Intrinsic and extrinsic spindle orientation mechanisms in mammalian epidermis

摘要 干细胞分裂的适当控制对于组织形态发生和稳态是至关重要的。当失调时， 一方面可能导致发育不全和干细胞衰竭，另一方面可能导致组织过度生长和癌症。 但是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，而且可以在空间上控制。 定向细胞分裂（OCD）是后者的一个例子，对于干细胞和祖细胞， 分裂轴可以决定细胞命运结果并影响组织结构。在复层上皮中，如 在表皮中，基底祖细胞在上皮平面内或垂直于上皮平面分裂。证据 表明平面分裂通常是自我更新的对称细胞分裂（SCD），而垂直分裂是自我更新的对称细胞分裂（SCD）。 细胞分裂是分化的不对称细胞分裂（ACD）。我们实验室以前的工作表明， 是由极性和纺锤体方向蛋白的复合体引导的--聚集在关键的支架上 蛋白LGN（Gpsm 2）-其不对称地定位于顶端细胞皮层。最近，我们发现， para-AGS 3（Gpsm 1）似乎对抗LGN，并通过未知的途径促进SCD 机制此外，我们最近有了一个令人惊讶的发现，即分裂方向在细胞分裂过程中并不固定。 中期，如先前所认为的，但在有丝分裂的晚期阶段可以进一步细化。在这个过程中， 我们称之为“末期校正”，大约三分之一的基底细胞以斜角进入后期， 重新定向为平面或垂直。我们已经了解到，细胞-细胞粘附-特别是， 粘附连接的机械感测组件对于发生末期校正是重要的，并且 可以独立于LGN运行。这表明，除了内在的线索，如LGN复合体， 诸如局部组织微环境的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ACD的分子控制，但在理解如何控制ACD方面存在重大知识差距。 在正常发育和先天性皮肤病中，定向的分裂形成组织结构 如大疱性表皮和外胚层发育不良。具体而言，本提案的目标是 更好地理解1）什么调节SCD以及如何在SCD/ACD之间做出选择 (SA1)2）细胞-细胞粘附、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-慢病毒超声引导基因失活和基因表达（LUGGIGE）- 我们将利用它来产生基因丢失和人类中发现的特定突变的小鼠模型， 疾病结合皮肤外植体的离体成像和体内蛋白质组学方法来表征 LGN和AGS 3相互作用组使用TurboID，这些全面的研究将提供深入了解细胞- 决定分裂方向的内在和外在线索，以及它们在正常表皮细胞分裂过程中如何运作。 生长以及起泡和发育不良的皮肤病。

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.