DETERMINATION OF MICROTUBULE POLARITY BY CRYOELECTRON MICROSCOPY

Background: Microtubules are tubular polymers of tubulin dimers, which are arranged head-to-tail in protofilaments that run lengthwise along the microtubules, giving them an overall structural polarity. Many of the functions of microtubules depend on this polarity, including dire cted intracellular transport and chromosome segregation during mitosis . The determination of microtubule polarity for lengthwise views of mi crotubules observed by electron microscopy has not previously been pos sible. Here, we present methods for directly determining the polarity of individual microtubules imaged by cryo-electron microscopy. Results : When observed in vitreous ice by cryo-electron microscopy, microtubu les with skewed protofilaments show arrowhead moire patterns, We have used centrosome nucleated microtubules to relate the directionality of the moire patterns to microtubule polarity, We show that the arrowhea ds point towards the plus end of microtubules with protofilaments havi ng a right-handed skew, and towards the minus end of microtubules with protofilaments having a left-handed skew, We describe two methods for determining the handedness of the protofilament skew. The first metho d uses two or more tilted views. The second method involves analysis o f the diffraction patterns of the microtubule images. Conclusions: It is now possible to determine directly the polarity of in vitro assembl ed microtubules from cryo-electron micrographs. This will be helpful i n a number of types of studies, including studies of the three-dimensi onal structure of microtubules interacting with motor proteins, as kno wledge of the polarity of the microtubule is essential to understand m otor directionality.