The role and regulation of polarised secretion in the development of Candida albicans hyphae

白色念珠菌是一种导致女性阴道炎(鹅口疮)的真菌。它也是医院重症监护病房中常见的、往往是致命的血液感染的原因。它的一个生物学特征是它能够在单细胞酵母和丝状生长形式之间切换，这对它的致病性很重要。丝状细胞由长管状细胞组成，称为菌丝，完全从它们的顶端生长。这一提议旨在了解导致这种极端形式的极化生长的分子机制。从长远来看，这可能有助于开发更好的药物，不仅可以对抗白色念珠菌的感染，还可以对抗由其他真菌引起的严重感染，这也显示了这种生长模式。到目前为止，我们的实验室已经表明，在白色念珠菌的菌丝中，顶端存在一种名为Spitzenkörper的结构(来自德国的顶体)。人们认为，含有新菌丝生长原料的膜结合小泡被运输到Spitzenkörper，在那里积累形成供应中心，将小泡输送到菌丝尖端。我们正在解决的问题是，是什么负责控制小泡流向Spitzenkörper？对萌芽酵母酿酒酵母的研究对我们有所帮助，事实证明，这是一个非常有用的模型，可以理解许多基本的细胞过程。在酿酒酵母中，极化生长不像白念珠菌菌丝那样极端，但分子细节已经解决。分泌的蛋白质在膜包裹的囊泡中穿过不同的隔室。最后一组隔室被称为高尔基体，囊泡从高尔基体沿着由肌动蛋白电缆组成的轨道传输到细胞表面。一旦它们到达细胞表面，它们在与质膜融合并释放其内容物之前，与一种名为胞囊的多蛋白质结构对接。肌动蛋白缆线是在极化生长的部位通过第二种多蛋白质结构形成的，这种结构被称为极化体。极化体和胞外囊泡的形成以及囊泡与胞外胞囊的对接是由一种名为CDC42的蛋白质促进的，它在控制芽的生长、形状和从母细胞中分离方面发挥着许多作用。我们的研究集中在特定调节高尔基体和外囊之间分泌囊泡运动的蛋白质。在酿酒酵母中，它们被鉴定为Ypt31(和非常相似的Ypt32)、Sec2、Sec4、Iki3和Msb3(以及非常相似的Msb4)。我们已经证明了Sec2在菌丝中的Spitzenkörper中积累，但它在酵母中没有显示任何特定的定位。我们还表明，Sec2在通过添加磷酸基团形成后受到修饰，这是一种众所周知的改变蛋白质性质的修饰。可能Sec2的磷酸化状态负责改变与细胞的位置。我们计划绘制精确的被磷酸化的氨基酸图，并通过将氨基酸改变为不能磷酸化的氨基酸来检验这一假设。我们还计划确定负责磷酸化的酶。同样，从酿酒酵母的研究中有一条线索表明，它可能是一种名为Cbk1的酶。我们将通过产生对某种药物特别敏感的Cbk1突变体来检验这一假设，这样我们就能够随意地断断续续地检测Cbk1，看看它的活性是否是菌丝中Sec2定位所必需的。我们还计划通过研究这些蛋白在细胞内的位置，并测试当编码它们的基因从基因组中删除时对菌丝生长的影响，来研究Ypt31、Iki3 Msb3的作用。最后，我们设计了一种细胞，在该细胞中，CDC42比正常细胞更活跃。这些细胞表现出计划外的菌丝样生长。我们将使用这些细胞来研究CDC42是否直接控制Sec2的活性。

Candida albicans is a fungus that is responsible for causing vaginitis (thrush) in women. It is also the cause of a common, and often fatal bloodstream infection in hospital intensive care units. A feature of its biology, which important for its pathogenicity, is its ability to switch between unicellular yeast and filamentous growth forms. The filamentous form consists of long tube-like cells, called hyphae, which grow exclusively from their tip. This proposal aims to understand the molecular mechanism responsible for this extreme form of polarised growth. In the long term this may help develop better drugs to fight not only C. albicans infections, but also serious infections caused by other fungi, which also show this pattern of growth. So far our laboratory has shown that in C. albicans hyphae a structure called a Spitzenkörper (from the German 'apical body') is present at the tip. It is thought membrane-bound vesicles that contain the raw materials for new hyphal growth are transported to the Spitzenkörper, where they accumulate to form a supply centre for the delivery of vesicles to hyphal tip. The problem we are addressing is what is responsible for controlling flow of vesicles to the Spitzenkörper? We are helped by research in the budding yeast Saccharomyces cerevisiae, which has proved a very useful model for understanding many fundamental cellular processes. In S. cerevisiae, polarised growth is not as extreme as in C. albicans hyphae, but the molecular detail has been worked out. Secreted proteins pass through various compartments in membrane-enclosed vesicles. The last set of compartments is called the Golgi, from which vesicles are transported to the cell surface along tracks consisting of actin cables. Once they arrive at the cell surface they dock with a multi-protein structure called the exocyst before fusing with the plasma membrane and releasing their contents. Actin cables are formed at sites of polarised growth by a second multi-protein structure called the polarisome. Formation of the polarisome and exocyst, and the docking of vesicles with the exocyst, is promoted by a protein called Cdc42, that plays many roles in controlling bud growth, shape and separation of the bud from the mother cell. Our research focuses on the proteins which specifically regulate secretory vesicle movement between the Golgi and the exocyst. In S. cerevisiae these have been identified as Ypt31 (and the very similar Ypt32), Sec2, Sec4, Iki3 and Msb3 (and the very similar Msb4). We have already shown that Sec2 accumulates in the Spitzenkörper in hyphae, but it does not show any specific localisation in yeast. We have also shown that Sec2 is subject to modification after it has formed by the addition of a phosphate group, a type of modification that well known to change the properties of a protein. It's possible that the state of phosphorylation of Sec2 is responsible for changing location with the cell. We plan to map the precise amino acid that is phosphorylated and test this hypothesis by changing the amino acid to one which can't be phosphorylated. We also plan to identify the enzyme responsible for the phosphorylation. Again there is a clue that from the S. cerevisiae research that it might be an enzyme called Cbk1. We will test this hypothesis by generating a mutant of Cbk1 that is specifically sensitive to a drug so we will be able to Cbk1 off and on at will to see if its activity is required for Sec2 localisation in hyphae. We also plan to investigate the roles of Ypt31, Iki3 Msb3 by investigating where these proteins are located within the cell and testing the effect on hyphal growth when the genes that encode them are deleted from the genome. Finally, we have engineered a cell where Cdc42 is more active than normal. These cells show unscheduled hyphal-like growth. We will use these cells to investigate whether Cdc42 directly controls the activity of Sec2.