We are interested in how biological information regulating gene expression is encoded, transmitted, and interpreted. In addressing this question, we focus on Toll signaling, a pathway for transducing information within cells that is conserved from insects to humans and that has diverse roles in development and innate immunity. Our studies focus on the fruit fly Drosophila melanogaster, with which we can readily generate mutations that disrupt pathway activity; monitor and manipulate gene activity; and map out signaling modules and regulatory circuitry using molecular, biochemical, and bioinformatic techniques. In this way, we have defined the structure and organization of a trimeric signaling complex, elucidated function-specific evolutionary adaptations, and identified novel pathway functions and effectors.
Signal Transduction in Disease and Development
The signal transduction pathway defined by the Toll transmembrane receptor is a critical component of the Drosophila innate immune response to microbial infection. Upon exposure to a fungal pathogen, wild-type fruit flies express an array of genes encoding antimicrobial peptides, including Drosomycin, a potent anti-fungal agent. The transcription factor activated by Toll in this setting is the Drosophila Immunity Factor (DIF), which belongs to the NF-kB protein family. In mammals, Toll-like receptors (TLR’s) activate NF-kB as a critical step in innate immune response to infection. Furthermore, aberrant Toll signaling underlies a number of debilitating inflammatory disorders. The function of Toll in innate immunity is thus broadly conserved and of interest from both a fundamental and clinical perspective.
In Drosophila, Toll signaling also establishes the dorsoventral axis of the embryo. Localized activation of the transmembrane receptor Toll leads to the graded nuclear translocation of the transcription factor Dorsal, another NF-kB family member. By activation of ventral-specific loci and repression of dorsal-specific loci, the Dorsal gradient establishes subdivides the dorsoventral axis.
The same signaling pathway is used in immunity and development. Prior to signaling, the target NF-kB protein, either DIF or Dorsal, is bound in the cytoplasm by an inhibitor, Cactus, that blocks nuclear translocation. Following generation of the active Toll ligand Spätzle by upstream signals, Toll transduces signals leading to degradation of Cactus, freeing the NF-kB protein to direct gene expression.
Currently, our studies of Toll signaling are centered on three research areas:
1. Dissecting the Mechanism of Signal Transduction. Toll-mediated inactivation of the inhibitor Cactus requires signal transduction by MyD88, Tube, and Pelle. We have demonstrated that these three proteins function in signaling by forming a trimeric protein complex. We have used molecular genetic, biochemical, and biophysical approaches to define and mutate the precise sites of interaction in the trimer, generating powerful reagents for our ongoing dissection of the signaling mechanism.
We have extended our studies to explore how formation of the death domain trimer leads to phosphorylation and degradation of the inhibitor protein, Cactus. In particular, we have recently identified Pelle as the protein kinase required for Cactus inactivation.
2. An Informatics-Based Approach to Decoding Global Transcriptional Control. The Toll pathway operates in parallel with a second signaling system –the Imd pathway–to govern innate immune responses. Both systems rely on NF-kB related transcription factors to regulate gene expression. Applying a molecular genetic approach, we have demonstrated that cis-acting control elements act as a specificity code for the response to either or both pathways. Using our understanding of these elements to inform bioinformatic studies, we have mapped out the global system of control for the major innate immune responses in flies.3. Exploring Evolutionary Pathway Adaptations to Embryonic Patterning. Although the Toll pathway acts in innate immunity in a wide range of animals, it has been specifically adapted to pattern formation in the insects. There it must function in a syncytial environment and be regulated in time and space in a manner distinct from that required for defensive responses. Through identification of pathway components from a variety of insects and functional assays of chimeric proteins, we are defining the domains and activities that have undergone modification to fulfill these specialized functions in the context of conservation of the basic signaling mechanism.
Busse M.S., Arnold C.P., Towb, P., Katrivesis J., and Wasserman S.A. (2007) A κB sequence code for pathway-specific innate immune responses. EMBO J. 26, 3826-3835.
Towb, P., Sun, H., and Wasserman S.A. (2009) Tube is an IRAK-4 homolog in a Toll pathway adapted for development and immunity. J. Innate Immunity 1, 309-321.
Lindsay, S.A., and Wasserman, S.A. (2013) Conventional and non-conventional Drosophila Toll signaling. Dev. Comp. Immunol., in press.
Clemmons A.W., and Wasserman, S.A. (2013) Combinatorial effects of transposable elements on gene expression and phenotypic robustness in Drosophila melanogaster. G3: Genes, Genomes, Genetics, in press.Daigneault, J., Klemetsaune, L., and Wasserman S.A. (2013) The IRAK homolog Pelle is the functional counterpart of IkB Kinase in the Drosophila Toll pathway. PLoS One, in press.
Steven Wasserman received his Ph.D. from MIT and was a postdoctoral fellow at UC Berkeley. He has been the recipient of a Lucille P. Markey Scholarship in Biomedical Sciences, a David and Lucile Packard Fellowship in Science and Engineering, and a Distinguished Teaching Award from the UCSD Academic Senate.