The G protein signaling pathway is very important in relaying information for growth and development. The main objectives of this dissertation are to: 1. Generate novel image processing tools with the aid of video bioinformatics, 2. Decipher the role of G protein signaling on development and growth through phenotypic analysis and localization studies, and 3. Confirm functions amongst proteins implicated in the Erk1/2 MAPK pathways in Neurospora crassa.
In Chapter 2, I used video bioinformatics and image processing tools to develop algorithms that allowed for the analysis of vegetative cell compartment size, conidia size, and growth rate. I used the hyphal compartment size program and found that Δgna-1 and Δric8 had smaller length and diameter, whereas Δrgs-3, Δrgs-4, and Δrgs-5 had larger compartment sizes. Morphological assays were also conducted on the G signaling components. The results in Chapter 2, suggests that Δgna-1 and Δric8 regulate cell compartment size and growth rate in N. crassa. In Chapter 3, the phenotypic analysis of available G protein coupled receptor (GPCR) mutants were performed in a collaborative manner, with a majority of contributions by undergraduate students at UCR. All available GPCR mutants were phenotyped using classical morphological assays, as well as tested on chemicals for resistance or sensitivity on that tested chemical. Publically available gene expression data was obtained in order to compare expression patterns of the GPCR’s during growth and development. The results obtained in Chapter 3 improved our knowledge on GPCRs, especially on the Pth-11 related pathogenic receptors. In Chapter 4, the Gα subunits were fluorescently tagged, and their subcellular localization assayed during early development. In Chapter 4, my results demonstrated that all three Gα proteins localize to the plasma membrane in ungerminated conidia and young germlings, with GNA-1 also localizing on septa, and GNA-3 localizing on distinct patches on germlings. Lastly, Chapter 5 examined downstream effectors, specifically the MAK-1 and MAK-2, mitogen-activated protein kinase (MAPK) cascades in two G protein signaling component mutants, ric8 and ste50. The experiments revealed that ste50 is necessary for phosphorylation of MAK-2 in 16 hr VM liquid cultures and MAK-1 and MAK-2 in 6 day old VM plate cultures. However, MAK-1 and MAK-2 were phosphorylated to wild-type levels in cultures grown on low nitrogen medium (SCM) in Δste50 mutants. The objective of this dissertation was to shed light and expand the knowledge on G protein signaling components and their effects on growth and development in N. crassa.