RNA localisation during the development of hyphae in the human pathogen Candida albicans

真菌白色念珠菌是一种主要的人类病原体。它是妇女阴道炎的病原体。此外，它还可能导致新生儿和某些重症监护患者等脆弱患者发生危及生命的血流感染，特别是那些正在接受癌症化疗，免疫抑制剂治疗或导管插入的患者。其生物学的一个显著特征是其能够作为单细胞酵母或以称为菌丝和假菌丝的丝状形式生长。菌丝是由长而薄的细胞组成的链，只从菌丝顶端生长。这种转变生长形式的能力对毒力很重要。人们认为菌丝可以穿透表皮屏障，而酵母可以在血流中循环，在肾脏和心脏瓣膜中造成致命的二次感染。菌丝生长仅限于其尖端;我们的研究重点是负责这种高度极化的生长形式的分子机制。如果我们理解了这些机制，就有可能通过找到阻断它们的方法来开发更好的抗真菌药物。然而，这是一个长期目标，不太可能作为本研究的直接结果实现。到目前为止，我们已经证明了一种称为Spitzenkörper的特殊结构位于尖端。据认为，膜结合的分泌囊泡，其中含有新的细胞壁和膜的原料，被运送到Spitzenkörper，在那里它们积累，然后移动到细胞表面。因此，我们首先需要了解负责Spitzenkörper中分泌囊泡聚集的机制。我们的研究集中在一种名为Sec 2 p的蛋白质上，它与分泌囊泡相关，并在它们向极化生长位点的运动中起着关键作用。该建议是由以下考虑引起的：当酵母细胞切换到菌丝生长时，细胞核保留在母酵母细胞中，母酵母细胞可以与菌丝尖端有相当大的距离，在菌丝尖端处许多蛋白质需要聚集以促进极化生长。我们想知道编码新蛋白质合成信息的mRNA分子是否可以特异性地从细胞核（在那里它们从DNA转录）运输到菌丝顶端，在那里它们被翻译成新蛋白质。为了验证这一想法，我们进行了一项初步实验，旨在检测Sec 2 p和mRNA分子之间的直接物理相互作用，并发现情况确实如此。我们现在希望鉴定附着在Sec 2 p上的单个mRNA分子，并解决以下问题：1）这些mRNA分子编码的蛋白质在菌丝生长中的作用是什么？2)编码这些蛋白质的mRNA被运输到菌丝顶端是否重要？3)mRNA与Sec 2 p直接相互作用还是间接相互作用/也就是说，mRNA与另一种蛋白质相互作用，该蛋白质是包括Sec 2 p的分子复合物的一部分？4)mRNA是在分泌囊泡的外面运输的吗？5)运送mRNA分子的分子马达的性质是什么？马达运动的沿着轨道的性质是什么？拟议的研究利用了最新的基于基因组的技术以及最先进的显微镜。此外，这是白念珠菌细胞生物学和分子遗传学专家Peter Sudbery教授与细胞内mRNA运动研究的世界领导者Stuart Wilson博士之间的合作提案。

The fungus Candida albicans is a major human pathogen. It is the causative agent of vaginitis in women. In addition, it can cause life-threatening blood stream infections in vulnerable patients such as the new born and certain intensive care patients, especially those undergoing cancer chemotherapy, immunosuppressant therapy or catheterisation. A striking feature of its biology is its ability to grow as a unicellular yeast or in filamentous forms called hyphae and pseudohyphae. Hyphae are chains of long thin cells that grow exclusively from the hyphal tip. This ability to switch growth forms is important for virulence. It is thought that the hyphae can penetrate epidermal barriers while the yeast can circulate in the blood stream to establish lethal secondary infections in the kidneys and heart valves. Hyphal growth is restricted to their tip; our research focuses on the molecular mechanisms responsible for this highly polarised form of growth. If we understood these mechanisms, it might be possible to develop better anti-fungal drugs by finding ways to block them. However, this is a long term goal that is unlikely to be realised as a direct result of this research. So far, we have shown that a special structure called a Spitzenkörper is located at the tip. It is thought that membrane-bound secretory vesicles, which contain the raw materials for the new cell wall and membranes, are transported to the Spitzenkörper where they accumulate before being moving on to the cell surface. Thus we first need to understand the mechanisms responsible for the aggregation of secretory vesicles in the Spitzenkörper. Our research has focussed on a protein called Sec2p, which is associated with secretory vesicles and plays a key role in their movement to sites of polarised growth. This proposal is prompted by the consideration that as a yeast cell switches to hyphal growth the nucleus remains in the mother yeast cell which can be a considerable distance from the hyphal tip where many proteins need to aggregate to facilitate polarised growth. We wondered whether the mRNA molecules, which encode the information for the synthesis of new proteins, may be specifically transported from the nucleus, where they are transcribed from DNA, to the hyphal tip where they are translated into new proteins. To test this idea we carried out a preliminary experiment designed to detect a direct physical interaction between Sec2p and mRNA molecules, and found that this was indeed the case. We now wish to identify the individual mRNA molecules attached to Sec2p and resolve such questions as: 1) What is the role of the proteins encoded by these mRNA molecules in hyphal growth? 2) Is it important that mRNAs encoding these proteins are transported to the hyphal tip? 3) Do the mRNAs interact directly with Sec2p or is the interaction indirect / that is, do the mRNAs interact with another protein that is part of a molecular complex that includes Sec2p? 4) Are the mRNAs transported on the outside of secretory vesicles? 5) What is the nature of the molecular motors that transport the mRNA molecules and the nature of the tracks along which the motors move? The proposed research exploits the latest genome-based technologies as well as state-of-the-art microscopy. Moreover, this is a collaborative proposal between Professor Peter Sudbery an expert in Candida albicans cell biology and molecular genetics and Dr Stuart Wilson a world leader in the study of mRNA movement within cells.