Self-signalling during cell fusion in filamentous fungi

丝状真菌细胞融合过程中的自我信号传导

• 批准号: BB/E010741/1
• 负责人: Nick Read
• 金额: $ 63.02万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE010741%2F1
• 关键词: Self; signalling; during; cell; fusion

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.

尽管人们对基因不同的细胞（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“自融合”知之甚少。自融合是霉菌（丝状真菌）菌落的一个决定性特征。了解丝状真菌粗糙脉孢菌中的细胞融合为了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），形成芽管，芽管生长并最终发育成成熟的真菌菌落。这些分生孢子还产生称为分生孢子吻合管（CAT）的短特化细胞，它们相互生长并融合。这一过程可分为三个阶段：CAT诱导、CAT归巢和CAT融合。粗糙脉孢菌是第一个对其基因组进行完全测序的丝状真菌，结果表明它拥有约 10,000 个基因。通过将这些基因与其他生物体测序基因组中的已知基因进行比较，可以预测这些基因编码的许多蛋白质的可能功能。脉孢菌的大约 10,000 个基因中的每一个都被删除以产生“敲除突变体”。在拟议的研究中，我们将筛选约 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细胞内信号转导受损的突变体。 CAT 归巢将使用我们开发的一种新颖的检测方法进行评估，其中涉及使用“激光镊子”。这项技术利用光创建“力场”，使人们能够用 CAT“捕获”分生孢子等细胞。使用我们的激光镊子归巢测定，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。我们使用这种技术作为一种明确的方法来确定突变体的 CAT 是否可以彼此归巢。 CAT 尖端相互吸引的原因是每个 CAT 尖端产生的化学引诱剂。不能相互归巢的突变体在 CAT 化学引诱剂的产生或感知方面存在缺陷。我们知道，参与 CAT 诱导、归巢和可能的融合过程的一种细胞内信号传导途径是所谓的 MAP 激酶途径。它由三种蛋白质组成，这些蛋白质被未知信号激活，然后通过磷酸化过程依次相互激活。它们的激活最终导致参与 CAT 诱导、归巢和可能的融合的其他过程的调节。我们最近发现其中一种 MAP 激酶位于 CAT 尖端内。我们将通过对我们荧光标记的三种 MAP 激酶进行成像来扩展这项研究。在分析了 CAT 诱导、归巢和融合过程中 MAP 激酶的定位和行为后，我们将在突变体中对它们进行荧光标记，这些突变体是我们在之前的突变体筛选中鉴定出的参与 CAT 诱导、归巢或融合的突变体。我们将寻找那些正常 MAP 激酶定位和行为已被破坏的突变体。这将为我们提供关于细胞外 CAT 诱导剂/趋化剂和 MAP 激酶途径之间发生的“上游”信号传导过程的线索。在鉴定出突变时会破坏 CAT 诱导、归巢或融合的 CAT 基因后，我们将对它们编码的 CAT 蛋白进行荧光标记，并分析它们的亚细胞定位和行为。最后，我们将通过用激光镊子操作其他 CAT 尖端来观察荧光标记的 MAP 激酶和 CAT 蛋白的细胞内分布是否会受到其他 CAT 尖端接近程度的影响。这将为迄今为止未知的 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。

项目成果

The ham-5, rcm-1 and rco-1 genes regulate hyphal fusion in Neurospora crassa.

• DOI: 10.1099/mic.0.040147-0
• 发表时间: 2010-09
• 期刊: Microbiology (Reading, England)
• 作者: Aldabbous MS;Roca MG;Stout A;Huang IC;Read ND;Free SJ
• 通讯作者: Free SJ

Heterokaryon incompatibility is suppressed following conidial anastomosis tube fusion in a fungal plant pathogen.

• DOI: 10.1371/journal.pone.0031175
• 发表时间: 2012
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Ishikawa FH;Souza EA;Shoji JY;Connolly L;Freitag M;Read ND;Roca MG
• 通讯作者: Roca MG

Septins are important for cell polarity, septation and asexual spore formation in Neurospora crassa and show different patterns of localisation at germ tube tips.

• DOI: 10.1371/journal.pone.0063843
• 发表时间: 2013
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Berepiki A;Read ND
• 通讯作者: Read ND

The Antifungal Activity of the Penicillium chrysogenum Protein PAF Disrupts Calcium Homeostasis in Neurospora crassa

• DOI: 10.1128/ec.00050-10
• 发表时间: 2010-09-01
• 期刊: EUKARYOTIC CELL
• 作者: Binder, Ulrike;Chu, Meiling;Marx, Florentine
• 通讯作者: Marx, Florentine