Molecular principles of spindle orientation complex organization and function

多细胞生物的发育是一个复杂的过程，它从根本上依赖于产生许多不同细胞类型的能力，这些细胞类型最终导致具有独特功能的成年组织。细胞多样性的创造是干细胞的任务，干细胞的分裂可以产生不同的、不相同的子细胞，这些子细胞能够承担不同的命运。这种所谓的不对称细胞分裂（ACD）过程是正常发育和生存所必需的。因此，了解ACD的细胞和分子机制具有广泛和重要的生物学意义，特别是考虑到这一过程在所有多细胞生命中的保护。该项目的目标是了解控制不对称干细胞分裂的特定方面的保守蛋白复合物如何被调节以确保适当的发育。虽然许多将被研究的特定成分是已知的，但关于它们在分子水平上如何起作用仍然存在重要的知识空白。该项目将特别关注特定蛋白质复合物如何在细胞内组装并最终实现合作功能。这些事件的破坏可导致组织发育缺陷，影响成体生物的生存能力和功能。这些研究将利用普通果蝇作为模型遗传生物，其对ACD重要的基因对人类高度保守。更广泛的影响活动包括研究的内在价值，因为所有多细胞生命都涉及ACD。这项工作还将为本科生和研究生提供培训，并为代表性不足的群体提供机会，激励他们参与更多样化的科学界。不对称细胞分裂（ACD）是一个进化保守的过程，它允许祖干细胞产生不相同的子细胞，最终在发育过程中使细胞命运多样化。适当的ACD需要皮质极性和纺锤体定位复合体之间的协调作用，而不耦合的缺陷会破坏组织发育和体内平衡。该项目的目标是了解尽管与细胞极性因素相互作用，但保守的Pins/Mud纺锤体取向复合物如何在细胞皮层组织和功能。我们提供的初步数据表明，Pins/Mud复合相在体外分离成致密的液滴。这是通过新发现的Mud和肌动蛋白结合蛋白Hts之间的相互作用促进的，我们也发现在果蝇神经干细胞中，这种相互作用是极化的。我们假设Hts/Pins/Mud复合物形成簇状生物凝聚体，屏蔽竞争相互作用，以确保不对称分裂干细胞的纺锤体定向精度。为了验证这一假设，我们将使用功能丧失遗传学方法首先确定Hts极性是如何在神经干细胞中建立的，以及它如何影响核心极性和纺锤体取向成分的定位。接下来，我们将使用定量荧光成像方法来确定相分离Pins/Mud液滴在没有和存在Hts的情况下的物理性质。最后，我们将使用基因组编辑和高分辨率显微镜来直接确定相分离在体内Pins/Mud复合物的组装和功能中的作用。该项目的结果将为进化保守的纺锤体定位复合体的生物物理特性和细胞功能提供见解，并解决长期以来在ACD分子基础方面的知识空白。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Development of multicellular organisms is a complex process that rests fundamentally on the ability to generate many diverse cell types that ultimately result in adult tissues with unique functions. The creation of cell diversity is tasked to stem cells, whose divisions can produce distinct, non-identical daughter cells capable of assuming different fates. This so-called asymmetric cell division (ACD) process is essential to proper development and viability. Understanding the cellular and molecular mechanisms involved in ACD, therefore, has broad and significant biological importance, particularly considering the conservation of this process across all multicellular life. The goal of this project is to understand how conserved protein complexes that control specific aspects of asymmetric stem cell divisions are regulated to ensure proper development. Although many of the specific components that will be investigated are known, important knowledge gaps remain with respect to how they function at the molecular level. The project will focus particularly on how specific protein complexes assemble and ultimately achieve a cooperative function within the cell. Disruption of these events can lead to defects in tissue development that impact the viability and function of adult organisms. These studies will utilize the common fruit fly as a model genetic organism, whose genes important for ACD are highly conserved to humans. The Broader Impact activities include the intrinsic merit of the research as all multicellular life involves ACD. The work will also provide training for undergraduate and graduate students, with opportunities provided to underrepresented groups to inspire their involvement in a more diverse scientific community.Asymmetric cell division (ACD) is an evolutionarily conserved process that allows progenitor stem cells to produce non-identical daughter cells that ultimately diversify cell fates during development. Proper ACD requires coordinated function between cortical polarity and spindle positioning complexes, and defects that uncouple these disrupt tissue development and homeostasis. The goal of this project is to understand how the conserved Pins/Mud spindle orientation complex organizes at the cell cortex and functions despite competing interactions with cell polarity factors. We provide preliminary data demonstrating that the Pins/Mud complex phase separates into dense liquid droplets in vitro. This is facilitated through a newly-identified interaction between Mud and the actin-binding protein Hts, which we also show to be polarized in Drosophila neural stem cells in vivo. We hypothesize that Hts/Pins/Mud complexes form clustered biological condensates shielded from competing interactions to ensure spindle orientation precision in asymmetrically dividing stem cells. To test this hypothesis, we will use loss-of-function genetic approaches to first determine how Hts polarity is established in neural stem cells and how it impacts localization of core polarity and spindle orientation components. Next, we will use quantitative fluorescence imaging approaches to determine the physical properties of phase separated Pins/Mud droplets in the absence and presence of Hts. Finally, we will use genome editing and high-resolution microscopy to directly ascertain the role of phase separation in the assembly and function of Pins/Mud complexes in vivo. Results from this project will provide insights into the biophysical properties and cellular function of an evolutionarily conserved spindle positioning complex and resolve long-standing knowledge gaps in the molecular underpinnings of ACD.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.