Self-signalling during cell fusion in filamentous fungi

尽管人们对基因上不同的细胞(例如动物的精子和卵子)之间的融合了解很多，但对基因相同的细胞之间的“自我融合”却知之甚少。自我融合是霉菌(丝状真菌)群体的一个重要特征。了解丝状真菌粗糙脉孢菌中的细胞融合为理解动植物和微生物中的细胞融合提供了一个模型。粗糙脉孢菌产生无性孢子(分生孢子)，形成生殖管，然后生长并最终发育成成熟的真菌菌落。这些分生孢子还产生被称为分生孢子吻合管(CATS)的短小的特化细胞，它们彼此生长并融合。这一过程可分为三个阶段：猫诱导、猫归巢和猫融合。粗糙脉孢菌是第一个完成基因组测序的丝状真菌，其基因组已被证明含有约10,000个基因。通过将这些基因与其他生物基因组测序中的已知基因进行比较，已经预测了这些基因编码的许多蛋白质的可能功能。脉孢子虫的大约10,000个基因中的每一个都被删除，以产生“敲除突变体”。在这项拟议的研究中，我们将筛选大约100个这些敲除突变体，以确定哪些在CAT诱导、归巢和/或融合方面存在缺陷。通过光学显微镜筛选的突变体将是那些在细胞内信号转导方面受到损害的突变体。我们将使用我们开发的一种新方法来评估猫咪的归巢情况，这种方法涉及到使用“激光镊子”。这项技术利用光来创造一个“力场”，让人们能够将分生孢子等细胞“捕获”在猫身上。使用我们的激光镊子定位试验，我们可以捕获单个分生孢子，并将其相对于另一个分生孢子移动。我们使用这项技术作为一种明确的方法来确定突变的猫是否可以彼此回家。猫尖相互吸引的结果是每一只猫尖产生的一种化学吸引剂。不能彼此亲近的突变体在猫趋化物质的生产或感知方面存在缺陷。在CAT的诱导、归巢和可能的融合过程中，我们所知道的一个细胞内信号通路就是所谓的MAP激酶通路。它由三种蛋白质组成，它们被未知信号激活，然后通过磷酸化过程相继相互激活。它们的激活最终导致对CAT诱导、归巢和可能的融合等其他过程的调控。我们最近发现，这些MAP中的一种在猫的尖端变得局部化。我们将通过成像我们已经荧光标记的三个映射激酶来扩展这项研究。在分析了MAP激酶在CAT诱导、归巢和融合过程中的定位和行为后，我们将在我们之前的突变筛选中确定参与CAT诱导、归巢或融合的突变体中对它们进行荧光标记。我们将寻找的是那些正常的MAPK定位和行为被打乱的突变体。这将为我们提供线索，了解发生在细胞外CAT诱导剂/化学诱导剂和MAP激酶途径之间的“上游”信号过程。在确定了当发生突变时会扰乱CAT诱导、归巢或融合的CAT基因后，我们将荧光标记它们编码的CAT蛋白，并分析它们的亚细胞定位和行为。最后，我们将通过激光镊子操纵其他CAT尖端，看看荧光标记的MAP激酶和CAT蛋白在细胞内的分布是否受到其他CAT尖端接近的影响。这将为一种迄今未知的CAT趋化剂的细胞外梯度影响参与CAT诱导和CAT归巢的细胞内机制的动态组织提供证据。

Although much is known about fusion between genetically non-identical cells (e.g. sperm and egg in animals), little is known about 'self fusion' between genetically identical cells. Self fusion is a defining feature of the colony of moulds (filamentous fungi). Understanding cell fusion in the filamentous fungus Neurospora crassa provides a model for understanding cell fusion in animals and plants and microbes. Neurospora crassa produces asexual spores (conidia) which form germ tubes that grow and eventually develop into the mature fungal colony. These conidia also produce short specialized cells called conidial anastomosis tubes (CATs) which grow towards each other and fuse. This process can be divided into three stages: CAT induction, CAT homing and CAT fusion. Neurospora crassa was the first filamentous fungus to have its genome completely sequenced and as a result it has been shown to possess ~ 10,000 genes. The likely function of many of the proteins encoded by these genes has been predicted by comparing these genes with known genes in the sequenced genomes of other organisms. Each of Neurospora's ~ 10,000 genes is being deleted to produce 'knockout mutants'. In the proposed study we will screen ~ 100 of these knockout mutants to determine which are defective in CAT induction, homing and/or fusion. Mutants to be screened by light microscopy will be those compromised in intracellular signaling. CAT homing will be assessed using a novel assay we have developed which involves the use of 'laser tweezers'. This technology uses light to create a 'force field' which allows one to 'trap' cells such as conidia with CATs. Using our laser tweezer homing assay we can trap an individual conidium and move it relative to another conidium. We use this technique as an unambiguous method to determine whether the CATs of a mutant can home towards each other. This attraction of CAT tips towards each other results from a chemoattractant which each CAT tip produces. Mutants that cannot home towards each other are defective in the production of the CAT chemoattractant or in its perception. One intracellular signalling pathway which we know is involved in the process of CAT induction, homing and possibly fusion, is the so-called MAP kinase pathway. This is comprised of three proteins which are activated by an unknown signal and then successively activate each other by a process of phosphorylation. Their activation ultimately leads to the regulation of other processes involved in CAT induction, homing and possibly fusion. We have recently found that one of these MAP kinases becomes localized within the tips of CATs. We will extend this study by imaging the three MAP kinases which we have fluorescently tagged. Having analysed the localization and behaviour of the MAP kinases during CAT induction, homing and fusion we will fluorescently tag them in mutants that we have identified in our previous mutant screen as being involved in CAT induction, homing or fusion. What we will be searching for will be those mutants in which the normal MAP kinase localization and behaviour have been disrupted. This will give us clues as to the 'upstream' signalling processes which occur between the extracellular CAT inducer/chemoattractant and the MAP kinase pathway. Having identified CAT genes that, when mutated, disrupt CAT induction, homing or fusion, we will fluorescently tag the CAT proteins they encode and analyse their subcellular localization and behaviour. Finally, we will see whether the intracellular distribution of fluorescently tagged MAP kinases and CAT proteins is influenced by the close proximity of other CAT tips by manipulating them with laser tweezers. This will provide evidence for extracellular gradients of a so far unidentified CAT chemoattractant influencing the dynamic organization of the intracellular machinery involved in CAT induction and CAT homing.