Self-signalling during cell fusion in filamentous fungi

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.

Self fusion between conidia and conidial germlings via conidial anastomosis tubes (CATs) in the model filamentous fungus, Neurospora crassa provides a simple and extremely experimentally amenable system to study self-signalling during cell fusion. The first part of the project will involve screening ~ 100 knockout mutants generated by the NIH Neurospora genome grant to determine which are defective in CAT induction, homing and fusion. Mutants to be screened will include those compromised in signalling, especially GPCRs, G-proteins, two-component signalling, phospholipase C, protein kinase C, calcium signaling, and cAMP signalling. Laser tweezers will form a critical part of the CAT homing assay. The second part will involve analyzing the dynamic subcellular distribution of three different fluorescently labelled MAP kinases in the MAP kinase pathway involved in regulating CAT induction, homing and possibly fusion. The third part will involve identifying and analysing the signalling pathways which are upstream of MAP kinase signaling by using the MAP kinase reporter system developed in the second part, and expressed in the different mutant backgrounds identified in the first part as being defective in CAT induction, homing or fusion. The fourth part will involve the live-cell analysis of the dynamic subcellular distribution of different fluorescently labelled CAT proteins identified in parts two and three as being important in CAT induction, homing and fusion. The final part will involve determining whether the intracellular distribution of fluorescently tagged MAP kinases and CAT proteins is influenced by the close proximity of other conidia or CAT tips manipulated by laser tweezers. This will provide evidence for extracellular gradients of a so far unidentified CAT chemoattractant influencing the dynamic organization of the intracellular signalling machinery.