The Arabidopsis TRM1-TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes

Abstract

Land plant cells assemble microtubule arrays without a conspicuous microtubule organizing center like a centrosome. In Arabidopsis thaliana, the TONNEAU1 (TON1) proteins, which share similarity with FOP, a human centrosomal protein, are essential for microtubule organization at the cortex. We have identified a novel superfamily of 34 proteins conserved in land plants, the TON1 Recruiting Motif (TRM) proteins, which share six short conserved motifs, including a TON1-interacting motif present in all TRMs. An archetypal member of this family, TRM1, is a microtubule-associated protein that localizes to cortical microtubules and binds microtubules in vitro. Not all TRM proteins can bind microtubules, suggesting a diversity of functions for this family. In addition, we show that TRM1 interacts in vivo with TON1 and is able to target TON1 to cortical microtubules via its C-terminal TON1 interaction motif. Interestingly, three motifs of TRMs are found in CAP350, a human centrosomal protein interacting with FOP, and the C-terminal M2 motif of CAP350 is responsible for FOP recruitment at the centrosome. Moreover, we found that TON1 can interact with the human CAP350 M2 motif in yeast. Taken together, our results suggest conservation of eukaryotic centrosomal components in plant cells.

Figure 1.

The Last 33 C-Terminal Residues of TRM1 Are Sufficient for Interaction with TON1. Summary of TRM1–TON1 interactions as determined by yeast two-hybrid analyses between TRM1 fragments and full-length TON1. Growth on selective medium was visually noted from no significant growth (−) to full-growth (+). The numbered boxes in the TRM1 protein depicted at the top designate the M1-M6 motifs. aa, amino acids. [See online article for color version of this figure.]

Figure 2.

Six Motifs Define a Superfamily of 34 Proteins in Arabidopsis. Maps of the 34 predicted TRM polypeptides, with occurrence and position of the motifs shown on the right. The eight groups of TRM proteins were defined by multiple alignment procedures, manually curated, and submitted to neighbor-joining phylogenetic analysis (unrooted NJ tree) and bootstrap validation (1000 trials); only strongly supported nodes are represented here (see Supplemental Data Set 1 online). The Arabidopsis Genome Initiative (AGI) gene number, calculated size (in residues), and pI of each predicted protein are indicated. Black bars indicate TRMs isolated from the two-hybrid screen using TON1 as bait. [See online article for color version of this figure.]

Figure 3.

TRM1 Interacts with TON1 in Vivo. (A) Immunoblot analysis of TRM1 protein levels in Arabidopsis tissues. Protein expression was tested in rosette leaves (Lr), cauline leaves (Lc), stem (S), opening flowers (F), and flower buds (FB) of Columbia-0 (Col-0) plants. Rosette leaves from the loss-of-function mutant allele Salk_034645 (also named lng2-2 in Lee et al., 2006) correspond to the negative control. (B) Coprecipitation experiments using GFP-trap beads were performed on flower buds extracts from Col-0 plants and from plants expressing GFP under the control of the 35S promoter or the genomic GFP-gTON1 fusion construct. Coprecipitated proteins were then analyzed by immunoblotting using the indicated antibodies. TRM1 is only copurified in coprecipitates of GFP-gTON1 extracts, demonstrating TRM1–TON1 interaction in vivo.

Figure 4.

TRM1 Is a Microtubule-Associated Protein. (A) to (F) GFP-TRM1 labels microtubules in Arabidopsis petal epidermal cells. In (A) and (B), Arabidopsis petal epidermal cells expressing the ProTRM1:GFP-TRM1 construct are shown. In elongated cells from the petal claw (A), GFP-TRM1 fluorescence is present at the cortex as a filamentous labeling organized in parallel arrays perpendicular to the cell elongation axis. In rounded cells from the abaxial side of the petal blade (B), GFP-TRM1 labeled randomly organized cortical filamentous structures. In an Arabidopsis mCherry-β-tubulin6 line ([C] and [D]), the overall cortical microtubule organization in elongated cells from the petal claw (C) and in rounded cells from the abaxial side of the petal blade (D) is similar to the GFP-TRM1 labeling shown in (A) and (B). However, GFP-TRM1 appeared as dots aligned along filaments ([A] and [B]), whereas mCherry-tubulin is evenly distributed along microtubules ([C] and [D]). Coalignment of GFP-TRM1 (red) with microtubules (green) was demonstrated in cells coexpressing the ProTRM1:GFP-TRM1 construct and the mCherry-β-tubulin6 marker (F). In (E), the GFP-TRM1 fluorescence alone is shown. (E) and (F) correspond to petal epidermal elongated cells. All micrographs are projections of Z-stack confocal images. Bars = 10 μm. (G) TRM1 cosediments with microtubules in vitro. Cosedimentation experiments were performed with 0.5 μM TRM1 in the presence (+) or absence (−) of 0.5 μM microtubules. Proteins present in the supernatant (S) and the pellet (P) after centrifugation were separated on a SDS-PAGE gel stained with Coomassie blue. The intensity of each TRM1 band was measured and expressed as the percentage of the total amount of TRM1 input.

Figure 5.

Mapping the TRM1 Microtubule-Interacting Domain. (A) Schematic representation of TRM1. The position of each motif is indicated. The charge plot of the protein is shown in black, and points above the protein represent positively charged (basic) domains and the ones below negatively charged (acidic) domains. (B) A series of truncated fragments of TRM1 were cloned in translational fusion with GFP, as N- and C-terminal fusions, under the control of the 35S promoter and expressed transiently in N. benthamiana epidermal cells. N- and C-terminal fusions gave comparable results in these experiments. Dark-gray fragments labeled microtubules structures, whereas white ones gave a cytoplasmic staining. MT, microtubule. (C) Colocalization with microtubules was confirmed by coexpression of the microtubule marker GFP-α-tubulin6 with each TRM1 fragment fused to RFP. An example of such colocalization in N. benthamiana jigsaw puzzle leaf cells is shown, where the minimal TRM1^342-586 fragment (red) colocalized with GFP-α-tubulin6 (green). The right panel corresponds to the overlay of both signals. Bar = 20 μm. (D) The TRM1^342-586 binds microtubules in vitro. Cosedimentation experiments were performed with 0.5 μM TRM1^342-586 in the presence (+) or absence (−) of 0.5 μM microtubules. Proteins present in the supernatant (S) and the pellet (P) after centrifugation were separated on an SDS-PAGE gel stained with Coomassie blue. The intensity of each TRM1^342-586 band was measured and expressed as the percentage of the total amount of TRM1^342-586 input.

Figure 6.

Not All TRM Proteins Are Microtubule-Associated Proteins. (A) to (F) TRM proteins were expressed as N-terminal GFP fusions in N. benthamiana leaf epidermal cells. GFP-TRM1 (A), GFP-TRM2 (B), GFP-TRM8 (C), and GFP-TRM25 (E) all labeled cortical microtubule arrays, whereas GFP-TRM20 (D) and GFP-TRM26 (F) displayed a cytoplasmic fluorescence. All micrographs are projections of Z-stack confocal images. Bars = 10 μm. (G) and (H) TRM8 (G) and TRM26 (H) microtubule (MT) cosedimentation assays. Each cosedimentation was performed with 0.5 μM TRM proteins in the presence (+) or absence (−) of 0.5 μM microtubules. Proteins present in the supernatant (S) and the pellet (P) after centrifugation were separated on an SDS-PAGE gel stained with Coomassie blue. The intensity of each TRM band was measured and expressed as the percentage of the total amount of TRM input. TRM8 directly binds to microtubules in vitro, whereas TRM26 does not. [See online article for color version of this figure.]

Figure 7.

TRM1 Targets TON1 to Microtubules through the TRM1 M2 Motif. (A) to (C) Pro35S-driven expression of GFP-TON1 (A), GFP-TRM1 (B), and TRM1^1-827-GFP (C) in N. benthamiana leaf epidermal cells. In these typical jigsaw puzzle cells, the cytoplasm is restricted to the cell’s periphery by the large central vacuole penetrated by cytoplasmic strands. (A) GFP-TON1 fluorescence accumulated diffusely in the cytoplasm and cytoplasmic strands (arrowhead). (B) and (C) GFP-TRM1 and TRM1^1-827-GFP both labeled microtubule arrays. (D) to (I) Coexpression of GFP-TON1 with TRM1-RFP ([D] to [F]) or TRM1^1-827-RFP ([G] to [I]) in tobacco leaf epidermal cells. Note that the GFP-TON1 signal is recruited to cytoskeletal structures only in cells expressing TRM1 (e.g., in the top right cell in [D] to [F]), which does not express TRM1-RFP, as judged from lack of RFP fluorescence, and the GFP-TON1 signal remains diffusely in the cytoplasm. In (G) to (I), an M2-deleted version of TRM1 (TRM1^1-827) is unable to recruit GFP-TON1, which remained in the cytoplasm. (J) to (L) Colocalization experiments of RFP-TON1 with the GFP-α-tubulin6 microtubule marker (GFP-TUA6) in tobacco leaf cells. Leaves were coinfiltrated with three different constructs: GFP-α-tubulin6, the RFP-TON1 construct, and an untagged version of TRM1. In cells where TRM1 is expressed as revealed by RFP-TON1 recruitment to cytoskeletal structures, the RFP-TON1 signal colocalized with the GFP-α-tubulin6 microtubule marker. (M) to (O) Coexpression of GFP-TON1 and TRM1-RFP at lower expression levels shows a punctate staining reminiscent of TRM1 and TON1 localization in Arabidopsis. To decrease expression levels of the TON1 fusion, we used the GFP-gTON1 construct. To decrease expression levels of the TRM1 fusion, agrobacteria carrying the TRM1-RFP construct were resuspended in infiltration buffer to an OD₆₀₀ of 0.05 (instead of 0.5). All micrographs are projections of Z-stack confocal images. Bars = 20 μm.

Figure 8.

The CAP350 M2 Motif Interacts with TON1. (A) CAP350 proteins contain the M2, M3, and M4 motifs of TRM proteins. Here, a map of human CAP350 where gray boxes indicate coiled-coil regions is shown. Positions of the M3, M4, and M2 motifs are indicated, as well as the CAP-Gly domain of CAP350. Below are regions of CAP350 implicated in microtubule binding, centrosome localization, and interaction with FOP (Yan et al., 2006; Hoppeler-Lebel et al., 2007). This last region corresponding to the C-terminal 48 amino acids of CAP350 coincides precisely with the predicted M2 motif. (B) The CAP350 M2 motif interacts with TON1. We tested the ability of the C-terminal 48 amino acids of CAP350 to interact with Arabidopsis TON1 in a yeast two-hybrid assay: A clear positive interaction was observed, demonstrating the functionality of CAP350 M2 as a TON1 binding motif. ∅, self-activation tests of the constructs; AD, activation domain; BD, binding domain; nt, not tested. [See online article for color version of this figure.]