Cellular Signaling Mechanisms in Neurospora crassa

Author(s): Ghosh, Arit | Advisor(s): Borkovich, Katherine A. | Abstract: Cellular signal transduction mechanisms are regulated at multiple different stages during an organism’s life cycle as well as life span. Environmental stress and starvation responses are extremely well coordinated by cell surface receptors and internal scaffolding molecules that are routing the signal to the effector proteins such as transcription factors. It is through these transcription factors that the cell will then regulate gene expression. With the help of this study, my co-authors and me have tried to elucidate a signal transduction network, which explains various facets of cellular signaling in the model filamentous fungus – Neurospora crassa. The first chapter has elucidated the role of serine/threonine and tyrosine phosphatases in growth and development in Neurospora. In addition, the chapter also shows that deletion of certain phosphatases lead to sensitivities to chemicals inducing osmotic stress, oxidative stress, cytoskeletal defects or ROS accumulation. Nine phosphatase mutants are also listed to have elevated levels of the active phosphorylated form of p38 mitogen activated protein kinase (OS-2 in Neurospora) which is a critical regulator for counteraction to osmo-stress as well as an important regulator of female sexual development. Two other interesting genes – NCU04600 (pph-8) and NCU08380 (csp-6) are also described in this chapter for their unique phenotypes. The second chapter deals with the role of the important scaffolding protein and RACK1 homolog – CPC-2 in regulation of amino acid starvation mechanisms in Neurospora known as cross pathway control. CPC-2 is found to regulate the bZIP transcription factor – CPC-1 via modulation of post-translational modifications on CPC-1 during amino acid starvation. This transcription factor is in turn, integral towards de-repression of amino acid biosynthetic genes under amino acid starvation conditions. This chapter provides mechanistic details on how CPC-2 is able to regulate CPC-1 protein and thereby affect cross pathway control. In chapter III, the focus is shifted towards translational regulation via heterotrimeric G proteins and the guanine-exchange factor RIC-8 to build up a novel finding revealing that G proteins and RIC-8 are an integral part of the ribosome. Elevated phospho-eIF2α levels in the G protein and ric-8 mutants suggest that global translation is greatly reduced in these strains. Poly-RNA-seq analyses of gna-1, gnb-1 and ric-8 mutants reveal certain ribosomal proteins as well as elongation factors and two serine/threonine kinases – stk-18 and stk-43 are greatly affected in polysomal co-migration by these gene deletions. In addition, deletion of ric-8 leads to loss of PKC protein from the polysomes, which suggests critical translational control of PKC via RIC-8. This thesis has thus aimed to expand on current knowledge on these abovementioned topics and laid the groundwork for future advances in understanding these cellular signaling mechanisms in Neurospora crassa.