The functional significance of microtubule quaternary structure

微管（MTs）是长而细长的α-β微管蛋白二聚体聚合物，形成有丝分裂纺锤体、轴突和细胞骨架。微管蛋白亚基头尾相连形成原丝（pfs），通常13个pfs横向相连形成MT。MT是“塑料”聚合物，也就是说微管蛋白可以形成不同的横向和纵向曲率结构。这种可塑性是由于微管蛋白二聚体之间连接的灵活性而成为可能，并可能赋予微管独特的机械性能。然而，在控制微管晶格的四级结构方面，结构可塑性对细胞构成了挑战。**为什么微管具有四元结构？例如，40年前我们就知道真核mt几乎总是有13个pfs，但为什么呢？13 pfs有什么特别之处？在过去的5年里，我的实验室利用单分子生物物理学成功地研究了MT生物学。但是对MT细胞骨架的全面了解需要结构数据。在这项拨款申请中，我们接受了电子显微镜（EM）的挑战，以回答该领域三个长期存在的问题。确定13pf MT的进化基础。*在13pf MT中，pfs相对于MT的长轴是直的。pfs过多或过少的MT在其pfs中有一个超扭曲，导致运动蛋白在细胞内运输过程中绕MT旋转。这种螺旋的缺点可能解释了大多数细胞对13pf mt的偏好。这个想法在该领域流传了多年，但没有得到实验验证。线虫线虫提供了一个独特的机会来解决这个问题，因为秀丽隐杆线虫的mt是11pf而不是13pf。秀丽隐杆线虫的mt也有直的pfs吗？我们建议使用冷冻电镜（cryo-EM）来确定秀丽隐杆线虫mt的三维结构。如果秀丽隐杆线虫进化出了11个缺乏超捻的MT，这将证明直pfs是MT结构的进化驱动力。**2。确定自发成核的机理基础。* mt在细胞提取物或体外自发成核时形成一定厚度。有些少于13个pfs，而有些则更多。我们不明白自发成核的过程，也不明白为什么自发成核不能产生均匀的13pf mt .毕竟，mt为什么不能形成更完美的晶格呢？这种可变性从何而来？我们建议使用自发成核的数学模型来发展假设的基础上的pf数分布。然后，这些假设将通过EM测量pf数分布进行检验。**3。研究MT端部的结构塑性。与晶格相比，MT端具有明显的结构特征。在新兴市场或新兴经济体中，结局并不总是生硬的。相反，末端有时是“片状”的，一些pfs延伸到其他pfs之外，向外弯曲，并变平。微管以这种方式生长的原因尚不清楚。在微管末端具有弯曲或“片状”的四元结构是否有优势？我们将使用单分子生物物理学和EM来解释为什么微管末端是弯曲的和“片状”的。我们建议使用药物治疗来改变微管末端的曲率，以研究微管生长的后果。**综上所述，本次资助将解决三个长期存在的关于微管四元结构功能意义的问题。在这些项目结束时，我们将理解为什么真核微管有13个pfs，为什么它们自发成核时没有13个pfs，以及为什么微管末端的结构与晶格不同。

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.