A complex of two centrosomal proteins, CAP350 and FOP, cooperates with EB1 in microtubule anchoring

Abstract

The anchoring of microtubules (MTs) to subcellular structures is critical for cell shape, polarity, and motility. In mammalian cells, the centrosome is a prominent MT anchoring structure. A number of proteins, including ninein, p150Glued, and EB1, have been implicated in centrosomal MT anchoring, but the process is far from understood. Here we show that CAP350 and FOP (FGFR1 oncogene partner) form a centrosomal complex required for MT anchoring. We show that the C-terminal domain of CAP350 interacts directly with FOP and that both proteins localize to the centrosome throughout the cell cycle. FOP also binds to EB1 and is required for localizing EB1 to the centrosome. Depletion of either CAP350, FOP, or EB1 by siRNA causes loss of MT anchoring and profound disorganization of the MT network. These results have implications for the mechanisms underlying MT anchoring at the centrosome and they attribute a key MT anchoring function to two novel centrosomal proteins, CAP350 and FOP.

Figure 1.

CAP350 localizes to the centrosome and is phosphorylated in M phase. (A) Schematic representation of CAP350, indicating the CAP_Gly motif and the domain (residues 2115-2643 aa) used for antibody production. (B) IF staining in U2OS cells of endogenous CAP350 (left) and γ-tubulin (middle). DNA (right) was stained with DAPI. Insets shows enlargement of the centriolar staining of CAP350 and γ-tubulin at the poles. Bar, 10 μm. (C) CAP350 is modified in M phase. HeLa S3 were released from a nocodazole block, and samples were taken for Western blot analysis at the time points indicated (T0-T4.0 in hours). For comparison, asynchronously growing cells (Asy) were analyzed in parallel. (D) CAP350 is phosphorylated during mitosis. CAP350 was immunoprecipitated from asynchronously growing (Asy) or nocodazole-arrested (M) HeLa cells and treated with (+) or without (-) calf intestinal phosphatase (CIP) in the presence (+) or absence (-) of phosphatase inhibitors. Samples were separated by 6% SDS-PAGE and probed by Western blotting.

Figure 2.

Depletion of CAP350 causes disorganization of the MT network. (A) Western blotting shows effective siRNA-mediated depletion of CAP350 in HeLa S3 cells. Cells were transfected with GL2 or CAP350-specific oligonucleotide duplexes for 72 h. Equal amounts of protein were separated by SDS-PAGE and probed by Western blotting with anti-CAP350 antibody (top) and anti-α-tubulin antibody for loading control (bottom). (B and C) IF staining of CAP-350 depleted (bottom) and control (top) HeLa S3 (B) and A549 cells (C) with antibodies to α-tubulin. Note that CAP350-depleted cells lack radial MT arrays focused on the centrosome. Bar, 10 μm.

Figure 3.

Depletion of CAP350 affects MT anchoring. HeLa S3 cells were transfected with GL2 (A), CAP350 -2 (B), and pericentrin A/B siRNA (C) oligonucleotide duplexes. They were then subjected to MT regrowth assays and fixed at the time points indicated. CAP350 and pericentrin were visualized with appropriate antibodies (inset in C shows positive staining for pericentrin in GL2-treated control cells) and MTs were stained with FITC-labeled anti-α-tubulin antibody. Bar, 10 μm. (D-F) Transfected cells were classified according to their failure to nucleate MTs (white bars), or nucleate MTs and then form asters (gray bars) or nonfocused MT networks (black bars). Histograms show results from three independent experiments, counting 300 cells each, and error bars indicate SDs.

Figure 4.

FOP is a centrosomal protein and phosphorylated in mitosis. (A) Schematic representation of FOP, indicating the LisH domain (residues 69-102). (B) Subcellular localization of FOP at various cell cycle stages. U2OS were labeled with anti-FOP antibody (top) and DAPI to visualize DNA (bottom). Bar, 10 μm. (C) Western blotting of total HEK293T, U2OS, and HeLa S3 cell extracts with affinity purified anti-FOP antibody. Cell extracts, 15 μg, were used for each lane. (D) FOP was immunoprecipitated from asynchronously growing (Asy) or nocodazole-arrested (M) HeLa cells and treated with (+) or without (-) calf intestinal phosphatase (CIP) in the presence (+) or absence (-) of phosphatase inhibitors. Samples were separated by 12% SDS-PAGE and probed by Western blotting.

Figure 5.

CAP350 interacts with FOP in vivo. (A) FOP interacts with a C-terminal fragment of CAP350. Immunoprecipitation experiments were performed on lysates from 293T cells after cotransfection with the indicated plasmids. Antibodies used were anti-myc (lanes 3 and 4) and anti-FLAG (lanes 5 and 6), coupled to beads. Immunoprecipitated proteins were then analyzed by Western blotting, using the indicated antibodies, and whole cell lysates were analyzed in parallel (lanes 1 and 2). In a separate experiment, anti-FLAG immunoprecipitates were also separated by 6% SDS-PAGE and probed with anti-CAP350 antibody to detect endogenous CAP350 (lanes 9-12). (B) Endogenous FOP interacts with endogenous CAP350. HEK293T cell extracts were immunoprecipitated with control rabbit IgG (lane 2) or goat IgG (lane 5) and anti-FOP (lane 3) or anti-CAP350 antibody (lanes 6). Immunoprecipitates were subject to Western blotting with anti-FOP (top) or anti-CAP350 (bottom) antibodies; whole cell lysates are shown in lanes 2 and 4.

Figure 6.

Mapping the interaction domain between CAP350 and FOP. (A) Summary of FOP interactions, as determined by yeast two-hybrid analyses with different C-terminal constructs of CAP350, and of centrosome localizations, as determined by IF microscopy. (B) Yeast two-hybrid analyses of interactions between the indicated C-terminal CAP350 fragment and FOP constructs. The wild-type and mutant FOP constructs analyzed are indicated schematically on the left and their ability to localize to the centrosome on the right. The abbreviation FOP(3M) stands for FOPV74FL82QE97K. The central panel shows the results of growing transformed yeast cells on selective medium (QDO), to test for interactions with the CAP350 bait. BD, binding domain; AD, activation domain.

Figure 7.

Effect of CAP350 depletion on FOP localization and vice versa. (A, B, and D) FOP targeting to centrosome requires CAP350, whereas spindle association of CAP350 requires FOP. HeLa S3 cells were treated for 72 h with the GL2 control duplex (A), a CAP350-specific duplex (B), or a FOP-specific duplex (D) and then interphase and mitotic cells were analyzed by IF microscopy, using the antibodies indicated. Bar, 10 μm. (C) Western blotting shows effective depletion of FOP by 72-h treatment with two different siRNA oligonucleotide duplexes. Equal amounts of protein were separated by SDS-PAGE and probed by Western blotting with anti-FOP antibody (top) and anti-α-tubulin antibody as a loading control (bottom).

Figure 8.

FOP interacts with EB1 and is required for its centrosome localization. (A) U2OS cells were transfected with FLAG-tagged FOP and costained with anti-FLAG and either anti-γ-tubulin or anti-EB1 antibodies. Bar, 10 μm. (B) FOP interacts with EB1 in vivo. HEK293T cells were transfected for 36 h with GFP-tagged EB1 and FLAG-tagged FOP (lanes 1 and 4) or empty FLAG vector (lanes 2 and 3). After lysis, immunoprecipitations were performed with anti-FLAG antibodies coupled to beads and proteins were subject to SDS-PAGE and transferred to membranes. Western blots were probed with anti-FLAG (bottom) or anti-GFP (top) antibodies. (C) Yeast two-hybrid analysis of the interaction between EB1 and various FOP constructs. The wild-type and mutant FOP constructs analyzed are indicated schematically on the left and their ability to localize to MT plus ends, as determined by transient transfection, on the right. The abbreviation FOP(3M) stands for FOPV74FL82QE97K. The central panel shows the results of growing transformed yeast cells on selective medium (QDO), to test for interactions with the CAP350 bait, and on nonselective medium (-LW) to control for viability. BD, binding domain; AD, activation domain. (D) U2OS cells were transfected with a FOP-specific siRNA duplex for 96 h, treated for 4 h with 6 μg/ml nocodazole, and then subjected to IF microscopy, using anti-FOP (left), anti-EB1 (middle), and anti-CAP350 (right) antibodies. Bar, 10 μm.

Figure 9.

Depletion of FOP or EB1 causes disorganization of the MT network. (A and B) HeLa S3 cells were transfected for 72 h with the indicated siRNA duplexes and then subjected to IF staining with the indicated antibodies. Note that both FOP- and EB1-depleted cells lack radial MT arrays focused on the centrosome. Bar, 10 μm. (C) COS7 cells were transfected for 48 h with the myc-tagged C-terminal CAP350 fragment, a domain known to bind FOP (Figure 6), and then stained with anti-myc (left), anti-FOP (middle), and anti-α-tubulin antibodies. Note the transfected cells, but not neighboring controls, lack radial MT arrays. Bar, 10 μm.

Figure 10.

Depletion of FOP affects MT anchoring. HeLa S3 cells were transfected with GL2 (A) or a FOP-specific siRNA oligonucleotide duplex (B). They were then subjected to MT regrowth assays and fixed at the time points indicated. FOP was visualized with anti-FOP antibodies and MTs were stained with FITC-labeled anti-α-tubulin antibody. Bar, 10 μm. (C) Transfected cells were classified according to their failure to nucleate MTs (white bars), or nucleate MTs and then form asters (gray bars) or nonfocused MT networks (black bars). Histograms show results from three independent experiments, counting 300 cells each, and error bars indicate SDs.