The primary objectives of the proposed project will be to analyse the role of cytosolic free calcium ([Ca2+]c) in Neurospora crassa for regulating: 1. The initiation of cell polarity (during hyphal branch formation). 2. The maintenance of cell polarity (during hyphal tip growth). 3. The reorientation of cell polarity (during hyphal tip growth in an applied electrical field).
Imaging, quantitation and experimental manipulation of Ca2+ dynamics in living hyphae will be central to the project. The research will involve using:
1. Pressure microinjection of Ca2+-sensitive dyes and caged probes. 2. Confocal imaging of Ca2+-sensitive dyes.
3. Ratio imaging of ([Ca2+]c).
4. Experimental manipulation of Ca2+signalling using photoactiveatable caged probes.
5. Analysis of polarity mutants impaired in chitin synthase and protein kinase activities.