The role of calcium in the tropisms of Candida albicans hyphae

许多细胞只在末端生长/这是一种细胞发育模式，需要在细胞表面的一个点上输送新的膜和组装新的细胞壁。这种两极分化的发育形式在神经细胞、花粉管和许多其他真核细胞类型中都很常见，尤其是真菌菌丝。顶端生长的菌丝也必须朝向营养物质、氧气、适当的交配伙伴或其他细胞，远离有毒化合物和不可渗透的物体。因此，尖端的生长必须与尖端定向的能力相结合。我们知道，位于细胞顶端的细胞骨架和几个重要的蛋白质复合体需要以这样一种方式组织细胞，即携带生长所需的膜和蛋白质的分泌小泡被输送到顶端质膜并与之融合。我们不知道细胞骨架和这些蛋白质复合体是如何被调节的，以使不断增长的菌丝尖端能够被操纵并对环境线索做出反应。这项研究计划建立在我们最近观察到的基础上，这些观察表明，菌丝的转化能力需要生长介质中的钙离子和几种钙通道蛋白的供应。这些见解体现在我们的假设中，即菌丝定位所需的分子机制是由菌丝顶端钙离子的局部摄取来调节的。在我们的实验中，我们使用了人类致病真菌白色念珠菌的菌丝定向反应作为我们的模型系统。这是因为我们可以从基因上操纵这种微生物来创造突变和蛋白质标记的菌株，这些突变和蛋白质标记的菌株可以用来解决我们的假设，而且因为菌丝向向性在这种真菌的致病生活方式中很可能是重要的。我们还为这个项目从正在研究尖端生长过程的同事那里收集了广泛的突变体和菌株，但没有将尖端取向作为一个单独的现象。我们已经开发了两种易于处理的分析方法，使我们能够观察和量化菌丝取向反应。首先，我们可以测量有多少菌丝在遇到细胞下方的脊状突起时偏向新的生长轴(接触指导或向着性)。其次，我们可以通过将菌丝放在电场中并观察它们朝向阴极的方向(电流趋向性)来推翻所有内源和外源的指导线索。到目前为止，我们的研究结果表明，钙离子对两种转向反应都是至关重要的，这表明转向机制是相同的，即使对不同的取向也是如此。我们将从几个方面检验这一假设，即菌丝取向受钙离子的调节。[I]我们将使用荧光标记的YFP和CFP融合来定位菌丝中的钙通道复合体蛋白质，并观察当菌丝经历亲水性和亲电性排列时，该复合体和其他与末端生长相关的蛋白质复合体(极化体、外囊、CDC42和Arp2/3复合体)中蛋白质的细胞定位。[ii]我们将确定来自细胞存储的钙离子是否也对支持趋向性也很重要。[III]我们将描述钙离子依赖的过程，这些过程可以将钙信号转化为趋向性生长反应。[IV]最后，我们将确定趋向性生长对白念珠菌菌丝侵入宿主组织能力的意义。

Many cells grow only at their ends / a mode of cell development that requires delivery of new membrane and assembly of new cell wall at a single point of the cell surface. This polarised form of development is common to nerve cells, pollen tubes and many other eukaryotic cell types, most notably fungal hyphae. Tip growing hyphae also have to be steered towards nutrients, oxygen, appropriate mating partners or other cells, and away from toxic compounds and around inpenetratable objects. Therefore tip growth has to be coupled to the ability to orient the tip. We know that the cytoskeleton and several important protein complexes located at the cell tip are required to organise the cell in such a way that secretory vesicles, which carry membrane and proteins for growth, are delivered to and fuse with the apical plasma membrane. We do not know how the cytoskeleton and these protein complexes are regulated to enable the growing hyphal tip to be steered and to respond to environmental cues. This research programme rests on the foundation of our recent observations that have shown that the ability of a hypha to turn requires a supply of calcium ions in the growth medium and several calcium channel proteins. These insights are embodied in our hypothesis that the molecular machinery required for hyphal orientation is regulated by local uptake of calcium ions at the hyphal apex. In our experiments we use hyphal orientation responses of the human pathogenic fungus Candida albicans as our model system. This is because we can genetically manipulate this organism to create mutations and protein-tagged strains that can be used to address our hypothesis, and because hyphal tropisms are likely to be important in the pathogenic life style of this fungus. We have also collected a wide range of mutants and strains for this project from colleagues who are investigating the tip growth process, but have not considered tip orientation as a separate phenomenon. We have developed two tractable assays that allow us to observe and quantify hyphal orientation responses. Firstly, we can measure how many hypha become deflected to a new axis of growth as the encounter a ridge underlying the cell (contact guidance or thigmotropism). Secondly we can override all endogenous and exogenous guidance cues by placing hyphae in an electrical field and observing them reorienting towards the cathodic pole (galvanotropism). So far our findings show that calcium ions are vital for both tropic responses suggesting that the steering machinery is the same, even for different tropisms. We will test the hypothesis that hyphal orientation is regulated by calcium ions in several ways. [i] We will localise the calcium channel complex proteins in the hyphae using fluorescently labelled YFP and CFP- fusions and observe the cellular localisation of proteins in this complex and other tip-growth related protein complexes (polarisome, exocyst, Cdc42 and Arp2/3 complexes) as hyphae undergo thigmotropic and galvanotropic alignments. [ii] We will determine whether calcium ions that originate from cellular stores are also important in supporting tropic orientations. [iii] We will characterise calcium-ion dependent processes that could translate calcium signals into tropic growth responses. [iv] Finally, we will determine the significance of tropic growth for the ability of C. albicans hyphae to invade host tissues.