The function and assembly of the lamin-B spindle sheath

描述(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的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基于微管的马达蛋白如动力蛋白和动蛋白EG5所产生的力，并适当地调节微管的动力学。我们对含层粘连蛋白B的有丝分裂膜网络(我们称之为层粘连蛋白B纺锤体鞘)的研究表明，这种纺锤体相关结构调节着纺锤体的形态和纺锤体的取向。由于Lamin-B与动力蛋白调节剂Nudel和微管解聚酶MCAK相互作用，我们假设包围纺锤体微管体的Lamin-B纺锤鞘通过MCAK和/或Nudel作为屏障限制微管在纺锤体边界内的生长。此外，我们认为，围绕着纺锤体极点的层蛋白-B纺锤体鞘调节星形微管，以确保适当地寻找和捕获皮质纺锤体定向线索。我们将使用我们生成的许多分析和工具在目标1和目标2中测试这些想法。我们以前已经证明RanGTP、微管和动力蛋白都是Lamin-B纺锤体鞘组装所必需的。最近，我们发现Lamin-B在有丝分裂中与几个核孔蛋白结合。在目标3中，我们将测试假设，即层蛋白-B和核孔蛋白之间的相互作用是组装纺锤体鞘所必需的。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要贡献。 与公共健康相关：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量上。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，围绕纺锤体微管组织的含层粘连蛋白B的膜网络在纺锤体的形态发生中起着作用。根据新发现的分子手柄，我们建议进一步剖析层粘连蛋白-膜网络与纺锤体微管相互作用以促进其组织的机制，以及这如何反过来调节细胞分裂中的纺锤体方向。这项提案涉及一个相对较少受到关注的细胞分裂领域，其目的是揭示关于纺锤体组装机制的新见解，这是以前从未考虑过的。由于有丝分裂的含层蛋白的膜网络包含信号分子和细胞命运决定因素，我们相信了解这种结构在细胞分裂过程中如何与纺锤体微管相互作用将为探索阻止不受控制的细胞分裂的治疗靶点开辟新的途径。

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.