GTP avoidance in Tetrahymena thermophila requires tyrosine kinase activity, intracellular calcium, NOS, and guanylyl cyclase

Guanosine 5'-triphosphate (GTP) is a chemorepellent in Tetrahymena thermophila that has been shown to stimulate cell division as well as ciliary reversal. Previous studies have proposed that GTP avoidance is linked to a receptor-mediated, calcium-based depolarization. However, the intracellular mechanisms involved in GTP avoidance have not been previously documented. In this study, we examine the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Using behavioral assays, enzyme immunosorbent assays, Western blotting, and immunofluorescence, we present data that implicate a tyrosine kinase, phospholipase C, intracellular calcium, nitric oxide synthase (NOS) and guanylyl cyclase in GTP signaling. The tyrosine kinase inhibitor genistein eliminates GTP avoidance in Tetrahymena in behavioral assays. Similarly, pharmacological inhibitors of phospholipase C, NOS, and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP. Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia, suggesting that this enzyme activity could be directly involved in ciliary reversal

Membrane protein structure, function, and dynamics: a perspective from experiments and theory

Membrane proteins mediate processes that are fundamental for the flourishing of biological cells. Membrane-embedded transporters move ions and larger solutes across membranes; receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. We present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function, and dynamics of membrane proteins.JTD, IA, and MR used the computational resources of the Modeling Facility of the Department of Chemistry, University of California Irvine funded by NSF Grant CHE-0840513 for this work. A-NB was supported in part by the Marie Curie International Reintegration Award IRG-26920.TWA was supported by ARC DP120103548, NSF MCB1052477, DE Shaw Anton (PSCA00061P; NRBSC, through NIH RC2GM093307), VLSCI (VR0200), and NCI (dd7). BA and SV acknowledge the support by ERC advanced Grant No. 268888. ZC and PG would like to acknowledge Reference Framework (NSRF) 2011–2013, National Action ‘‘Cooperation,’’ under grant entitled ‘‘Magnetic Nanoparticles for targeted MRI therapy (NANOTHER),’’ with code ‘‘11RYM-1-1799.’’ The program is cofunded by the European Regional Development Fund and national resources. Part of the calculations presented herein were performed using resources of the LinkSCEEM-2 project, funded by the EC under FP7 through Capacities Research Infrastructure, INFRA-2010-1.2.3 Virtual Research Communities, Combination of Collaborative Project and Coordination and Support Actions (CPCSA) under Grant agreement no. RI-261600. GB was supported in part by NSF grant MCB1330728 from the National Science Foundation and Grant PO1GM55876-14A1 from the National Institutes of Health. LD received funding from EU FP7 (PIOF-GA-2012-329534). LD, and MLK used the computational resources of Temple University, supported by the National Science Foundation through major research instrumentation grant number CNS-09-58854. JS acknowledges support from the Instituto de Salud Carlos III FEDER (CP12/03139