Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends

Abstract

Microtubule (MT) plus end-tracking proteins (+TIPs) specifically recognize the ends of growing MTs. +TIPs are involved in diverse cellular processes such as cell division, cell migration, and cell polarity. Although +TIP tracking is important for these processes, the mechanisms underlying plus end specificity of mammalian +TIPs are not completely understood. Cytoplasmic linker protein 170 (CLIP-170), the prototype +TIP, was proposed to bind to MT ends with high affinity, possibly by copolymerization with tubulin, and to dissociate seconds later. However, using fluorescence-based approaches, we show that two +TIPs, CLIP-170 and end-binding protein 3 (EB3), turn over rapidly on MT ends. Diffusion of CLIP-170 and EB3 appears to be rate limiting for their binding to MT plus ends. We also report that the ends of growing MTs contain a surplus of sites to which CLIP-170 binds with relatively low affinity. We propose that the observed loss of fluorescent +TIPs at plus ends does not reflect the behavior of single molecules but is a result of overall structural changes of the MT end.

Figure 1.

Fast FRAP analysis. (A–A′′′) Time-lapse imaging of COS-7 cells transiently expressing GFP–CLIP-170 (every 10th frame is shown). Two GFP–CLIP-170–labeled MT ends are indicated by arrows. In A, one of these is about to traverse a 210 × 210-nm ROI (red rectangles). After 2.25 s (A′′′), this MT end has traversed the ROI. (B) Fluorescence intensity in an ROI of 210 × 210 nm as a GFP–CLIP-170–labeled MT end traverses. (C and G) Mean fluorescence decay of nonbleached, GFP–CLIP-170-labeled (C) or EB3-GFP–labeled (G) MT ends (k_decay is indicated). (D–D′′′) COS-7 cells transiently expressing GFP–CLIP-170 were imaged as in A. An area of 256 × 3 pixels (indicated in D′) was bleached every 7.5 s, occasionally resulting in bleaching of MT end–bound GFP–CLIP-170. Rectangles 1–4 are shown enlarged underneath D–D′′′. Fluorescence intensity was measured in ROIs of 210 × 210 nm (red rectangles). (E) Fluorescence intensity of a bleached GFP–CLIP-170–labeled MT end (black arrow indicates bleach; black line indicates mean fluorescence decay). (F and H) Mean fluorescence recovery of GFP–CLIP-170 (F) and of EB3-GFP (H) on MT ends (k_recovery is indicated).

Figure 2.

Influence of temperature on GFP–CLIP-170 behavior. (A) GFP–CLIP-170 comets analyzed in stably expressing 3T3 cells (13 MT ends analyzed at 37°C, 19 at 32°C, and 14 at 27°C). (B) Mean fluorescence decay of GFP–CLIP-170–labeled MT ends in stably expressing 3T3 cells measured at 37°C (black; 13 ends analyzed) and at 27°C (red; 12 ends analyzed). A fit (see Materials and methods) yields a half-life of GFP–CLIP-170 fluorescence of 1.35 s at 37°C and 2.67 s at 27°C. (C) Mean fluorescence decay of GFP–CLIP-170–labeled MT ends in transiently transfected COS-7 cells at 37°C (black) and 27°C (red). See Table I for values. (D and E) Fast FRAP analysis at 27°C in MEFs transiently transfected with GFP–CLIP-170 (D) or GFP–CLIP-170XmnI (E). Fluorescent recovery was measured in the cytoplasm (blue) and on MT ends (red). For k_recovery values, see Table I. Error bars indicate SEM.

Figure 3.

Analyzing GFP–CLIP-170 on MT ends with FCS. (A) Confocal image of a COS-7 cell transiently transfected with GFP–CLIP-170. The plus sign indicates the location of FCS measurement. Bar, 5 μm. (B) Intensity track of FCS measurement in a transfected COS-7 cell. Peaks of fluorescence are occasionally detected. The dotted red line indicates the exponential fluorescence decay; the bottom double-headed arrows indicate cytoplasmic fluorescence; the top double-headed arrows indicate peak fluorescence. (C) Comparison of the number of cytoplasmic and peak-bound GFP–CLIP-170 particles. Values as depicted in B were measured, and the number of particles was determined for 110 peaks. A scatter plot of these values is best approximated by the curve (indicated by the red line) Y = Y_max × (1 − e^−kx).

Figure 4.

Fast exchange model. (A) MT polymerization generates a large number of binding sites (orange ellipses), which disappear with single-order reaction kinetics. Thus, as time progresses, less binding sites are present within the depicted rectangle. (B) Dimeric CLIP-170 exchanges rapidly on binding sites irrespective of the position on the MT end. Several interactions with CLIP-170 molecules can occur during the lifetime of a binding site. The equilibrium between cytoplasmic and MT end–bound CLIP-170 (reaction a) might be determined by posttranslational modifications (reaction c), conformational changes, and/or protein–protein interactions. As we find CLIP-170 exchange on MT ends distal of sites of MT polymerization, copolymerization of CLIP-170 with tubulin (reaction b) does not explain the cometlike distribution of +TIPs. However, it is not excluded (hence the stippled arrow), and modified forms of CLIP-170 (indicated by the purple ellipses) might bind tubulin with higher affinity.