Multimerized +TIP Domains Confer Plus End Tracking In Vivo

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Having determined structures and in vitro activities of +TIP domains, we next tested the functions of these domains in living cells with regard to plus end tracking activity.  A single CH domain from EB1 or Cap-Gly domain from CLIP-190 fused to GFP did not plus end track when transfected into Drosophila S2 cells (Figure 6).  However, artificial dimerization of these domains with a leucine zipper enabled clear plus end tracking along microtubules (Figure 6; Movies S2 and S3). We also tested the ability of the TOG domains from Msps to confer plus end localization in S2 cells.  Consistent with previous findings (Popov et al., 2001), we found that full length Msps fused to GFP could plus end track, but a truncation encompassing TOG1-4 did not and instead was diffusely localized in the cell (Figure 6) as was a leucine zipper dimerized TOG1-2 construct (not shown).  This result, unlike the above result for EB1 and CLIP-170, suggests a more complex plus end tracking requirement for XMAP215 than simply binding multiple tubulin monomers and suggests a possible synergistic role for the C-terminal domain (CTD) to cooperate with the TOG domains for microtubule plus end localization.  Analysis of Msps TOG1-4 fused to a leucine zipper however, resulted in microtubule decoration.  This construct raised the functional number of TOG domains to eight indicative that TOG domains have an inherent affinity for the microtubule lattice, but the bona-fide microtubule bind and release mechanism employed by XMAP215 family +TIPs likely requires a carefully titrated affinity mediated by five TOG domains and the CTD in metazoans and four TOG domains and two C-terminal tubulin binding domains in the functionally homodimeric yeast counterparts.

To obtain further evidence for a role of dimerization in plus end tracking, we fused the human EB1 CH domain to either FRB or FKBP domains, transfected both constructs into HeLa cells and then induced dimerization in real time by addition of the natural product rapamycin (which binds to and crossbridges FRB and FKBP (Banaszynski et al., 2005; Choi et al., 1996)).  When either construct alone was transfected into HeLa cells, only diffuse localization was observed either in the presence or absence of rapamycin (data not shown).  When both constructs were transfected simultaneously in the absence of rapamycin, a diffuse cytoplasmic localization was again observed.  However, when rapamycin was added, the GFP-tagged proteins rapidly relocalized to the tips of growing microtubules (Figure 7; Movies S5A and B).  Identical results were obtained with a similar set of experiments chemically dimerizing Cap-Gly domain 1 of CLIP-170 (CLIP-170_3-210-FRB) to Cap-Gly domain 2 (CLIP-170_129-350-FKBP) in HeLa cells (Figure 6B; Movie S6).  These results provide strong evidence for the role of dimerization of +TIP tubulin binding domains in the localization to microtubule plus ends.

Given the similar minimal requirement for two tubulin binding domains for plus end tracking by EB1 and CLIP-170, we next asked whether a heterodimer of these two unrelated structural domains might enable plus end tracking. To address this, we co-transfected EB1_1-187-FRB and CLIP-170_3-210-FKBP into cells.  Remarkably, rapamycin addition also induced a relocalization of these proteins to microtubule tips (Figure 7, Movie S7).  These results indicate that two structurally unrelated +TIP tubulin binding scaffolds can cooperate together to localize to growing microtubule plus ends.