The roles of C. elegans patched family genes in cell division

这个项目的重点是细胞生物过程称为胞质分裂。当一个细胞分裂成2个子细胞时，它首先复制它的遗传物质，这样每个子细胞就继承了与其亲本相同数量的染色体和DNA含量。随后，复制的遗传物质均匀地分配在两个新的子细胞之间。在细胞分裂的最后阶段，当一个收缩出现时，亲本细胞分裂成两个子细胞-胞质分裂的过程。因此，很明显，理解胞质分裂的机制是细胞生物学中的一个基本问题，并对细胞增殖、发育生物学和癌症产生影响，如下所述。我们已经确定了一个基因，命名为ptr-2（补丁相关），这是所需的细胞质分裂的模式生物，线虫C。优雅C.线虫是一种小型透明的非致病性蠕虫，由于其具有许多实验优势，包括能够以低成本大量生长，因此被广泛用于生物医学研究。蠕虫从卵到成虫的生命周期仅需3天，这使得快速分析基因突变的影响成为可能。这种蠕虫是第一种具有完全测序基因组的多细胞动物，因此已知超过70%与人类疾病相关的基因也存在于蠕虫中。然而，对基因功能的解释在蠕虫中往往被简化，因为它只有约1000个细胞。使用一种称为RNA介导干扰（RNAi）的技术，我们已经证明ptr-2的缺失或缺失会导致早期C. elegans胚胎有胞质分裂缺陷。通常检测到胞质分裂缺陷，因为细胞具有多个核，这是由于在每次细胞分裂时未能将亲本细胞划分为子细胞而产生的。ptr-2参与这一过程特别令人感兴趣，因为它与另一个基因有关，该基因编码一种名为Patched（Ptch）的蛋白质。在人类细胞中，Ptch是一种肿瘤抑制因子，因为Ptch活性的丧失会导致家族性和散发性癌症; Ptch的突变是皮肤癌的主要原因。Ptch活性的缺陷也可能导致发育异常。因此，从细胞生物学的角度以及从人类发育和疾病的角度来看，理解Ptch蛋白如何发挥功能是重要的。为了了解ptr-2如何影响胞质分裂，我们建议采取遗传学方法，首先分析ptr-2的缺失如何影响胞质分裂。通过确定细胞分裂的哪些方面受到ptr-2缺失的干扰，我们可以应用遗传学逻辑来推断ptr-2在细胞中的正常作用。为了使这种分析成为可能，我们已经获得了一个缺乏ptr-2基因的遗传突变体。通过确定PTR-2蛋白在细胞中的位置，可以获得其他可以帮助我们理解PTR-2如何影响胞质分裂和细胞分裂的线索。蛋白质定位可以在使用抗体的固定细胞中进行，或者在使用荧光报道分子的活细胞中进行，所述荧光报道分子例如来自水母的与PTR-2融合的绿色荧光蛋白。PTR-2：：GFP融合可以通过照射适当波长的光在细胞中检测。这种技术的优点是，我们可以通过显微镜延时记录在整个细胞分裂周期的过程中跟踪PTR-2：：GFP的行为。一旦我们知道了PTR-2在细胞中的行为，我们就可以对可能与PTR-2相互作用的其他蛋白质的身份进行预测和测试。这个项目的结果是，我们将扩大我们的细胞分裂的细胞生物学知识，也获得了补丁蛋白的生化活性的更好的理解。

This project focuses on a cell biological process called cytokinesis. When a cell divides into 2 daughter cells, it first replicates its genetic material, so that each daughter cell inherits the same number of chromosomes and DNA content as its parent. Subsequently, the duplicated genetic material is evenly partitioned between what will become the 2 new daughter cells. During the terminal stages of cell division, the partitioning of the parental cell into daughter cells is completed when a constriction arises that physically separates the parental cell into two daughter cells - the process of cytokinesis. Thus, it is clear that understanding the mechanisms underlying cytokinesis is a fundamental problem in cell biology, and has consequences with regard to cell proliferation, developmental biology and cancer, as discussed below. We have identified a gene, named ptr-2 (for patched-related), which is required for cytokinesis in the model organism, the nematode C. elegans. C. elegans is a small transparent non-pathogenic worm, which is a widely used in biomedical studies because it possesses a number of experimental advantages, including the ability to grow large numbers at low cost. The lifecycle of the worm from egg to adult takes only 3 days, which makes it possible to analyse the effects of genetic mutation very rapidly. The worm was the first multicellular animal with a fully sequenced genome, so it is known that over 70% of genes associated with human disease are also present in the worm. However, the interpretation of gene function is often simplified in the worm because it only has ~1000 cells. Using a technique known as RNA mediated interference (RNAi), we have shown that the depletion or absence of ptr-2 causes cells of the early C. elegans embryo to have a cytokinesis defect. Cytokinesis defects are usually detected because a cell has multiple nuclei arising from the failure to partition the parental cell into daughter cells at each cell division. The involvement of ptr-2 in this process is of particular interest, because it is related to another gene, which encodes a protein named Patched (Ptch). In human cells, Ptch is a tumour suppressor, because the loss of Ptch activity causes both familial and sporadic carcinomas; mutations in Ptch are the leading cause of skin cancer. Defects in Ptch activity can also lead to developmental abnormalities. Hence an understanding of how the Ptch protein functions is important from both a cell biological standpoint and also from the perspective of human development and disease. To understand how ptr-2 affects cytokinesis, we are proposing to take a genetic approach by first analysing how the absence of ptr-2 affects cytokinesis. By determining what aspects of cell division are perturbed by the absence of ptr-2, we can then apply genetic logic to infer the normal role of ptr-2 in the cell. To make this analysis possible, we have obtained a genetic mutant that lacks the ptr-2 gene. Other clues that can help us to understand how ptr-2 affects cytokinesis and cell division will be obtained by determining where the PTR-2 protein is found in the cell. Protein localisation can be performed in either fixed cells using antibodies or in living cells using a fluorescent reporter, such as the green fluorescent protein from the jellyfish, fused to PTR-2. The PTR-2::GFP fusion can be detected in cells by shining light of the appropriate wavelength. This technique has the advantage that we can follow the behaviour of PTR-2::GFP by microscopic time-lapse recording during the course of an entire cell-division cycle. Once we know how PTR-2 behaves in the cell, we can then make and test predictions regarding the identity of other proteins that could interact with PTR-2. The outcome of this project is that we will expand our knowledge of the cell biology of cell division and also gain an improved understanding of the biochemical activities of Patched proteins.