The function and assembly of the lamin-B spindle sheath

描述（由申请人提供）：本提案旨在研究围绕纺锤体微管的有丝分裂膜网络如何调节纺锤体形态发生和纺锤体方向。有丝分裂纺锤体形态的组装和维持需要平衡由基于微管的马达蛋白（如动力蛋白和驱动蛋白Eg 5）产生的力，以及微管动力学的适当调节。我们的研究的核纤层蛋白B的有丝分裂膜网络，我们称之为核纤层蛋白B的纺锤体鞘，已经表明，这种纺锤体相关的结构调节纺锤体的形态和方向。由于层粘连蛋白-B与动力蛋白调节器NudEL和微管解聚酶MCAK相互作用，我们假设层粘连蛋白-B的纺锤体鞘，围绕身体的纺锤体微管功能作为一个障碍，限制微管生长的纺锤体边界内通过MCAK和/或NudEL。此外，我们建议，层蛋白B纺锤体鞘周围的纺锤体极调节星形微管，以确保适当的搜索和捕获的皮质纺锤体方向线索。我们将在目标1和目标2中使用我们生成的一些分析和工具来测试这些想法。我们以前已经表明，RanGTP，微管，动力蛋白都需要的层蛋白B纺锤体鞘组件。最近，我们发现层蛋白B在有丝分裂中与几种核孔蛋白结合。在目标3中，我们将测试的假设，核层蛋白B和核孔蛋白之间的相互作用所需的纺锤体鞘的组装。这些研究将对理解非微管细胞结构如何调节有丝分裂中纺锤体的组装和取向做出重大贡献。 公共卫生相关性：大多数关于纺锤体组装的研究都集中在纺锤体微管内的活动和力上。然而，许多观察表明，纺锤体微管外的结构也有助于维持纺锤体的形态和位置。我们的研究发现，一个层蛋白B的膜网络组织周围的纺锤体微管在纺锤体形态发生中发挥作用。随着新发现的分子手柄，我们建议进一步剖析的机制，层蛋白膜网络与纺锤体微管相互作用，以促进其组织，以及如何这将反过来调节纺锤体方向在细胞分裂。这项建议解决了一个领域的细胞分裂，已收到相对较少的关注，它的目的是发现新的见解纺锤体组装机制，以前没有考虑。由于含有有丝分裂层蛋白的膜网络含有信号分子和细胞命运决定因素，我们相信，了解这种结构如何在细胞分裂过程中与纺锤体微管相互作用，将开辟新的途径，探索治疗靶点，以阻止不受控制的细胞分裂。

DESCRIPTION (provided by applicant): This proposal aims to study how the mitotic membrane network that surrounds the spindle microtubules regulates spindle morphogenesis and spindle orientation. The assembly and maintenance of mitotic spindle morphology requires balancing of forces generated by microtubule-based motor proteins such as dynein and the kinesin Eg5, and proper regulation of microtubule dynamics. Our studies of the lamin-B-containing mitotic membrane network, which we refer to as the lamin-B spindle sheath, have shown that this spindle-associated structure regulates spindle morphology and spindle orientation. Since lamin-B interacts with the dynein regulator NudEL and the microtubule depolymerase MCAK, we hypothesize that the lamin-B spindle sheath that surrounds the body of the spindle microtubules functions as a barrier to limit microtubule growth within the spindle boundary through MCAK and/or NudEL. Moreover, we propose that the lamin-B spindle sheath surrounding the spindle poles regulates the astral microtubules to ensure proper search and capture of the cortical spindle orientation cues. We will test these ideas in Aim 1 and Aim 2 using a number of assays and tools we have generated. We have shown previously that RanGTP, microtubules, and dynein are all required for the lamin-B spindle sheath assembly. More recently, we have found that lamin-B binds to several nucleoporins in mitosis. In Aim 3, we will test the hypothesis that the interactions between lamin-B and nucleoporins are required for the assembly of the spindle sheath. These studies will make a significant contribution toward understanding how non-microtubule cellular structures regulate spindle assembly and orientation in mitosis. PUBLIC HEALTH RELEVANCE: Most studies of spindle assembly have focused on activities and forces within the spindle microtubules. However, many observations have shown that structures outside of spindle microtubules also contribute toward maintaining spindle morphology and position. Our studies have uncovered that a lamin-B-containing membrane network organized around the spindle microtubules plays a role in spindle morphogenesis. With the newly identified molecular handles, we propose to further dissect the mechanism by which the lamin- membrane network interacts with spindle microtubules to facilitate their organization and how this would in turn regulate spindle orientation during cell division. This proposal addresses an area of cell division that has received relatively little attention and it aims to uncover novel insights about spindle assembly mechanisms that had not been considered previously. Since the mitotic lamin-containing membrane network contains signaling molecules and cell fate determinants, we believe that understanding how this structure interacts with spindle microtubules during cell division will open new avenues to explore therapeutic targets for halting uncontrolled cell divisions.