Nanometer targeting of microtubules to focal adhesions

Abstract

Although cell movement is driven by actin, polarization and directional locomotion require an intact microtubule cytoskeleton that influences polarization by modulating substrate adhesion via specific targeting interactions with adhesion complexes. The fidelity of adhesion site targeting is precise; using total internal reflection fluorescence microscopy (TIRFM), we now show microtubule ends (visualized by incorporation of GFP tubulin) are within 50 nm of the substrate when polymerizing toward the cell periphery, but not when shrinking from it. Multiple microtubules sometimes followed similar tracks, suggesting guidance along a common cytoskeletal element. Use of TIRFM with GFP- or DsRed-zyxin in combination with either GFP-tubulin or GFP-CLIP-170 further revealed that the polymerizing microtubule plus ends that tracked close to the dorsal surface consistently targeted substrate adhesion complexes. This supports a central role for the microtubule tip complex in the guidance of microtubules into adhesion foci, and provides evidence for an intimate cross-talk between microtubule tips and substrate adhesions in the range of molecular dimensions.

Figure 1.

TIRFM reveals microtubule tips and focal adhesions within 150 nm of the dorsal cell surface. CAR fibroblasts were transfected with either GFP-tubulin (A and C) or GFP-zyxin (B and D). Live-cell images were taken either with standard wide field epi-illumination (A and B) or TIRFM (C and D). For clarity, TIRFM images are shown in reverse contrast. Bar, 10 μm. For C, see also Video 1 and Video 2.

Figure 2.

Approach of microtubule plus end tips to the cell surface. (A) CAR fibroblasts transfected with GFP-tubulin and imaged by TIRFM (as in Fig. 1 C) were false colored to highlight the change in fluorescence intensity along the microtubule. No pixels were saturated. Bar, 10 μm. (B) The intensity of four microtubules (labeled 1–4 in A) was measured along the microtubules starting near the extreme plus end (near number). Based on an exponential drop-off in fluorescence intensity as an inverse exponential away from the surface (1/e = penetration depth) the relative distance of the microtubules to the cell surface was calculated and plotted as a line trace (0 = plus end). Traces were also taken in regions not containing microtubules to correct for the local background (not depicted). Note that close contact of the microtubule plus end with the cell surface over several micrometers; gradually, the intensity drops as the microtubules enter deeper into the cell (right side). In this temporal “snapshot,” there are both polymerizing and depolymerizing microtubules, and the extreme plus end often “lifts off” before the microtubule depolymerizes (see Video 1 and Video 2). Most microtubules approach the surface at a shallow angle of <5°, although occasionally the angle of attack was observed as steep as ∼10°, e.g., in the region marked with a red asterisk.

Figure 3.

Observation of microtubule plus ends by TIRFM indicates that microtubules lift away from the substrate during shrinkage, and shows microtubule tracking along common paths. (A–C) Higher magnification views of individual time-lapse frames from Fig. 1 C. (A) Growing microtubule plus ends as observed by TIRFM. The asterisk marks the position of the microtubule ends in the first frame, and the arrows follows the microtubule growth in subsequent frames. Note that the microtubules “dip down” and form a close contact with the cell cortex during microtubule elongation, as seen by the difference in fluorescent intensity along the microtubule. (B) A representative example of a shrinking microtubule. Note the loss of close contact of the microtubule with the cell matrix as it shrinks. (C) Microtubules were observed to follow common tracks appearing to piggy-back along each other, sometimes with three or more following a common track. The arrows mark microtubules that follow common tracks (see also Video 1 and Video 2).

Figure 4.

Nanometer targeting of microtubules to focal adhesions revealed by TIRFM. Video frames in TIRFM of CAR fibroblasts double transfected with DsRed-zyxin and GFP-tubulin. Pseudocolors have been used to facilitate visualization of adhesions (zyxin, green) and microtubules (tubulin, red). Single-channel GFP-tubulin images are shown in black and white. (A) An overview of the edge of the cell showing microtubule-adhesion targeting (top) and, on the bottom, the approach of a number of microtubules tips to the substrate in positions colocalizing with adhesion sites (arrows). (B and C) Other examples of microtubules dipping down to adhesion complexes; time shown in seconds (see also Videos 3–5).

Figure 5.

Polymerization of microtubule tips into adhesion sites. (A) Confocal video frames of CAR fibroblast transfected with DsRed-zyxin and GFP–CLIP-170. Different arrowhead pairs mark the tips of three microtubules en route to an adhesion complex. Times are in seconds. (B) Video images of a cell transfected as in A, but imaged instead by TIRFM, showing the close proximity of polymerizing microtubule tips to the dorsal cell surface (note that the high contrast TIRFM has much less background and microtubule plus ends (green) are seen in the evanescent field). (C) A merge of five time points (over 20 s) from the boxes in B shows the microtubules polymerizing toward the adhesion markers (red). (D) Dual-color TIRFM imaging of a cell transfected with GFP-tubulin and DsRed-zyxin. Cell region is similar to the one shown in B (see also Videos 6–8). (E) Working model of the guidance of microtubules into focal adhesions. Top part shows side view of guidance scenario highlighting the striking differences in Z-axis position of microtubule plus ends during polymerization (Pol.) and depolymerization (de-Pol.). Microtubule polymerization is directed down to an adhesion site and into the evanescent wave (EW) by the docking of the microtubule tip complex onto an actin track. Depolymerization away from the adhesion site is associated with the loss of the tip complex and the lifting of the microtubule away from the track and the substrate. Depolymerizing microtubules can go through rescue and reenter the polymerizing cycle to retarget the adhesion site through dynamic instability. Bottom part shows boxed region scaled to approximate molecular dimensions. The focal adhesion (FA) and axis of the microtubule (∼24 nm in diameter) are drawn 15 and 50 nm away from the substrate, respectively. The numerous known tip complex proteins (TCx) likely decorate the microtubule surface much more extensively than depicted. Coupling between microtubules and actin (a) is effected by a coupler complex (Cr) that is part of the microtubule tip complex, as proposed elsewhere (Small and Kaverina, 2003). The coupler could be an unconventional myosin or a microtubule–actin cross-linker (see text). Microtubules may be captured (hook) at the adhesion site. ECM, extracellular matrix.