Intrinsic and extrinsic spindle orientation mechanisms in mammalian epidermis

摘要 干细胞分裂的适当控制对组织形态发生和动态平衡至关重要。当监管失调时，它 一方面会导致发育不全和干细胞枯竭，另一方面会导致组织过度生长和癌症。 但有丝分裂不仅仅是简单的增殖，因为细胞分裂不仅可以在时间上控制，而且在空间上也可以控制。 定向细胞分裂(OCD)是后者的一个例子，对于干细胞和祖细胞，在 分裂轴可以决定细胞命运的结果，并影响组织结构。在复层上皮中，如 表皮，基本祖细胞要么在上皮平面内分裂，要么垂直于上皮平面分裂。证据 表明平面分裂通常是自更新的对称细胞分裂(SCD)，而垂直分裂 分裂是分化的不对称细胞分裂(ACDs)。我们实验室之前的研究表明，ACDs 由极性和纺锤体取向蛋白的复合体引导--聚集在关键的支架上 蛋白LGN(Gpsm2)-不对称地定位于顶端细胞皮质。最近，我们发现 Paralog AGS3(Gpsm1)似乎与LGN相反，并通过未知的方式促进SCD 机制。此外，我们最近有了一个令人惊讶的发现，在 中期，与以前认为的一样，但可以在有丝分裂的后期进一步提纯。在这个过程中， 我们称之为“末期矫正”，大约三分之一的基底细胞以倾斜的角度进入后期，但随后 重定向为平面或垂直。我们已经了解到细胞间的粘连--具体地说， 附着物连接的机械传感组件--对于进行末期校正很重要，以及 可以独立于LGN运行。这表明，除了LGN复合体等内在线索外， 局部组织微环境等外在因素影响最终的分裂轴。尽管我们和 其他人已经了解了ACDs的分子控制，主要的知识空白存在于理解如何 在正常发育和先天性皮肤病中，定向分裂塑造组织结构 如大疱性表皮松解症和外胚层发育不良。具体地说，这项提案的目标是 更好地了解1)监管SCD的内容以及如何在SCD/ACD之间做出选择 (SA1)，2)细胞-细胞黏附、细胞-基质和局部细胞密度如何影响分裂方向和命运决定 (SA2)。为了实现这些目标，我们将利用创新方法的组合，以我们的快速、 高通量技术-慢病毒超声引导的基因失活和基因表达(LUGGIGE)- 我们将利用它来建立人类基因缺失和特定突变的小鼠模型 疾病。结合皮肤外植体的体外成像和体内蛋白质组学方法来表征 使用TurboID的LGN和AGS3交互作用，这些全面的研究将提供对细胞- 决定分裂方向的内在和外在线索，以及它们在正常表皮中的运作方式 在生长以及水泡和发育不良皮肤病中。

ABSTRACT Proper control of stem cell division is critical for tissue morphogenesis and homeostasis. When dysregulated, it can lead to hypoplasia and stem cell exhaustion on the one hand, or tissue overgrowth and cancer on the other. But mitosis is more than simple proliferation, as cell division can be controlled not only in time but also in space. Oriented cell divisions (OCDs) are an example of the latter, and for stem and progenitor cells, choices between division axes can dictate cell fate outcomes and impact tissue architecture. In stratified epithelia such as the epidermis, basal progenitors divide either within the plane of the epithelium, or perpendicular to it. Evidence suggests that planar divisions are generally self-renewing symmetric cell divisions (SCDs) while perpendicular divisions are differentiative asymmetric cell divisions (ACDs). Previous work from our lab has shown that ACDs are directed by a complex of polarity and spindle orientation proteins—converging on the critical scaffolding protein LGN (Gpsm2)—which localize asymmetrically at the apical cell cortex. More recently, we have found that the paralog AGS3 (Gpsm1) seems to oppose LGN, and functions in promoting SCDs through an unknown mechanism. In addition, we recently made the surprising discovery that division orientation is not fixed during metaphase, as previously thought, but can be further refined during late stages of mitosis. In this process, which we term “telophase correction,” roughly one-third of basal cells enter anaphase at oblique angles, but then reorient to either planar or perpendicular. We have learned that cell-cell adhesions—specifically, the mechanosensing components of the adherens junction—are important for telophase correction to occur, and can operate independently of LGN. This demonstrates that in addition to intrinsic cues such as the LGN complex, extrinsic factors such as the local tissue microenvironment influence the final division axis. Despite what we and others have learned about the molecular control of ACDs, major knowledge gaps exist in understanding how oriented divisions shape tissue architecture both during normal development and in congenital skin diseases such as epidermolysis bullosa and ectodermal dysplasia. Specifically, the objectives of this proposal are to develop a better understanding of 1) what regulates SCDs and how the choice between SCD/ACD is made (SA1), 2) how cell-cell adhesion, cell-matrix, and local cell density impact division orientation and fate decisions (SA2). To achieve these goals, we will leverage a combination of innovative approaches, centered on our rapid, high-throughput technique—lentiviral ultrasound-guided gene inactivation and gene expression (LUGGIGE)— which we will utilize to generate mouse models of both gene loss and of specific mutations found in human diseases. Combined with ex vivo imaging of skin explants and in vivo proteomic approaches to characterize the LGN and AGS3 interactomes using TurboID, these comprehensive studies will provide insights into the cell- intrinsic and extrinsic cues that determine division orientation, and how they operate during normal epidermal growth and in blistering and dysplastic skin diseases.