The bending of sliding microtubules imaged by confocal light microscopy and negative stain electron microscopy

Abstract

Individual microtubules can be visualised by confocal microscopy in reflection mode; when associated with a glass surface, they show up as black lines against the bright reflection from the surface. The high contrast imaging allows details of the behaviour of sliding microtubules to be studied easily. Taxol-stabilised microtubules sliding over kinesin-coated surfaces are normally straight, but can bend into tight loops if the leading end sticks to the surface. Some remain curved after release and move in circles. In such cases, the microtubule lattice must have become stably deformed. Electron microscopy of microtubules fixed during sliding shows no gross rearrangement of the subunit lattice and indicates that microtubule bending is mainly achieved by increased twisting of the longitudinal protofilaments around the microtubule.

Fig. 1

Confocal laser scanning image of microtubules, stabilized with taxol and free of associated proteins, adhering to the surface of a glass coverslip. Individual microtubules, interfering with reflection from the surface, produce black or white lines in the image. Averaged over 50 scanning frames and computer enhanced. Specimens in Pipes buffer, pH 6.9. Scale bar, 4 μm.

Fig. 2

Series of individual scanning frames recorded on video and then played back through the confocal frame board; frames at 4 s intervals have been selected. Specimen in Hepes buffer, pH 7.4. Diffraction ripples on the right of each image come from a small central spot of light reflected from the eye-piece. White scale bar, 6 μm

Fig. 3

Confocal series, similar to Fig. 2. The long microtubule in the centre of (A) was moving in a straight line until its leading end became attached to the glass (B–C). After pronounced bending (D), the tubule broke (E). A major portion re-straightened and slid off in a straight line (F). A smaller fragment remained curved and moved in a circle (arrows in G–H). Meanwhile, a similar fragment (x) produced by an earlier event rotated until its path changed (F). Hepes buffer, pH7.4. Time intervals shown are unequal. White scale bar, 6 μm.

Fig. 4

Confocal image series showing an arc-shaped microtubule moving around a circle. ×marks the leading end. Hepes buffer, pH 7.4. 3–4 s between images. Scale bar, 3 μm.

Fig. 5

Confocal series in which an arc-shaped microtubule was seen moving initially in a circle (A–C). The original leading end stuck to the surface, producing a break (D). The rear portion became straight and slid off in a straight line (F). Hepes buffer, pH 7.4. Time intervals unequal, chosen to show particular events. Scale bar, 6 μm.

Fig. 6

Electron micrograph of a microtubule bent almost into a circle with a radius of 500 nm. Specimen in Hepes buffer, pH 7.4, before staining. Inset shows a confocal light microscope image including a similarly bent microtubule that was observed rotating. White scale bar, 100 nm for EM image, 1 μm for confocal image.

Fig. 7

Electron micrograph of parts of a curved and a straight microtubule on a kinesin coated plastic surface. Kinesin molecules, appearing as fine rods (indicated by arrow-heads), are not easily distinguished against the irregular background of negative stain. Hepes buffer, pH 7.4. Scale bar, 100 nm.