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方向的分子基础。有丝分裂纺锤体沿着预定轴的排列是许多情况下正常细胞功能所必需的，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）的不对称分裂期间，细胞命运决定簇定位于细胞的相对两极，使得它们分离成离散的子细胞。决定子的正确分布和随后的命运特化要求有丝分裂纺锤体与极性轴精确对齐。我们建议调查这一过程中重建纺锤体的方向在培养的细胞，通常不定向的纺锤体。在这种情况下，建立规范的主轴定位将使我们能够确定哪些组件是足够的主轴方向，我们建议检查这些组件的生化和结构，以确定其作用机制。在我们的初步工作中，我们已经成功地极化果蝇S2细胞使用的粘附蛋白Echinoid，并已发现，表达的Echinoid蛋白，其中的细胞质部分已被替换为域的伴侣的不可见（针）蛋白可以稳健地定向纺锤体的方式类似于神经母细胞。我们正在使用生物化学，生物物理学和细胞生物学方法的组合来研究我们在纺锤体方向重建中识别的分子的功能，包括Pin。 公共卫生相关性：在细胞分裂过程中，染色体通过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进行适当的组织组织化、分化或防止肿瘤形成。在这项工作中，我们正试图确定所需的纺锤体位置控制的细胞机器，试图重新创建这个过程中的细胞类型，通常不定向其纺锤体。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了解疾病状态的机制。

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.