Super-Resolution Imaging of Slowly Depolymerizing Microtubules Reveals No Curved Protofilaments with a Lifetime Longer than Seconds

Microtubules are 25 nm diameter, micrometer long, cylindrical protein polymers involved in cell division, cell organization, and intracellular transport among other roles. Microtubule dynamics, that is, how microtubules grow and shrink (polymerization, catastrophe, depolymerization, and rescue) plays a role in each of these functions. Microtubule depolymerization (shrinkage), in particular, is proposed to involve a structural transition from straight protofilaments during microtubule polymerization to curled protofilaments at microtubule plus-ends during shrinkage. Direct evidence of this curved structure (sometimes called ‘ram's horns’) comes from static electron microscopy images and from unresolved intensity increases in light microscopy. As of now, however, no dynamic images of depolymerizing microtubules have directly resolved this ram's horns structure.We use STORM super-resolution microscopy on a custom-built microscope (the Warwick Open Source Microscope, WOSM) to image microtubules, in vitro, with a spatial resolution better than 20 nm and a temporal resolution of 10 seconds. We slow microtubule depolymerization to a similar timescale using sub-stoichiometric concentrations of paclitaxel or kinesin binding in rigor. Movies of these slowly depolymerizing microtubules show no curved protofilaments at microtubule plus ends. Instead, depolymerizing microtubules remain straight objects with a diameter of 25 nm. Our interpretation of these negative results are that the ram's horns structures have a lifetime shorter than seconds. An alternative hypothesis is that partial stabilization of microtubules is concomitant with a straightening of protofilaments at the shrinking end. This alternative suggests that proposed coupling mechanisms for force generation by depolymerizing microtubules must accommodate straight, rather than curved, protofilaments.