Potential mechanisms of microtubule plus-end tracking. (A) Motor-driven transport: CLIP-170 (red) is physically moved toward the microtubule tip by motors (blue). Fungal CLIP-170 homologues Bik1p and Tip1p appear to track by this mechanism; (B) “surfing”: CLIP-170 (red bars) diffuses (“surfs”) along a tip-specific conformation or another plus-end tracking protein (bright green). Note that CLIP-170 is represented by bars rather than circles because this model likely requires multiple binding sites; (C) end-loading: CLIP-170 (red) binds specifically at the microtubule plus-end and then dissociates shortly thereafter. CLIP-170 is stationary on the microtubule and would not be observed to move toward either the plus- or minus-end of the microtubule. Loading at the tip could occur by preferential binding of CLIP-170 to the cap (“cap-recognition”), or by preassociation with unpolymerized tubulin followed by copolymerization. Dissociation could occur by CLIP-170 having an intrinsically weak affinity for the “older” GDP polymer or be induced by a regulatory event that reduces the affinity of CLIP-170 for microtubules.

Dynamic behavior of CLIP-170 in vivo. (A) CLIP-170 constructs used in this work. As previously described by Scheel et al. H1 acts as a monomer and H2 as a dimer; (B and C) Cos7 cells were transfected with GFP-CLIP-170 (B) or GFP-CLIP-170-H1 (C), imaged by streaming video (0.2 s/frame; see movies in Supplementary Data), and the behavior of CLIP-170 on individual microtubules was examined by making kymographs; (D) Cartoon depicting the pattern expected if CLIP-170 were moving along a microtubule via motors. The white line indicates the plus-end of the microtubule, the gray arrow predicts what would be observed if CLIP-170 were moving toward the plus-end and the white arrow predicts what would be observed if CLIP-170 is moving away from the microtubule plus-end; (E–G) Kymographs of comets in cells transfected with GFP-CLIP-170 (E) and GFP-CLIP-170-H1 fragment (F and G). The kymographs depict position on the x-axis (increasing to the right) and time on the y-axis (increasing toward the bottom). Notice that both bright and dim heterogeneities form vertical lines indicating that they maintain their position (they do not move to the left or right) while they decay over time. (G) An example of GFP-H1 behavior during microtubule rescue. The microtubules corresponding to the kymographs are indicated by arrows in B and C and in the corresponding movies (see Supplementary Data); (H) cartoon interpreting the kymograph of the rescue event (2G). The microtubule seen in white is shrinking for several seconds. Suddenly CLIP-170 (green) appears at the plus-end of the shrinking microtubule at approximately the same time that the microtubule is rescued. The most important aspect of the CLIP-170 appearance is that it is not seen traveling to the plus-end on the microtubule but instead appears de novo from the cytosol.

CLIP-170 can distinguish between GTP- and GDP-like microtubules. Cosedimentation assays were used to analyze CLIP-170 binding to microtubules. Microtubules stabilized with Taxol were used to mimic the GDP lattice of the microtubule. Microtubules stabilized with the slowly hydrolyzed GTP analog GMPCPP were used to mimic the GTP tubulin in the cap. The binding of 2 μM CLIP-170 fragments was measured as a function of microtubule concentration. Three separate experiments were performed and are indicated by squares, triangles, and circles. The fraction of CLIP-170 bound (in the pellet) was plotted against the concentration of unbound polymerized tubulin, and the three experiments then simultaneously fit to the bimolecular binding curve to obtain the apparent K_d (see Materials and Methods for details). Both H1 and H2 bind GDP microtubules with a strong affinity, 0.5 ± 0.1 μM (A) and 0.2 ± 0.1 μM (B), respectively. However, each construct binds the GTP-like GMPCPP microtubules more tightly, with respective K_d values of 0.15 ± 0.3 μM (C) and 0.12 ± 0.02 μM (D).

CLIP-170 binds tubulin tightly and with slow kinetics. (A) Coimmunoprecipitation experiment visualized by western blot with the polyclonal antibody against tubulin, T13. Coimmunoprecipitations were performed with 4D3, a mAb against CLIP-170 (lane 1) or a mAb against myc (lane 2). Loading controls from these experiments are shown in lane 3 (4D3) and lane 4 (anti-myc); (B and C) the tubulin binding ability of both H1 and H2 were analyzed by SPR (Biacore) as described in Materials and Methods. H1 (B) and H2 (C) show nearly identical equilibrium binding abilities for soluble tubulin with respective K_d values of 0.033 ± 0.004 and 0.037 ± 0.004 μM for GTP tubulin (gray) and 0.055 ± 0.008 and 0.054 ± 0.009 μM for GDP tubulin (black). Three replicates were performed at each tubulin concentration and the variation is shown by error bars. Representative experiments for binding to GTP tubulin are shown as insets; (D) SPR experiments optimized to determine dissociation kinetics show that CLIP-170 releases both GDP tubulin dimers (dark gray) and GTP tubulin dimers (light gray) slowly. Fitting of both data sets as single phase dissociations (see Materials and Methods for details) showed that GTP tubulin and GDP tubulin dissociate with k_off values of 3.5 × 10^-3± 2 × 10^-4 s^-1 and 4.4 × 10^-3± 5 × 10^-4 s^-1, respectively.

Fluorescence anisotropy confirms the cosedimentation and Biacore experiments. Equilibrium binding affinities of CLIP-170 for GTP-like microtubules, GDP-like microtubules, and GTP tubulin were determined by measuring the anisotropy of labeled H1 as a function of tubulin/microtubule concentration and fitting to a rectangular hyperbola (see Materials and Methods for details). In the case of these analyses, unlike those in Figure 3, H1 binding is plotted as a function of total microtubule concentration because the technique allows us to use H1 at a concentration that is less than the K_d (0.1 μM). This results in a situation where the concentration of free ligand is approximately equal to the concentration of total ligand. (A) H1 binds GDP-like (Taxol) microtubules with a 0.67 ± 0.08 μM affinity; (B) H1 binds GTP-like (GMPCPP) microtubules with a 0.40 ± 0.05 μM affinity; (C) H1 binds GTP tubulin with a 0.26 ± 0.04 μM affinity; (D) binding kinetics were studied by adding tubulin (1 μM, blue); 2 μM, red; or 4 μM green) to labeled H1. Anisotropy was read before the addition of tubulin, 5 s after the tubulin was added, and every 15 s thereafter. The first 60 s of binding are depicted in the inset. Data were fit as a single-phase association and analysis of the kinetic data are presented in Table 1.

Binding of CLIP-170 to microtubules is characterized by slow kinetics in vitro and fast kinetics in vivo. (A and B) Linescan analysis of kymographs that were generated from the microtubules indicated in Figure 2 was used to measure the decay of CLIP-170 fluorescence (A) or H1 fluorescence (B) at a particular position on the microtubule over time. Fitting the decay as a single-phase dissociation gave k_off values of 0.21 s^-1 and 0.16 s^-1 for both CLIP-170 and H1, respectively. Note that though these single measurements appear to give different rates, the average values obtained from the full analysis (see text) show that H1 and CLIP-170 have indistinguishable off rates in our experiments; (C and D) the rate of H1 association with microtubule polymer was measured by fluorescence anisotropy. Microtubules, 5 μM (blue), 10 μM (red), and 20 μM (green), stabilized by either Taxol (C) or GMPCPP (D) were added to 0.1 μM labeled H1. The anisotropy was measured before microtubule addition, 5 s after microtubule addition, and every 15 s thereafter; insets show the first 100 s of binding. Data sets were fit as a two phase association (see Materials and Methods for details) and analysis of this data are presented in Table 2.

Model for the plus-end tracking mechanism of CLIP-170. (1) CLIP-170 binds dimeric tubulin; (2) CLIP-170 copolymerizes onto the growing microtubule plus-end; (3) CLIP-170 remains bound to the microtubule without moving toward either the plus-end or the minus end; (4) CLIP-170 dissociates from the microtubule with the help of other cellular factors.