Microtubule regulation by isotype expression and by small molecules.

天然产物历来是大多数微管（MT）靶向小分子的来源，这些小分子的特性使它们成为有用的药物。这仍然适用于我们在本研究中使用的大多数但不是所有化合物。这些包括临床上确定的MT活性药物秋水仙碱、考布他汀、长春新碱、紫杉醇等。几乎所有这些试剂首先在临床前研究中开发，所述临床前研究包括化合物对微管蛋白聚合成微管的影响以及这些化合物对细胞行为的影响的体外研究，特别是检查化合物通过对包含有丝分裂纺锤体的MT阵列的影响破坏有丝分裂的能力。事实上，在实验室中快速生长的细胞培养物中引起有丝分裂停滞的能力通常被认为是这些药物的主要机制的测定。 通常，微管和微管阵列的调节以及小分子对这些阵列的影响的研究取决于显微镜的工具，用于纯化蛋白质的体外测定，特别是用于解决细胞内微管阵列的测定。因此，我们与专注于荧光方法和先进显微镜应用的实验室合作。一个这样的应用是超分辨率显微镜，这实际上是一类使用光学显微镜实现增强空间分辨率的方法，直到几年前还被认为是不可能的。一种这样的方法是STED显微术，受激发射损耗显微术。这种方法可能需要高功率的光，导致荧光团的光漂白。为了最大限度地减少这种情况并优化分辨率，我们开发了一种简单的方法来检查STED显微镜中的功率优化，该方法可以在显微镜工作台上轻松完成。 我们对靶向微管蛋白/微管的小分子的研究重点是利用我们对微管蛋白结构的了解来鉴定可能成为有用药物的新分子，并对现有的有效药物进行改造，以延长其有效性或更好地靶向它们在体内，以最大限度地减少对旁观者组织的副作用。我们以前已经开发了将连接臂连接到一些已知药物的化学方法，今年通过展示如何将连接臂连接到有效的天然产物药物spongistatin 1而不失去效力来扩展这项工作。 这一成就将允许这种药物（已知最有效的细胞毒性化合物之一）连接到抗体或其他赋予亲和力的实体上，这些实体将使强效药物靶向预期靶点，如肿瘤或转移灶。虽然spongistatin使微管不稳定，但其他药物使它们稳定（如紫杉醇，商品名Taxol），我们也在寻找这些药物的新变体。其中两个最有前途的是结构类似物zampanestrin和dactylestrin。它们结合在微管蛋白的同一位置;赞潘鲁肽共价结合，而dactyllavin非共价结合。我们已经制备并检查了这些化合物的构象类似物，以更好地了解它们的稳定效力和共价反应的意义。 我们也追求微管蛋白二聚体的基本结构生物学。这些知识是理解小分子如何调节微管蛋白以及微管蛋白结合结构域如何调节与其他蛋白质的相互作用所必需的。微管蛋白生物学中最基本的步骤是异二聚体的组装，我们先前描述了使用分析性超离心和荧光偏振的研究，以表明二聚体的形成是一个可逆的，质量作用驱动的过程。我们还研究了微管蛋白羧基末端尾肽的作用，其介导许多蛋白质与微管蛋白和/或微管的相互作用。我们已经扩展了这些研究，表明二聚体解离和单体交换控制的平衡常数，不同的数量级之间的微管蛋白从不同的生物来源。这些包括来自人细胞的微管蛋白与来自不同生物体（包括原生动物寄生虫）的微管蛋白的比较，以及来自不同组织的微管之间的微管蛋白，其代表微管同种型的不同表达水平。这项工作也证明了非结合表面在蛋白质-蛋白质相互作用中的重要性。除了异源二聚体的形成之外，还调节α和β微管蛋白的同种型的表达以促成异源二聚体。表达的组织特异性强有力地表明，同种型对特定细胞或组织类型具有特异性的功能，但这方面的证据并不丰富。我们报告了β微管蛋白同种型在骨骼肌中的表达改变对杜氏肌营养不良症（DMD）和常用的mdx小鼠肌肉营养不良模型的肌肉功能的影响。微管阵列是已知的DMD和mdx骨骼肌纤维被破坏，在这里，我们表明，过度表达的微管蛋白β 6同种型（基因= tubb 6）的结果在微管解体中看到营养不良的肌肉。β 5同种型的过表达没有这种作用。

Natural products have historically been the source of most of the microtubule (MT)-targeting small molecules whose properties have allowed them to become useful drugs. That remains true of most but not all of the compounds that we have used in this study. These include the clinically established MT-active drugs colchicine, combretastatin, vincristine, taxol, and others. Almost all such agents were developed first in pre-clinical research that included in vitro studies of the effect of the compounds on polymerization of tubulin to microtubules as well as the effect of such compounds on cell behavior, especially examining the ability of the compounds to disrupt mitosis through effects on the MT arrays that comprise the mitotic spindle. Indeed the ability to cause mitotic arrest in rapidly growing cell cultures in the laboratory is often considered to be an assay of the principal mechanism of these drugs. Often study of the regulation of microtubules and microtubule arrays and the effects of small molecules on those arrays depends on tools of microscopy, both for in vitro assays with purified proteins and especially for assays that address microtubule arrays inside cells. For this reason we have collaborated with labs that focus on fluorescence methods and advanced microscope applications. One such application is super-resolution microscopy, which is actually a class of methods for achieving enhanced spatial resolution using light microscopy, that until a few years ago was thought to be not possible. One such method is STED microscopy, Stimulated Emission Depletion Microscopy. This method can require high power of light, resulting in photobleaching of the fluorophores. To minimize this and optimize resolution we have developed a simple method to check power optimization in STED microscopy that can be readily accomplished at the microscopy bench. Our work with small molecules that target tubulin / microtubules has focused on using our knowledge of tubulin structure to identify new molecules that could become useful drugs, and also to take potent existing drugs and modify them to extend their usefulness or better target them in the body in order to minimize side effects on bystander tissues. We have previously developed chemistry to attach linker arms to some known drugs and this year extend that work by demonstrating how to attach linker arms to the potent natural product drug, spongistatin 1, without losing potency. This accomplishment will allow this drug, one of the most potent cytotoxic compounds known, to be attached to antibodies or other affinity-conferring entities that will target the potent drug to the intended target, such as a tumor or metastases. While spongistatin destabilizes microtubules, other drugs stabilize them (such as paclitaxel, commercial name Taxol), and we have also looked for new varients of these. Two of the most promising of these are the structural analogs zampanolide and dactylolide. These bind to the same place on tubulin; zampanolide binds covalently and dactylolide binds non-covalently. We have prepared and examined conformational analogs of these compounds to better understand their stabilizing potency and the significance of the covalent reaction. We have also pursued the basic structural biology of the tubulin dimer. This knowledge is required to understand how small molecules regulate tubulin and how tubulin binding domains regulate interaction with other proteins. The most basic step in tubulin biology is assembly of the heterodimer, and we previously described studies that used analytical ultracentrifugation as well as fluorescence polarization to show that dimer formation is a reversible, mass-action-driven process. We also examined the role of the tubulin carboxyl terminal tail peptides which mediate interaction of many proteins with tubulin and/or microtubules. We have extended those studies, showing that dimer dissociation and monomer exchange are controlled by equilibrium constants that differ by orders of magnitude between tubulins from different biological sources. These include tubulins from human cells compared to tubulins from different organisms, including protozoan parasites, and also between tubules from different tissues, which represent different expression levels of tubule isotypes. This work also demonstrated the importance of non-binding surfaces in protein-protein interactions. Beyond the formation of heterodimers is the regulation of expression of which isotypes of alpha and of beta tubulin contributes to the heterodimers. The tissue specificity of expression strongly indicates that isotypes contribute function that is specific to particular cell- or tissue types but the evidence for this is not abundant. We report the effect of altered expression of beta tubulin isotypes in skeletal muscle on muscle function in Duschenne Muscular Dystrophy (DMD) and the commonly used mdx mouse model of dystrophic muscle. Microtubule arrays are known to be disrupted in DMD and mdx skeletal muscle fibers, and here we show that over expression of the tubulin beta6 isotype (gene = tubb6) results in the microtubule disorganization seen in dystrophic muscle. Overexpression of the beta5 isotype has no such effect.