFP-AktPH, CFP-CAAX, and RFP-CLASP2. In the right two panels, the YFP/CFP ratio is shown by shades of gray. RFP-CLASP2 signals are shown in red.
(E) HeLa cells were serum starved for 72 hr and either fixed directly or incubated for 1 hr in medium containing serum and 0.1% DMSO, serum and 100 nM wortmannin, or serum and 100 μM LY Cells were fixed with methanol and stained with the mouse anti-LL5β antibody.
(F) Plots of average intensity of fluorescence of LL5β staining performed as described for Figure 6E. The average intensity was measured within a 1 μm2 box along the cell radius with subtracting the background. Measurements were performed in five different cell regions in 20 cells per treatment. SEM is shown by whiskers.
(G–I) Ratio of the peak value of average intensity of LL5β staining to the average intensity in the internal cytoplasm at a 10 μm distance from the peak along the same radius. SD is shown by whiskers. The quantification was performed for serum-starved cells stimulated by serum for the indicated time periods (G), for serum-starved cells stimulated by serum for 1 hr in the presence of 100 nM wortmannin or 100 μM LY (H), or for serum-grown cells treated with the same inhibitors for 1 hr (I). Values significantly different from those for the serum-stimulated cells (H) or for the control serum-grown cells (I) are indicated by asterisks (p < 0.001).
(J and K) Swiss 3T3 cells were grown to a monolayer, serum starved for 48 hr, and serum stimulated for 1 hr after scratching the monolayer. Cells were fixed with methanol and stained with a mixture of antibodies against CLASP1 and CLASP2 ([J], green in the overlay), mouse anti-LL5β antibody ([J], red in the overlay), rabbit anti-LL5β antibody ([K], green in the overlay), and mouse anti-ELKS antibody ([K], red in the overlay). White rectangles indicate the portion of the overlay shown enlarged on the right.
(L) Swiss 3T3 cells were grown to a monolayer, serum starved for 48 hr, and incubated for 1 hr in serum-free medium (serum starved), in serum-containing medium supplemented with either 0.1% DMSO (1 hr serum) or with one of the indicated inhibitors (100 nM wortmannin, 10 μM nocodazole). Cells were fixed with methanol and stained for LL5β. Bars, 10 μm.
Developmental Cell  , 21-32DOI: ( /j.devcel )
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8. Figure 7 Relationship between LL5β Clusters and FAs
(A) Swiss 3T3 cells were grown at low density in the presence of serum, fixed with methanol, and stained with the mouse anti-LL5β antibody (green in the overlay) and rabbit anti-FAK-pY397 antibody (FA marker, red in the overlay).
(B) Serum-grown HeLa cells were treated with 10 μM nocodazole for 1 hr, fixed with methanol, and stained with antibodies against LL5β (green in the overlay), FA marker vinculin (red in the overlay), and α-tubulin.
(C) HeLa cells were serum-starved for 72 hr and either fixed directly or incubated for 1 hr in the medium containing 10 μM nocodazole or 10 μM nocodazole and 100 nM wortmannin. The panel on the right shows serum-starved HeLa cells 72 hr after transfection with CLASP1+2 siRNAs. Cells were stained in the same way as in Figure 7A.
(D) Serum-grown cells were fixed with PFA and stained for the FA marker paxillin 72 hr after transfection with the indicated siRNAs. HeLa cells do not migrate and as a result, form groups where they remain in contact with each other. Cells located at the “free” edges of such groups are shown. Bars, 10 μm.
(E) Quantification of the area of individual FAs located at the “free” edges of the groups of serum-grown HeLa cells. Plots were constructed in the same way as in Figure 5D.
(F) A model for cortical MT attachment through CLASPs, LL5β, and ELKS. CLASPs can interact with the plus end of a growing MT and the same time make contact with the cell cortex through the association with LL5β. LL5β clusters at the membrane are formed with the participation of ELKS and are recruited to the membrane by PIP3 as well as other lipid and/or protein partners, which are indicated by question marks. As an alternative, CLASPs can form an MT tip bound complex with CLIP-170, which is not involved in LL5β-mediated membrane attachment. Functional interplay between MT ends, LL5β, and FAs is indicated by arrows.
Developmental Cell  , 21-32DOI: ( /j.devcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions