Evidence that an interaction between EB1 and p150(Glued) is required for the formation and maintenance of a radial microtubule array anchored at the centrosome

Abstract

EB1 is a microtubule tip-associated protein that interacts with the APC tumor suppressor protein and components of the dynein/dynactin complex. We have found that the C-terminal 50 and 84 amino acids (aa) of EB1 were sufficient to mediate the interactions with APC and dynactin, respectively. EB1 formed mutually exclusive complexes with APC and dynactin, and a direct interaction between EB1 and p150(Glued) was identified. EB1-GFP deletion mutants demonstrated a role for the N-terminus in mediating the EB1-microtubule interaction, whereas C-terminal regions contributed to both its microtubule tip localization and a centrosomal localization. Cells expressing the last 84 aa of EB1 fused to GFP (EB1-C84-GFP) displayed profound defects in microtubule organization and centrosomal anchoring. EB1-C84-GFP expression severely inhibited microtubule regrowth, focusing, and anchoring in transfected cells during recovery from nocodazole treatment. The recruitment of gamma-tubulin and p150(Glued) to centrosomes was also inhibited. None of these effects were seen in cells expressing the last 50 aa of EB1 fused to GFP. Furthermore, EB1-C84-GFP expression did not induce Golgi apparatus fragmentation. We propose that a functional interaction between EB1 and p150(Glued) is required for microtubule minus end anchoring at centrosomes during the assembly and maintenance of a radial microtubule array.

Figure 1

Schematic of EB1 deletion mutants used in this study. F/L, full-length. bZIP, basic leucine zipper motif.

Figure 2

Identification of protein–protein interaction regions in EB1. (A) EB1-GFP, a series of EB1-GFP deletion mutants, and luciferase were in vitro transcribed/translated using radiolabeled amino acids. The top panel shows an autoradiograph of the products of the translation resolved by SDS-PAGE. Minor bands are likely to have arisen from translational initiation at internal methionines in the EB1 cDNA. The reaction mix was incubated with GST-APC-C1 coupled to glutathione-Sepharose beads, and washed, and interacting species were resolved by SDS-PAGE and autoradiography. EB1-GFP and the deletion mutants EB1-ΔN1, -ΔN2, and -ΔN3 were all precipitated by GST-APC-C1. The negative control luciferase and the deletion mutants EB1-ΔC1, -ΔC2, and -ΔC3-GFP did not interact with GST-APC-C1. (B) GST, GST-EB1, and a series of EB1 deletion mutants fused to GST were coupled to glutathione-Sepharose beads and used in precipitations from cell extracts. The detection of β-catenin in precipitates from HCT116 cells indicates APC binding by GST-EB1, GST-EB1-ΔN2, and GST-EB1-ΔN3. No specific precipitation of β-catenin was obtained from extracts of SW480 cells; this blot is also shown grossly overexposed to reveal nonspecific binding. GST-EB1, GST-EB1-ΔN2, and GST-EB1-ΔN3 also precipitated p150^Glued from cell extracts. (C) Glutathione-Sepharose–bound GST, GST-EB1, and three additional EB1 deletion mutants were used to precipitate p150^Glued and β-catenin (demonstrating an interaction with APC) from HCT116 cell extracts. The only deletion mutant to precipitate p150^Glued from cell extracts was GST-EB1-C84. All three deletion mutants were capable of precipitating β-catenin.

Figure 3

Distribution of EB1-GFP and deletion mutants in transfected COS-7 cells. Cells were processed for immunofluorescence with an anti-GFP antibody 18 h after transfection. (A) Distribution of EB1-GFP. The cell on the left displays microtubule tip labeling (arrows). Quantitative analysis suggested that the cell on the right was expressing about fourfold higher levels of EB1-GFP than the cell on the left, and more extensive microtubule labeling was seen. (B) Cell expressing EB1-ΔN1-GFP. No microtubule association is seen, but the centrosome is stained (arrow). (C) Cells expressing EB1-ΔN2-GFP. The image was optimized for the cell expressing higher levels of the fusion protein. At low expression levels a diffuse cytoplasmic distribution is seen. At higher levels a centrosome-associated aggregate is observed (arrow). (D) Cells expressing EB1-ΔN3-GFP. A weak microtubule association and a centrosomal localization are seen (arrows). (E) Cells expressing EB1-ΔC1-GFP. A robust microtubule associated staining pattern is seen (arrows), although it appears less polarized toward microtubule tips than that seen for full-length EB1. (F) Cell expressing EB1-ΔC2-GFP. A similar microtubule association is again apparent (arrows). (G) Cells expressing EB1-ΔC3-GFP. A diffuse cytoplasmic staining pattern is seen. Bars in all but B, 20 μm. Bar in B, 10 μm.

Figure 4

(A) EB1 binds directly to p150^Glued. In vitro–translated GFP-p150 or GFP alone was incubated with purified, glutathione-Sepharose–bound GST-EB1 or GST alone. Protein complexes were harvested by centrifugation, washed, and analyzed by SDS-PAGE and autoradiography. GST-EB1 precipitated GFP-p150 but not GFP alone demonstrating an EB1-p150 interaction. No proteins were precipitated with GST alone. (B) Schematic representing the in vitro–translated p150^Glued deletion mutants used in subsequent experiments. (C) In vitro–translated p150 deletion mutants were incubated with purified, glutathione-Sepharose–bound GST-EB1 or GST alone. Protein complexes were harvested by centrifugation, washed extensively, and analyzed by SDS-PAGE and autoradiography. Significantly greater amounts of 6His-p150(1–811) and 6His-p150(1–330) were precipitated by GST-EB1 than GST alone, indicating that these deletion mutants specifically interacted with EB1. (D) Purified bacterially expressed 6His-p150(1–330) was incubated with purified, glutathione-Sepharose–bound GST-EB1 or GST alone. Protein complexes were harvested by centrifugation, washed, and analyzed by SDS-PAGE and Western blotting using an anti-His tag antibody. GST-EB1 precipitated 6His-p150(1–330), whereas GST did not, demonstrating a direct EB1-p150 interaction. The control protein was purified 6His-p150(1–330). (E) Purified bacterially expressed GST-EB1 or GST was incubated with purified 6His-p150(1–330). Protein complexes were harvested using Ni-²⁺ magnetic beads, washed, and analyzed by SDS-PAGE and Western blotting using anti-GST and anti-His tag antibodies. 6His-p150(1–330) was successfully precipitated using the magnetic beads. GST-EB1 coprecipitated with 6His-p150(1–330), whereas GST did not. The band detected with the anti-GST antibody at ∼45 kDa (immediately below the GST-EB1 band) represents a breakdown product of GST-EB1. No proteins were precipitated by the magnetic beads alone. This demonstrates a direct interaction between EB1 and p150.

Figure 5

EB1 does not simultaneously interact with dynactin and APC. Ni-²⁺-Sepharose–bound 6His-EB1 was used to precipitate proteins from an extract of HCT116 cells. 6His-EB1 was preincubated with either GST, GST-APC-C1, or buffer alone before precipitations were performed. Precipitates were washed and probed for the presence of the dynactin subunit p150^Glued. Preincubation of 6His-EB1 with GST-APC-C1 virtually abolished any interaction with p150^Glued, whereas preincubation with GST had no effect.

Figure 6

Overexpression of full-length EB1, but not EB1 deletion mutants stabilizes microtubules. Cells were transfected with EB1-GFP (A and B), EB1-ΔN1-GFP (C and D), and EB1-ΔC2-GFP (E and F), and coimmunostained with antibodies specific for GFP (A, C, and E) and acetylated tubulin (B, D, and F). EB1-GFP–induced microtubule bundles (A) showed significantly more intense staining for acetylated tubulin (B). No increase in acetylated tubulin staining was seen in cells transfected with any of the EB1-GFP deletion mutants. Bar, 20 μm.

Figure 7

EB1-ΔC1-GFP displaces endogenous EB1 from microtubules. (A) Epitope mapping of the Transduction Laboratories anti-EB1 mAb. Purified GST, GST-EB1, and all EB1-deletion mutants were Western blotted and probed with the EB1 mAb. The antibody detected full-length EB1 and EB1 proteins with N-terminal deletions. Deletion of the last 50 aa of EB1 (EB1-ΔC1) destroyed the antibody epitope, which lies within the last 84 aa of EB1 (EB1-C84). (B–D) EB1-ΔC1-GFP competes with endogenous EB1 for binding to microtubules. COS-7 cells overexpressing EB1-ΔC1-GFP (asterisk) were processed for immunofluorescence with antibodies to GFP (B) and EB1 (C). (D) The merged image. Microtubule tip labeling for endogenous EB1 was absent in cells overexpressing EB1-ΔC1-GFP. Bar, 15 μm.

Figure 8

Full-length EB1, but not EB1-ΔC1-GFP, competes CLIP-170 from microtubule tips. COS-7 cells overexpressing EB1-GFP (A–C, asterisks) and EB1-ΔC1-GFP (D–F, asterisks) were processed for immunofluorescence with antibodies to GFP (A and D) and CLIP-170 (B and E). (C and F) The merged images. Microtubule tip labeling for endogenous CLIP-170 was absent in cells overexpressing EB1-GFP but was unaffected in cells overexpressing EB1-ΔC1-GFP. Bars, 10 μm.

Figure 9

EB1-C84-GFP overexpression inhibits microtubule focusing and anchoring at centrosomes. COS-7 cells overexpressing EB1-C87-GFP were processed for immunofluorescence with antibodies to GFP (A, D, G, and J), p150^Glued (H and K), and/or tubulin (B, E, H, and K). Merged images are shown in C and F. EB1-C84-GFP localized to centrosomes (J, arrow), and its overexpression resulted in an unfocused microtubule array (compare transfected cell in A–C with adjacent cells). Loose cytoplasmic foci of microtubules were sometimes seen (E and I, arrows), with which p150^Glued was associated (H and I, arrow). Free cytoplasmic microtubules were also seen (E, I, and L, arrowheads), suggesting a defect in centrosomal microtubule anchoring. p150^Glued immunostaining at centrosomes was reduced in cells overexpressing EB1-C84-GFP, where a clear centrosomal structure was still identifiable (K, arrow). Bars, 20 μm.

Figure 10

Localization of centrosomes in cells expressing EB1-C84-GFP. COS-7 cells expressing EB1-C84-GFP were processed for immunofluorescence with antibodies to GFP (A and C, green), α-tubulin (B and D, blue), and γ-tubulin (B and D, red). Merged images of the α- and γ- tubulin staining are shown in B and D. Robust γ-tubulin immunostaining revealed a normal perinuclear localization for the centrosomes (arrowhead, A and B) in cells expressing moderate levels of EB1-C84-GFP where free cytoplasmic microtubules (arrows, B) and microtubule disorganization were clearly evident. At higher levels of EB1-C84-GFP expression, where the majority of microtubules were free in the cytoplasm and the radial microtubule array was completely destroyed, γ-tubulin immunostaining was often weaker, but the centrosomes could still be clearly identified. These were usually associated with one of the loose tangles of microtubules in these cells (arrows, C and D). Bars, 10 μm.

Figure 11

EB1-C50-GFP overexpression does not inhibit microtubule anchoring or focusing at centrosomes. COS-7 cells overexpressing EB1-C50-GFP were processed for immunofluorescence with antibodies to GFP (A and D), p150^Glued (E), and/or tubulin (B and F). A merged image is shown in C. EB1-C50-GFP localized to centrosomes in some (D, arrow) but not all cells (A). Its overexpression had little effect on microtubule focusing and anchoring at the centrosome (B and F). The localization of p150^Glued to centrosomes was unaffected in transfected cells (E, arrow). Bars, 20 μm.

Figure 12

EB1-C84-GFP overexpression inhibits centrosome assembly and microtubule regrowth after nocodazole treatment. COS-7 cells transfected with EB1-C84-GFP were treated with nocodazole. Cells were fixed after increasing recovery times after nocodazole washout, and microtubule regrowth was examined by immunofluorescence microscopy. (A, D, G, and J) GFP immunostaining; (B, E, I, and L) α-tubulin; (H) p150^Glued; and (K) γ-tubulin. (C) A merged image. In some cells EB1-C84-GFP expression completely abolished microtubule regrowth (A–C and G–I). This was associated with a failure to concentrate p150^Glued (H, arrows) and γ-tubulin (K, arrows) at a single intracellular focus. In transfected cells where microtubule regrowth was evident, an unfocused, disorganized microtubule array was seen (D–F and J–L). Regrowth was centered around a subset of γ-tubulin foci (K and L, arrowhead), but free cytoplasmic microtubules were often seen (K and L, small arrows), implying defective microtubule anchoring at nucleation sites. Representative images from a number of different experiments and recovery times are shown. (A–C) A 45-min recovery; (D–F and J–L) a 30-min recovery; and (G–I) a 15-min recovery. All of the described effects were seen in cultures processed up to 90 min after washing. α-Tubulin was detected with an Alexa 564 secondary antibody in the experiment in D–F, and Alexa 633 elsewhere. Bars, 20 μm.

Figure 13

EB1-C50-GFP and EB1-ΔC1-GFP overexpression have no effect on microtubule regrowth and focusing after nocodazole treatment. The ability of the microtubule cytoskeleton in cells transfected with EB1-C50-GFP (A–F) and EB1-ΔC1-GFP (G–I) to recover from nocodazole treatment was examined as described in Figure 12. (A, D, and G) GFP immunostaining; (B and H) p150^Glued; (E) γ-tubulin; and (C, F, and I) α-tubulin. Overexpression of these deletion mutants had no effect on microtubule regrowth and centrosomal focusing or on the recruitment of either p150^Glued (B and H, arrows) or γ-tubulin (E, arrow) to centrosomes. All cells shown in this figure were fixed 30 min after nocodazole washout. Bars, 20 μm.

Figure 14

EB1-C84-GFP and EB1-C50-GFP overexpression do not induce Golgi apparatus fragmentation. COS-7 cells transfected with EB1-C84-GFP (A–F) and EB1-C50-GFP (G–I) were fixed and immunostained with antibodies to GFP (A, D, and G), the Golgi 58K protein (B, E, and H), and α-tubulin (C, F, and I). Overexpression of these fusion proteins did not result in the dispersal of the Golgi apparatus from its normal perinuclear localization (arrows), even in cells where microtubule organization was clearly impaired (D–F). Note again the presence of unanchored and looped microtubules in cells overexpressing EB1-C84-GFP (i.e., F, arrowheads). Bars, 20 μm.