The dynamic behavior of microtubules is essential to their functions, as underlined by the large number of compounds that bind tubulin, alter dynamics and result in mitotic arrest. A growing number of cellular factors regulate this dynamic behavior during the cell cycle. Interestingly, dynamic instability is an instrinsic property of tubulin that can be observed in purified solutions and is linked to the binding and hydrolysis of GPT. Our studies of tubulin bound to taxol in polymerized, straight protofilaments, obtained by electron crystallography (Nogales et al., Nature 1998) established the structural basis of nucleotide exchange and polymerization-coupled hydrolysis (Nogales et al., Nature Struct Biol. 1998; Löwe et al., JMB 2001). An essential question to understand microtubule dynamics is whether the structure of tubulin is dictated by nucleotide, polymerization state, or both. My lab has recently obtained two structures corresponding to the start and end points in the polymerization and nucleotide hydrolysis cycles that illustrate the conformational consequences of the nucleotide state and how they relate to longitudinal and lateral assembly.

Using cryo-electron microscopy and a new iterative Fourier Bessel method (Wang and Nogales, JSB 2005) we have obtained a structure of GDP-tubulin in the absence of depolymerizers (Wang and Nogales, Nature 2005). This structure shows distinctive intra and inter dimer interactions and thus distinguishes the GTP and GDP interfaces. The bending of these interfaces in GDP-tubulin is incompatible with the formation of the lateral contacts in microtubules. So, how can binding of GTP result in the "straightening" of protofilaments observed in microtubules? To answer this question we have studied the self-assembly of GMPCPP-bound tubulin into helical ribbons that correspond to the structural intermediate in microtubule assembly that preceed microtubule closure into a tube (Wang and Nogales, Nature 2005). Our study supports the separation of the straightening process into two stages: one, nucleotide-dependent, that allows for lateral association into a curved sheet, and a later one that occurs upon microtubule closure. Most importantly, it provides a pseudo-atomic model of this sheet that illustrates a bimodal mechanism of lateral protofilament interaction preceding microtubule closure.

The flexibility of tubulin and the consequent versatility of its self-assembly can hardly be an accident. We propose that the polymorphism of assembly unique to tubulin reflects an exquisite tuning mechanism for the complex interaction of different microtubule intermediates with cellular factors that need to detect or make direct use of the growing or shortening state of microtubules to play functional roles at the right time and place in the cell. The characterization of the molecular interplay between tubulin polymers and cellular factors that affect tubulin assembly and/or are able to select tubulin polymerization states has only just begun, and promises to create new paradigms of microtubule cellular function, where tubulin polymers are not seen as passive platforms but as molecular machines capable of work by switching conformational and polymerization states. This is now a major area of research in my lab (see also Dam1 complex studies). In collaboration with Rebecca Heald, we are now pursuing the idea that the transient sheets at the end of growing microtubules may be the landing site for microtubule plus-end tracking proteins. Of additional interest are other cellular factors that interact more generally with stable microtubules, but concentrating on proteins or complexes essential during cell division. In this area we have just initiated a collaboration with Michael Rape to study the interaction of the APC with microtubules via a recently discovered microtubule-binding E2 enzyme.