The functional significance of microtubule quaternary structure

微管四级结构的功能意义

• 批准号: RGPIN-2014-03791
• 负责人: Brouhard, Gary
• 金额: $ 2.55万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=646332
• 关键词: functional; significance; microtubule; quaternary; structure

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.

微管（MT）是α-β微管蛋白二聚体的细长聚合物，其形成有丝分裂纺锤体、轴丝和细胞骨架。微管蛋白亚基首尾相连形成原丝（pfs），通常13个pfs横向相连形成MT。MT是“可塑性”聚合物，也就是说微管蛋白可以形成横向和纵向曲率不同的结构。这种可塑性是由微管蛋白二聚体之间的连接的灵活性，并可能给予微管其独特的机械性能。然而，在控制微管晶格的四级结构方面，结构可塑性给细胞带来了挑战。为什么微管具有它们所具有的四级结构？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，我的实验室成功地利用单分子生物物理学进行了MT生物学研究。但是对MT细胞骨架的全面理解需要结构数据。在这项拨款提案中，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。1.确定13 pf MT的进化基础。在13 pf MT中，pfs相对于MT的长轴是直的。具有太多或太少pfs的MT在其pfs中具有超扭曲，这导致马达蛋白在细胞内运输期间围绕MT螺旋。这种螺旋的缺点可以解释大多数细胞对13 pf MT的偏好。这个想法已经在这个领域流传了多年，但没有得到实验验证。线虫C. elegans提供了一个独特的机会来解决这个问题，因为C.线虫MT为11 pf而不是13 pf。秀丽线虫MT也有直型pfs？我们建议确定C的三维结构。elegans MT的冷冻电镜观察。如果C. elegans已经进化出了11 pf的MT，但缺乏超扭曲，这将是直pf是MT结构进化驱动力的证据。2.确定自发成核的机理基础。当在细胞提取物中或在体外自发成核时，MT以一定厚度范围形成。有些人有不到13个pfs，而有些人有更多。我们不理解自发成核的过程，以及为什么它不能产生均匀的13 pf MT。毕竟，为什么MT不形成更完美的晶格呢？这种可变性从何而来？我们建议使用一个数学模型的自发成核发展的基础上的pf数分布的假设。然后通过EM测量pf数量分布来检验这些假设。** 3.研究MT末端的结构可塑性。 *MT末端与晶格相比具有不同的结构特征。在不断增长的MT的EM中，末端并不总是钝的。相反，两端有时是“片状”的，一些pfs延伸到其他pfs之外，向外弯曲，变平。目前还不清楚为什么微管以这种方式生长。在微管末端有一个弯曲的或“片状”的四级结构是否有优势？我们将解决为什么微管末端弯曲和“片状”使用单分子生物物理学和EM。我们建议使用改变MT末端曲率的药物治疗来研究MT生长的后果。总之，这项资助将解决微管四级结构功能意义上的三个长期存在的问题。在这些项目的最后，我们将理解为什么真核微管有13个pfs，为什么它们在自发成核时没有13个pfs，以及为什么微管末端的结构与晶格不同。