Browsing by Subject "Evolution, Molecular"
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Item Evolution of Pharyngeal Feeding Behaviors in Free-Living Soil Nematodes(2007-05-22) Chiang, Jing-Tzyh Alan; Avery, LeonTo explore using nematodes as a model for studying behavioral evolution, I examined pharyngeal behaviors in free-living soil nematodes related to Caenorhabditis elegans. The nematode pharynx is divided into three regions: corpus, isthmus, and terminal bulb. Pharyngeal behaviors consist of stereotyped patterns of two motions: pumping and peristalses. I observed pharyngeal behaviors in multiple species, and constructed the following evolutionary model. In the ancestor of free-living soil nematodes, the pharynx had corpus pumping, isthmus peristalses, and terminal bulb pumping, each of which occurred independently. In the Rhabditidae family, the anterior isthmus switched to pumping, and anterior isthmus and terminal bulb pumping became coupled to corpus pumping. In the Diplogasteridae family, the terminal bulb switched to peristalses. In the Cephalobidae family, isthmus peristalses and terminal bulb pumping became coupled. And in the Panagrolaimidae family, the posterior isthmus switched to pumping. The above changes in isthmus and TB behaviors suggested corresponding changes in their neuronal regulation. Using laser ablations, I found that M4's function evolved significantly: M4 stimulated posterior isthmus peristalsis (Rhabditidae), isthmus/terminal bulb peristalsis (Diplogasteridae), isthmus peristalsis and terminal bulb pumping (Cephalobidae), and posterior isthmus/terminal bulb pumping (Panagrolaimidae). Yet, increased food activated M4 activity in all families. Thus, M4 appeared to be a general "food sensor" neuron, which was co-opted during evolution to perform different downstream functions. Additionally, M2 stimulated anterior isthmus peristalsis in the Panagrolaimidae. Using Caenorhabditis elegans, I investigated possible molecular/genetic causes for the above changes. Why is the terminal bulb unaffected by M4 in Caenorhabditis elegans and the Rhabditidae? I found that mutating slo-1 activated M4-terminal bulb stimulations, suggesting that alterations in synaptic transmission silenced M4-terminal bulb synapses. Also, why does M2 stimulate peristalses in the Panagrolaimidae, and M4 in the other families? I found ceh-28 important for M4 to stimulate peristalsis in Caenorhabditis elegans, but M2 also had the potential for ceh-28 expression. This suggested a genetic/molecular link between the two neurons, and ceh-28 related mechanisms may determine which neuron stimulates peristalsis. Overall, I characterized how pharyngeal behaviors evolved at the behavioral, neuronal, and genetic levels. These results suggested the utility of nematodes for studying behavioral evolution.Item A Global Experimental Analysis of Protein Function: a Case Study in the PDZ Domain(2011-02-01T19:36:33Z) McLaughlin, Richard Noel Jr.; Ranganathan, RamaA complete understanding of the energetic architecture of a protein can be achieved only with a comprehensive description of the interaction of every amino acid with every other amino acid. Many efforts to understand the apparent complexity of protein function have attempted to address this problem with limited mutagenesis studies. A global computational description of amino acid interactions, Statistical Coupling Analysis has shown the existence of a contiguous subset of positions within a protein that displays significant co-evolution, termed protein sectors. Limited mutagenesis studies have shown sectors to be networks of higher-order interaction crucial for protein function; however, a theory of such global scope requires validation with a global experiment. Here, we design an assay system that measures the cellular function of a PDZ domain in a high-throughput and quantitative manner. We perform a comprehensive single amino acid mutagenesis experiment to show that most positions in the protein are robust to most mutations, and the set of positions that shows sensitivity to mutation is enriched for sector positions. Further, we perform a global pairwise epistasis experiment in which we measure the way in which every amino acid mutation in the PDZ domain feels the effect of a second mutation at a key specificity and affinity determining position in the peptide ligand of the PDZ domain. We find that those positions that show strong non-additivity in the context of the peptide mutation are all contained within the PDZ sector. Further, these sector positions that display strong non-additivity all display the property of rapidly changing specificity upon mutation. That is, any mutation at these sector positions has a negative functional effect in the context of the endogenous peptide. However, these positions appear to be spring-loaded for change since these same mutations enhance function in the context of an alternative peptide. We hypothesize that proteins are robust as shown by their insensitivity to general mutation. However, proteins are simultaneously fragile as shown by their sensitivity to specific mutagenesis at sector positions. This fragility, however, is strongly coupled to evolvability as shown by the enhancement of alternative function endowed by these endogenously detrimental mutations.Item Low-Density Lipoprotein Receptors in Signaling Modulation and Development(2010-05-14) Dietrich, Martin Frederik; Herz, JoachimThe Low-Density Lipoprotein Receptor gene family is a group of ancient membrane receptors. Originally implied in cargo transport and development of atherosclerosis, the number of members and the diversity of functions have been greatly expanded. LRP1, LRP1b and LRP4 are gene family members that are implicated in the regulation of signaling pathways at the intracellular, extracellular and transcriptional level. These regulations confer viability, control the cellular proliferation at several molecular steps, and allow for proper organ formation by moderating and integrating cellular signaling pathways. / The use of knockin mutant mice has, for the first time, implicated the extracellular domains of LRP1b and LRP4 in signaling modulation in development. While the complete knockout of either receptor is embryonically lethal, the expression of a truncated receptor, spanning only the extracellular domain, confers viability and only a mitigated phenotype. / For LRP4, the difference is most visible in the kidney. The present LRP4 extracellular preserves thresholds critical for organogenesis, yet, the complete absence displays a subpenetrant phenotype of kidney agenesis. In this thesis work, results demonstrate the ability of the LRP4 extracellular domain to not only bind a broad variety of soluble ligands in the extracellular space, but further to influence the Wnt, and possibly others, signaling pathways that are required for kidney development. / In an osteoblast-specific model of LRP1 knock-out, the relationship between theLRP1and the PDGF receptor has been further investigated. LRP1 is known to negatively regulate the PDGF receptor. However, the exact mechanism(s) are not fully understood. In the wild-type, PDGF receptor beta binds directly to LRP1 upon ligand stimulation. LRP1 knockout leads to significant upregulation of the PDGF receptor beta at the protein level. The stimulation of the receptor with PDGF-BB, its corresponding ligand, leads to overactivation of the signaling pathway with both increased turnover and phosphorylation/activation of the receptor, demonstrated by cellular proliferation and p21 downregulation. In vivo, the LRP1 knockout leads to a bone-derived hyperproliferation with formation of tumors at the epiphysis. The in vitro experiments are supporting evidence, combined with previously published literature, to imply the LRP1/PDGF receptor pathway.Item Multi-Scale Structure and Dynamics of Visual Signaling in Drosophila Photoreceptor Cells(2012-07-16) Helms, Stephen Jess; Ranganathan, RamaA general problem in science today is how to understand complex systems. An emerging and promising approach makes the bold assumption that complex systems adhere to particular design principles. The power of this is that design principles by definition impose an intuitive nature on a system by presupposing purpose. Existing studies have fruitfully shown the application of engineering principles in biology, but biological systems have many distinct features, particularly due to evolution. In this work, I used Drosophila phototransduction, a well-studied sensory system renowned for its high performance, to search for evolutionary design principles. I focused on three levels of structure in the system: compartmentalization of molecules into microvilli, modularity of dynamic scaffolding by InaD, and functional integration within a single domain of InaD. Using rigorous quantitative measurement and theory with an evolutionary mindset, I uncovered intuitive, simplifying design principles at each level: Microvilli are used to build fast, homogeneous signaling compartments whose dimensions are constrained by these requirements. Dynamic scaffolding is a modular feature of InaD PDZs 4-5 which have been co-inherited in many scaffolds. Within PDZ5, ligand binding and oxidation of the domain are linked through pairwise coupling with a conformational equilibrium—a generic property found in all proteins—and not each other. These results show that this approach can be successful in revealing novel design principles in complex evolved systems. [Keywords: signaling, Drosophila, photoreceptor, vision, scaffolding, compartmentalization, modularity, systems biology, biophysics, structure]