The functional significance of microtubule quaternary structure

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Microtubules (MTs) are long, slender polymers of α-β tubulin dimers that form the mitotic spindle, axonemes, and the cytoskeleton. Tubulin subunits associate head-to-tail to form protofilaments (pfs), and typically 13 pfs are associated laterally to form the MT. MTs are "plastic" polymers, which is to say that tubulin can form structures that differ in their lateral and longitudinal curvature. This plasticity is made possible by flexibility in the links between tubulin dimers and may give microtubules their unique mechanical properties. Structural plasticity creates a challenge for cells, however, in terms of controlling the quaternary structure of the microtubule lattice.**Why do microtubules have the quaternary structure(s) they do? For example, we have known for 40 years that eukaryotic MTs almost always have 13 pfs, but why? What is special about 13 pfs? Over the past 5 years, my lab has successfully pursued MT biology using single-molecule biophysics. But a comprehensive understanding of the MT cytoskeleton will require structural data. In this grant proposal, we have taken up the challenge of elecron microscopy (EM) in order to answer three long-standing questions in the field.**1. Determine the evolutionary basis of the 13 pf MT.*In a 13 pf MT, the pfs are straight relative to the long-axis of the MT. MTs with too many or too few pfs have a supertwist in their pfs that causes motor proteins to spiral around the MT during intracellular transport. The disadvantages of such spiraling may explain the preference of most cells for 13 pf MTs. This idea has circulated in the field for years without experimental validation. The nematode worm C. elegans offers a unique opportunity to address this question, because C. elegans MTs are 11 pf rather than 13 pf. Do C. elegans MTs also have straight pfs? We propose to determine the 3D structure of C. elegans MTs using cryo-EM. If C. elegans has evolved 11 pf MTs that lack a supertwist, this would be evidence that straight pfs are the evolutionary driving force for MT structure.**2. Determine the mechanistic basis of spontaneous nucleation.*MTs form in a range of thicknesses when nucleated spontaneously in cell extracts or in vitro. Some have fewer than 13 pfs, whereas some have more. We do not understand the process of spontaneous nucleation and why it does not produce uniform 13 pf MTs. After all, why don't MTs form a more perfect lattice? Where is this variability coming from? We propose to use a mathematical model of spontaneous nucleation to develop hypotheses for the basis of pf number distributions. These hypotheses will then be tested by measuring pf number distributions by EM.**3. Investigate the structural plasticity of MT ends. *MT ends have distinct structural features compared to the lattice. In EM of growing MTs, the ends are not always blunt. Rather, the ends are sometimes "sheet-like", with some pfs extending beyond others, curving outward, and flattening. It remains unclear why microtubules grow this way. Is there an advantage to having a curved or "sheet-like" quaternary structure at microtubule ends? We will address why microtubule ends are curved and "sheet-like" using both single-molecule biophysics and EM. We propose to use drug treatments that alter the curvature at MT ends to investigate the consequences with respect to MT growth.**In summary, this grant will solve three long-standing problems on the functional significance of microtubule quaternary structure. At the end of these projects, we will understand why eukaryotic microtubules have 13 pfs, why they do not have 13 pfs when nucleated spontaneously, and why the structure of microtubule ends differs from the lattice.