Intrinsic and extrinsic mechanisms regulating asymmetric cell division in C. elegans

不对称细胞分裂是母细胞分裂产生两个采用不同细胞命运的子细胞的过程，是在发育过程中产生细胞多样性的关键机制。细胞内蛋白质的不对称定位和细胞信号都参与了这一过程。尽管进行了大量研究，但仍有许多悬而未决的问题。细胞信号如何极化不对称分裂的细胞？生物体如何整合细胞信号来调节细胞内决定因素的不对称定位。线虫非常适合解决这些问题，因为大多数细胞是由不对称的细胞分裂产生的。因为整个细胞谱系是已知的，所以可以在单个细胞的分辨率下分析细胞分裂的时间、位置和极性。*我的长期目标是阐明线虫细胞不对称分裂背后的分子和细胞机制。我们的分析集中在Wnt信号通路和Ham-1上，Ham-1是一种在许多分裂细胞中不对称定位于细胞皮质的蛋白质。在线虫中，Wnt信号调节许多不对称的细胞分裂。Wnt通路是一条保守的细胞信号通路，对发育至关重要。在我们之前的NSERC资助的研究中，我们表明DSH-2，一个关键的Wnt途径成分，在发育过程中调节不对称的神经母细胞分裂，从而促进神经元的多样性。我们进行了一次非常成功的遗传筛选，以确定与Wnt/DSH-2信号通路有关的基因。被确认的基因之一是hcf-1，一种转录辅助激活因子。这是一个新的发现，因为之前还没有在任何实验系统中描述过hcf-1在Wnt信号中的作用。我们进一步证明，HCF-1参与了Wnt转录因子POP-1/TCF的不对称定位。在本研究方案中，我们旨在确定HCF-1调节POP-1本地化的机制。初步结果表明，HCF-1可能影响POP-1的乙酰化，我们将进行实验来验证这一假说。我们克隆了第二个dsh-2相互作用基因，它编码一种新的蛋白质。我们将进行一系列遗传、分子和细胞生物学实验，以了解该蛋白如何调节依赖Wnt的不对称细胞分裂。我们还将利用全基因组测序来确定在我们的基因筛查中分离的其他突变的分子身份。最后，我们证明了另外两种线虫蓬乱的同系物，DSH-1和MIG-5，与Ham-1物理上相互作用。这种相互作用提供了细胞信号和细胞内蛋白质的不对称定位之间的关键联系。我们提出了实验来研究Wnt信号在HAM-1不对称定位中的作用。********

Asymmetric cell division, the process by which a mother cell divides to produce two daughter cells that adopt distinct cell fates, is a key mechanism that generates cellular diversity during development. Both the asymmetric localization of intracellular proteins and cell signaling contribute to this process. Despite numerous studies many outstanding questions remain. How does cell signaling polarize asymmetrically dividing cells? How does an organism integrate cell signaling to regulate the asymmetric localization of intracellular determinants. C. elegans is ideally suited address these questions since the majority of cells are generated by asymmetric cell division. Because the entire cell lineage is known, the timing, location and polarity of cell divisions can be analyzed at the resolution of single cells. *** My long-term goal is to elucidate the molecular and cellular mechanisms underlying asymmetric cell division in C. elegans. We have focused our analysis on the Wnt signalling pathway and HAM-1, a protein asymmetrically localized to the cell cortex in many dividing cells. In C. elegans, Wnt signaling regulates many asymmetric cell divisions. The Wnt pathway is a conserved cell-signaling pathway that is essential for development. In our previous NSERC funded research we showed that DSH-2, a key Wnt pathway component, regulates asymmetric neuroblast division during development and thus contributes to neuronal diversity. We undertook a highly successful genetic screen to identify genes that function with the Wnt/DSH-2 signaling pathway. One of the genes identified was hcf-1, a transcriptional co-activator. This was a novel finding since a role for hcf-1 in Wnt signalling has not been previously described in any experimental system. We proceeded to show that HCF-1 contributes to the asymmetric localization of the Wnt transcription factor POP-1/TCF. In this research proposal, we aim to determine the mechanism by which HCF-1 regulates POP-1 localization. Preliminary results indicates HCF-1 is likely affecting the acetylation of POP-1 and we will undertake experiments to test this hypothesis. We have cloned a second dsh-2 interacting gene and it encodes a novel protein. We will perform a combination of genetic, molecular and cell biological experiments to understand how this protein regulates Wnt dependent asymmetric cell division. We will also make use of whole genome sequencing to determine the molecular identity of additional mutations isolated in our genetic screen. Finally, we have shown that two other C. elegans Dishevelled homologs, DSH-1 and MIG-5, physically interact with HAM-1. This interaction provides a key link between cell signalling and the asymmetric localization of intracellular proteins. We propose experiments to investigate the role of Wnt signalling in the asymmetric localization of HAM-1. ********