Regulated spindle orientation during asymmetric cell division

描述(由申请人提供)：这项工作的长期目标是了解调节有丝分裂纺锤体取向的分子基础。有丝分裂纺锤体沿预定的轴排列是许多情况下细胞正常功能所必需的，包括分化、胚胎发生和器官发生。例如，在果蝇神经母细胞(中枢神经系统的前体)的不对称分裂过程中，细胞命运决定因素定位于细胞的相反两极，从而使它们分离成离散的子细胞。决定因素的正确分布，以及随后的命运指定，要求有丝分裂纺锤体与极轴精确对准。我们建议通过在正常情况下不定向纺锤体的培养细胞中重建纺锤体取向来研究这一过程。在这种情况下，建立调节的主轴定位将使我们能够确定哪些组件足够用于主轴定位，我们建议从生化和结构上检查这些组件，以确定它们的作用机制。在我们的初步工作中，我们成功地使用黏附蛋白Echinid来极化果蝇S2细胞，并发现Echinid蛋白的表达可以以类似于神经母细胞的方式牢固地定位纺锤体。我们结合使用生化、生物物理和细胞生物学的方法来研究我们在纺锤体定向重组中识别的分子的功能，包括Pins。 与公共卫生相关：在细胞分裂过程中，染色体被有丝分裂纺锤体分离成子细胞。在许多细胞中，纺锤体必须精确定位，以便适当的组织、分化或防止肿瘤形成。在这项工作中，我们试图通过尝试在一种不正常定向其纺锤体的细胞类型中重建这一过程，来识别纺锤体位置控制所需的细胞机制。由于失去准确的纺锤体定位与人类疾病有关，提高我们对控制这一过程的分子的理解将有助于我们对疾病状态机制的了解。

DESCRIPTION (provided by applicant): The long-term objective of this work is to understand the molecular basis of regulated mitotic spindle orientation. Alignment of the mitotic spindle along a predetermined axis is required for proper cell function in many contexts, including differentiation, embryogenesis, and organogenesis. For example, during the asymmetric division of Drosophila neuroblasts, precursors of the central nervous system, cell fate determinants localize to opposite poles of the cell such that they become segregated into discrete daughter cells. Proper distribution of determinants, and subsequent fate specification, requires that the mitotic spindle align precisely with the polarity axis. We propose to investigate this process by reconstituting spindle orientation in a cultured cell that does not normally orient the spindle. Establishing regulated spindle positioning in this context will allow us to determine which components are sufficient for spindle orientation, and we propose to examine these components biochemically and structurally to determine their mechanism of action. In our preliminary work we have successfully polarized Drosophila S2 cells using the adhesion protein Echinoid and have found that expression of Echinoid proteins in which the cytoplasmic portion has been replaced with domains from the Partner of Inscuteable (Pins) protein can robustly orient the spindle in a manner similar to neuroblasts. We are using a combination of biochemical, biophysical, and cell biological methods to investigate the function of molecules that we identify in our spindle orientation reconstitution, including Pins. PUBLIC HEALTH RELEVANCE: During cell division, chromosomes are separated into the daughter cells by the mitotic spindle. In many cells, the spindle must be precisely positioned for proper tissue organization, differentiation, or prevention of tumor formation. In this work, we are attempting to identify the cellular machinery required for spindle position control by attempting to recreate this process in a cell type that does not normally orient its spindle. As the loss of accurate spindle positioning is associated with human disease, improving our understanding of the molecules that control this process will contribute to our knowledge of the mechanisms of disease states.