MT-binding regions in GPT1/2
Although GPT1/2 do not exhibit any significant amino acid homology with functionally characterized proteins [21], we noticed that the middle (M) and C-terminal (C) regions of these proteins are enriched in the basic amino acid residues Arg, Lys, and His (18.9% in GPT1 and 17.9% in GPT2; Fig. 1a and b). By contrast, the N-terminal (N) regions are abundant in acidic Asp and Glu (22.2% in both GPT1/2). Thus, GPTs are polar proteins with short, negatively charged regions at their N-termini (pI values of 4.4 for GPT1 and 4.5 for GPT2) and longer, positively charged regions in their middles and C-termini (pI values of 10.7 for GPT1 and 10.6 for GPT2).
To identify MT-binding regions in these proteins, we fused full-length and truncated versions of GPT1 and GPT2 to GFP, and transiently expressed these fusions in onion epidermal cells, together with the red fluorescent MT marker tagRFP-MAP4 (Fig. 1). Dual color visualization of GPT1-GFP and tagRFP-MAP4 proved difficult, possibly due to GPT1 having a weak MT-binding capacity, competition between GPT1 and MAP4 for overlapping MT binding sites, or both. Therefore, the MT-binding capacity of GPT1-GFP was determined based on the presence of fine filaments (presumably representing cortical MTs decorated by GPT1-GFP). Colocalization of the GFP and RFP signals confirmed that full-length GPT2 localized to cortical MTs (Fig. 1f). The N-terminal (N) regions of GPT1 (1–180) and GPT2 (1–207) did not localize to MTs, whereas the N-terminal-deleted (M + C) (181–530 for GPT1, and 208–553 for GPT2) and the middle (M) (181–300 for GPT1, and 208–328 for GPT2) fragments were clearly localized to cortical MTs (Fig. 1f). The C-terminal (C) fragments (301–530 for GPT1, and 329–553 for GPT2) were somewhat associated with MTs, but to a lesser extent than the fragments containing the M region. These results from transient expression assays indicate that the positively charged basic amino acid residues (especially in the M region) mediate the binding of GPT1/2 to MTs in vivo.
MT co-sedimentation assay
To test whether GPT1/2 directly bind to MTs, we fused full-length GPT1 and GPT2 to maltose-binding protein (MBP), expressed the recombinant proteins in bacteria, and purified the proteins by affinity purification using amylose resins. Some degradation occurred during the purification, particularly in the case of MBP-GPT1. When 1 μM of purified proteins was incubated with MTs assembled from bovine brain tubulin and pelleted by ultracentrifugation, intact MBP-GPT1 and MBP-GPT2 were identified in the microtubule pellet fraction (P) (arrowheads in Fig. 2a). The partial degradation products of MBP-GPT1 (asterisk in Fig. 2a) were also pelleted, whereas further degraded MBP-GPT1 and MBP-GPT2 fragments were not. In the absence of MTs, MBP-GPT1 and MBP-GPT2 remained in the supernatant (S). MBP alone was also evaluated to verify that it is not responsible for binding of GPT1/2 to MTs. As shown in Fig. 2, MBP remained in the supernatant fraction after ultracentrifugation. These results show that GPT1/2 bind MTs directly.
To determine the stoichiometry and affinity of GPT toward MTs, various amounts of MBP-GPT2 were centrifuged with a constant amount of MTs that were polymerized from 1 μM tubulin heterodimer, and a binding curve was obtained (Fig. 2b). MBP-GPT2 bound to MTs in a concentration-dependent manner and saturated at a stoichiometry of approximately 2.5 +/− 0.9 mol of GPT2 per mol of tubulin dimer. The dissociation constant Kd was calculated to be 1.9 +/− 1.0 μM. However, if GPT2 associates with MTs in more than one binding mode with different affinities (as indicated below), this simple regression analysis does not provide true binding values.
Subcellular localization
To determine the subcellular localization of GTP1/2 and to monitor their dynamics in vivo, GFP was fused to either the N-terminus or the C-terminus of GPT1 and expressed under the constitutive cauliflower mosaic virus 35S promoter, because the 5′-regulatory region of GPT1 was recalcitrant to cloning, whereas GFP was fused to the C-terminus of GPT2 and expressed under its native promoter. Both N-terminal and C-terminal GFP fusions of GPT1 localized to MTs in transient expression assays using onion epidermal cells (Additional file 1), indicating that the location of GFP does not affect the subcellular localization of fusion proteins. The Arabidopsis MT marker line that expresses mCherry fluorescent protein fused to β-tubulin 6 (mCherry-TUB6) was used as a transformation host.
GFP-GPT1 and GTP2-GFP co-localized with mCherry-TUB6 and decorated the nuclear envelope and preprophase bands, mitotic spindles, and phragmoplasts of the mitotic cells of the root meristem (Fig. 3). GFP-GPT1 labeling differed slightly from mCherry-TUB6 labeling, particularly in expanding phragmoplasts. This observation prompted us to carefully examine the localization and dynamics of GPT1 and GPT2 in cortical MT arrays, where the dynamics of single MTs can be clearly visualized.
GPT1 and GPT2 are novel + TIPs
Time-lapse imaging of root epidermal cells using a spinning disk confocal microscope revealed that GFP-GPT1 and GPT2-GFP not only decorated the MT lattice, but also accumulated as particles on MTs (Fig. 4 and Additional files 2, 3 and 4). Double intensity plots with MTs (mCherry-TUB6) and GFP-labeled GPT1/2 showed that GPT1/2 are localized to the MT ends in a comet-like pattern, with the highest signal intensity at the MT ends and a gradual decline in signal further from the MT ends. This MT-end localization was more pronounced for GFP-GPT1 than for GPT2-GFP. The MT ends labeled with GFP were highly dynamic, indicating that GPT1/2 both label the plus ends of cortical MTs. GFP-GPT1 was associated with polymerizing plus ends, and dissociated as soon as catastrophe occurred (Fig. 4c). However, when the MTs started to polymerize again, GFP-GPT1 was immediately recruited to the growing ends (Fig. 4d). GPT2-GFP showed a similar but weaker labeling pattern when compared with GFP-GPT1 (Additional file 2). These results demonstrate that GPT1 and GPT2 preferentially recognize the plus ends of growing MTs.
Additional file 3: Movie S1. Dynamics of cortical MTs (magenta; mCherry-TUB6) and GFP-GPT1 (green) in epidermal cells of the roots of Arabidopsis seedlings. The image sequence corresponds to Fig. 4a. (AVI 14800 kb)
Additional file 4: Movie S2. Dynamics of cortical MTs (labelled magenta by mCherry-TUB6) and GPT2-GFP (green) in epidermal cells of the cotyledons of Arabidopsis seedlings. The image sequence corresponds to Additional file 2: Figure S2A. (AVI 11123 kb)
EB1 is a + TIP family member that recognizes the GTP-cap region of growing MTs [7]. To compare the labeling patterns of GPT1/2 with that of EB1, we stably co-expressed GFP-labeled GPT1 or 2 and mCherry-labeled Arabidopsis EB1b (EB1-mCherry) in Arabidopsis plants. EB1-mCherry showed strong plus-end labeling, as previously reported [11–15]. Many, but not all, EB1 particles co-localized with GFP-GPT1 (Fig. 5a; Additional file 5). When the relative fluorescent intensities of GFP and mCherry were plotted, the comet-shaped fluorescent signals of GFP-GPT1 and EB1-mCherry partially overlapped. In our confocal microscopy setup, the two emitted fluorescent signals from GFP and mCherry were sequentially detected after alternately exciting each fluorophore. When mCherry fluorescence was detected for 0.2 s, followed by the detection of GFP signal for 0.5 s, the highest GFP-GPT1 signal intensity was located approximately 0.2 μm closer to the MT end when compared with the highest EB1-mCherry signal (Fig. 5b, c). When the detection order was reversed, however, the highest EB1-mCherry signal intensity was located approximately 0.15 μm in front of the highest signal intensity of GFP-GPT1 (Fig. 5b, d). At the current spatial resolution, optical artifacts associated with our detection system make it difficult to conclusively determine whether EB1 and GPT1 completely co-localize at growing MT plus ends. In both observations, the fluorescent signal intensities of the EB1 comets decreased gradually and reached background levels at 1.5 μm behind the tips. By contrast, substantial levels of GFP-GPT1 signal (approximately 20% of the highest intensities) remained associated with the MT lattice behind the comet tails.
Additional file 5: Movie S3. Dynamics of EB1-mCherry (magenta) and GFP-GPT1 (green) in epidermal cells of the roots of Arabidopsis seedlings. In one image acquisition, mCherry fluorescence was first recorded for 0.2 s and then GFP fluorescence was recorded for 0.5 s. The image sequence corresponds to Fig. 5a. (AVI 13786 kb)
Tip-tracking of GPT does not require EB1 or SPR1
EB1 recognizes a structural feature of the MT GTP cap and directly binds to the plus ends of growing MTs both in vitro and in vivo [7]. Many animal + TIPs do not bind directly to the MT plus ends in vivo, but are recruited to this region by EB1. To test whether GPT accumulates at the MT plus end directly or through a hitchhiking mechanism involving interaction with EB1, we analyzed the tip-tracking behavior of GPT in the Arabidopsis eb1 null mutant. Because the Arabidopsis genome includes three EB1 genes that may function redundantly [14], a triple eb1 knockout mutant was used to study the expression of GFP-GPT1 and GPT2-GFP. GPT1/2 both localized to the plus ends of growing MTs, in the same pattern as observed in the wild-type background (Fig. 6a, b; Additional files 6, 7 and 8).
Additional file 7: Movie S4. Dynamics of GFP-GPT1 in root epidermal cells of the eb1 triple mutant in Arabidopsis. The image sequence corresponds to Fig. 6a. (AVI 14176 kb)
Additional file 8: Movie S5. Dynamics of GPT2-GFP in cotyledon epidermal cells of the eb1 triple mutant in Arabidopsis. The image sequence corresponds to Additional file 6: Figure S3A. (AVI 13792 kb)
SPR1 and its homologues are plant-specific MT-localized proteins that bind to both the growing plus ends and the MT lattice [17, 18]. Arabidopsis contains seven SPR1 homologues that may be redundant, and SPR1 contributes predominantly to the anisotropic growth of seedling roots [19]. Therefore, we used a spr1 null mutant to test whether SPR1 is required for the plus-end tracking behavior of GPT. Cortical MTs were labeled with mCherry-TUB6. In the spr1 mutant, GPT1/2 labeled growing MT plus ends in interphase epidermal cells, forming a comet-like pattern (Fig. 6c, d; Additional files 6, 9 and 10). The tip-tracking behavior of the GFP-labeled GPTs in the spr1 null mutant was indistinguishable from that in wild-type epidermal cells.
Additional file 9: Movie S6. Dynamics of cortical MTs (labelled magenta by mCherry-TUB6) and GFP-GPT1 (green) in root epidermal cells of the spr1 mutant in Arabidopsis. The image sequence corresponds to Fig. 6c. (AVI 8429 kb)
Additional file 10: Movie S7. Dynamics of cortical MTs (labelled magenta by mCherry-TUB6) and GPT2-GFP (green) in root epidermal cells of the spr1 mutant in Arabidopsis. The image sequence corresponds to Additional file 6: Figure S3C. (AVI 14930 kb)
These results demonstrate that EB1 and SPR1 are not required for the MT plus-end tracking function of GPT1 and GPT2.