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控有丝分裂纺锤体取向的分子基础。在许多情况下，有丝分裂纺锤体沿预定轴的排列是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，这样它们就被分离成离散的子细胞。决定因子的适当分布和随后的命运规范要求有丝分裂纺锤体与极性轴精确对齐。我们建议通过在培养细胞中重建纺锤体取向来研究这一过程，这种培养细胞通常不定向纺锤体。在这种情况下，建立受调节的主轴定位将使我们能够确定哪些成分对主轴定向是足够的，我们建议对这些成分进行生化和结构检查，以确定它们的作用机制。在我们的初步工作中，我们已经成功地使用粘附蛋白Echinoid极化果蝇S2细胞，并发现Echinoid蛋白的表达，其中细胞质部分被来自incuteable Partner （Pins）蛋白的结构域所取代，可以以类似于神经母细胞的方式稳固地定向纺锤体。我们正在使用生物化学、生物物理学和细胞生物学方法的结合来研究我们在纺锤体取向重构中发现的分子的功能，包括pin。

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.