Signal transduction, regulation, and developmental logic of EGFR signalling in C. elegans

RTKs-mediated signaling systems and the pathways with which they interact (e.g., those initiated by G protein-mediated signaling) involve a highly cooperative network that sense a large number of cellular inputs and then integrate, amplify, and process this information to orchestrate an appropriate set of cellular responses. The responses include virtually all aspects of cell function, from the most fundamental (proliferation, differentiation) to the most specialized (movement, metabolism, chemosensation). The basic tenets of RTK signaling system seem rather well established. Yet, new pathways and even new molecular players continue to be discovered. Although we believe that many of the essential modules of RTK signaling system are rather well understood, we have relatively little knowledge of the extent of interaction among these modules and their overall quantitative importance.

My research has encompassed the study of both positive and negative signaling by RTKs in C. elegans. I identified the C. elegans S0S-1 gene and showed that it is necessary for multiple RAS-mediated developmental signals. In addition, I demonstrated that there is a SOS-1-independent signaling during RAS-mediated vulval differentiation. By assessing signal outputs from various triple mutants, I have concluded that this SOS-1-independent signaling is not mediated by PTP-2/SHP-2 or the removal of inhibition by GAP-1/ RasGAP and it is not under regulation by SLI-1/Cb1. I speculate that there is either another exchange factor for RASor an as yet unidentified signaling pathway operating during RAS-mediated vulval induction in C. elegans.

In an attempt to uncover the molecular mechanisms of negative regulation of EGFR signaling by SLI-1/Cb1, I and two other colleagues codiscovered that RING finger domain of SLI-1 is partially dispensable for activity. This structure-function analysis shows that there is an ubiquitin protein ligase-independent activity for SLI-1 in regulating EGFR signaling. Further, we identified an inhibitory tyrosine of LET-23/ EGFR requiring sli-1(+)for its effects: removal of this tyrosine closely mimics loss of sli-1 but not loss of other negative regulator function.

By comparative analysis of two RTK pathways with similar signaling mechanisms, I have found that clr-1, a previously identified negative regulator of egl-15 mediated FGFR signaling, is also involved in let-23 EGFR signaling. The success of this approach promises a similar reciprocal test and could potentially extend to the study of other signaling pathways with similar signaling logic.

Finally, by correlating the developmental expression of lin-3 EGF to let-23 EGFR signaling activity, I demonstrated the existence of reciprocal EGF signaling in coordinating the morphogenesis of epithelia. This developmental logic of EGF signaling could provide a basis to understand a universal mechanism for organogenesis.

The logic of receptor-ligand interactions in the Notch signaling pathway

The Notch signaling pathway enables neighboring cells to coordinate developmental fates in diverse processes such as angiogenesis, neuronal differentiation, and immune system development. Although key components and interactions in the Notch pathway are known, it remains unclear how they work together to determine a cell's signaling state, defined as its quantitative ability to send and receive signals using particular Notch receptors and ligands. Recent work suggests that several aspects of the system can lead to complex signaling behaviors: First, receptors and ligands interact in two distinct ways, inhibiting each other in the same cell (in cis) while productively interacting between cells (in trans) to signal. The ability of a cell to send or receive signals depends strongly on both types of interactions. Second, mammals have multiple types of receptors and ligands, which interact with different strengths, and are frequently co-expressed in natural systems. Third, the three mammalian Fringe proteins can modify receptor-ligand interaction strengths in distinct and ligand-specific ways. Consequently, cells can exhibit non-intuitive signaling states even with relatively few components.

In order to understand what signaling states occur in natural processes, and what types of signaling behaviors they enable, this thesis puts forward a quantitative and predictive model of how the Notch signaling state is determined by the expression levels of receptors, ligands, and Fringe proteins. To specify the parameters of the model, we constructed a set of cell lines that allow control of ligand and Fringe expression level, and readout of the resulting Notch activity. We subjected these cell lines to an assay to quantitatively assess the levels of Notch ligands and receptors on the surface of individual cells. We further analyzed the dependence of these interactions on the level and type of Fringe expression. We developed a mathematical modeling framework that uses these data to predict the signaling states of individual cells from component expression levels. These methods allow us to reconstitute and analyze a diverse set of Notch signaling configurations from the bottom up, and provide a comprehensive view of the signaling repertoire of this major signaling pathway.

Sequence specific recognition and cleavage of DNA by synthetic molecules

DNA recognition is an essential biological process responsible for the regulation of cellular functions including protein synthesis and cell division and is implicated in the mechanism of action of some anticancer drugs. Studies directed towards defining the elements responsible for sequence specific DNA recognition through the study of the interactions of synthetic organic ligands with DNA are described.

DNA recognition by poly-N-methylpyrrolecarboxamides was studied by the synthesis and characterization of a series of molecules where the number of contiguous N-methylpyrrolecarboxamide units was increased from 2 to 9. The effect of this incremental change in structure on DNA recognition has been investigated at base pair resolution using affinity cleaving and MPE•Fe(II) footprinting techniques. These studies led to a quantitative relationship between the number of amides in the molecule and the DNA binding site size. This relationship is called the n + 1 rule and it states that a poly-N methylpyrrolecarboxamide molecule with n amides will bind n + 1 base pairs of DNA. This rule is consistent with a model where the carboxamides of these compounds form three center bridging hydrogen bonds between adjacent base pairs on opposite strands of the helix. The poly-N methylpyrrolecarboxamide recognition element was found to preferentially bind poly dA•poly dT stretches; however, both binding site selection and orientation were found to be affected by flanking sequences. Cleavage of large DNA is also described.

One approach towards the design of molecules that bind large sequences of double helical DNA sequence specifically is to couple DNA binding subunits of similar or diverse base pair specificity. Bis-EDTA-distamycin-fumaramide (BEDF) is an octaamide dimer of two tri-N methylpyrrolecarboxamide subunits linked by fumaramide. DNA recognition by BEDF was compared to P7E, an octaamide molecule containing seven consecutive pyrroles. These two compounds were found to recognize the same sites on pBR322 with approximately the same affinities demonstrating that fumaramide is an effective linking element for Nmethylpyrrolecarboxamide recognition subunits. Further studies involved the synthesis and characterization of a trimer of tetra-N-methylpyrrolecarboxamide subunits linked by β-alanine ((P4)_(3)E). This trimerization produced a molecule which is capable of recognizing 16 base pairs of A•T DNA, more than a turn and a half of the DNA helix.

DNA footprinting is a powerful direct method for determining the binding sites of proteins and small molecules on heterogeneous DNA. It was found that attachment of EDTA•Fe(II) to spermine creates a molecule, SE•Fe(II), which binds and cleaves DNA sequence neutrally. This lack of specificity provides evidence that at the nucleotide level polyamines recognize heterogeneous DNA independent of sequence and allows SE•Fe(II) to be used as a footprinting reagent. SE•Fe(II) was compared with two other small molecule footprinting reagents, EDTA•Fe(II) and MPE•Fe(II).

Analysis of temporal structure in spike trains of visual cortical area MT

The temporal structure of neuronal spike trains in the visual cortex can provide detailed information about the stimulus and about the neuronal implementation of visual processing. Spike trains recorded from the macaque motion area MT in previous studies (Newsome et al., 1989a; Britten et al., 1992; Zohary et al., 1994) are analyzed here in the context of the dynamic random dot stimulus which was used to evoke them. If the stimulus is incoherent, the spike trains can be highly modulated and precisely locked in time to the stimulus. In contrast, the coherent motion stimulus creates little or no temporal modulation and allows us to study patterns in the spike train that may be intrinsic to the cortical circuitry in area MT. Long gaps in the spike train evoked by the preferred direction motion stimulus are found, and they appear to be symmetrical to bursts in the response to the anti-preferred direction of motion. A novel cross-correlation technique is used to establish that the gaps are correlated between pairs of neurons. Temporal modulation is also found in psychophysical experiments using a modified stimulus. A model is made that can account for the temporal modulation in terms of the computational theory of biological image motion processing. A frequency domain analysis of the stimulus reveals that it contains a repeated power spectrum that may account for psychophysical and electrophysiological observations.

Some neurons tend to fire bursts of action potentials while others avoid burst firing. Using numerical and analytical models of spike trains as Poisson processes with the addition of refractory periods and bursting, we are able to account for peaks in the power spectrum near 40 Hz without assuming the existence of an underlying oscillatory signal. A preliminary examination of the local field potential reveals that stimulus-locked oscillation appears briefly at the beginning of the trial.

Engineering nucleic acid mechanisms for regulation and readout of gene expression : conditional Dicer substrate formation and sensitive multiplexed northern blots

Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.

RNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.

Signal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.

Antitumor activity of Py-Im polyamides

Molecules that inhibit DNA dependent processes are the most commonly used agents for the treatment of cancer. The genotoxicity associated with their mechanisms of action, unfortunately, make them extremely toxic to the patient and cancer cells alike. The work presented in this thesis outlines the development of Py-Im polyamides as non-genotoxic DNA-targeted antitumor molecules that interfere with RNA polymerase II elongation. We initially characterized the pharmacokinetic profiles of two hairpin polyamides to establish their bioavailability in the serum and tissues after a single administration. We next determined the molecular mechanism that contributes to toxicity of a hairpin polyamide in human prostate cancer cells in cell culture and we demonstrated antitumor effects of the compound against LNCaP xenografts in mice. Finally, we conducted animal toxicity experiments on 4 polyamides that vary on the gamma-turn with respect to the substitution of amino and acetamide groups at the alpha and beta positions. From this study we identified a second generation compound that retains antitumor activity with significantly reduce animal toxicity. This work sets the foundation for the development of Py-Im polyamides as DNA targeted therapeutics for the treatment of advanced prostate cancer.

Structural and mechanistic motifs in membrane proteins: the three-dimensional modelling of rhodopsin, band 3, and the nicotinic acetylcholine receptor

Because so little is known about the structure of membrane proteins, an attempt has been made in this work to develop techniques by which to model them in three dimensions. The procedures devised rely heavily upon the availability of several sequences of a given protein. The modelling procedure is composed of two parts. The first identifies transmembrane regions within the protein sequence on the basis of hydrophobicity, β-turn potential, and the presence of certain amino acid types, specifically, proline and basic residues. The second part of the procedure arranges these transmembrane helices within the bilayer based upon the evolutionary conservation of their residues. Conserved residues are oriented toward other helices and variable residues are positioned to face the surrounding lipids. Available structural information concerning the protein's helical arrangement, including the lengths of interhelical loops, is also taken into account. Rhodopsin, band 3, and the nicotinic acetylcholine receptor have all been modelled using this methodology, and mechanisms of action could be proposed based upon the resulting structures.

Specific residues in the rhodopsin and iodopsin sequences were identified, which may regulate the proteins' wavelength selectivities. A hinge-like motion of helices M3, M4, and M5 with respect to the rest of the protein was proposed to result in the activation of transducin, the G-protein associated with rhodopsin. A similar mechanism is also proposed for signal transduction by the muscarinic acetylcholine and β-adrenergic receptors.

The nicotinic acetylcholine receptor was modelled with four trans-membrane helices per subunit and with the five homologous M2 helices forming the cation channel. Putative channel-lining residues were identified and a mechanism of channel-opening based upon the concerted, tangential rotation of the M2 helices was proposed.

Band 3, the anion exchange protein found in the erythrocyte membrane, was modelled with 14 transmembrane helices. In general the pathway of anion transport can be viewed as a channel composed of six helices that contains a single hydrophobic restriction. This hydrophobic region will not allow the passage of charged species, unless they are part of an ion-pair. An arginine residue located near this restriction is proposed to be responsible for anion transport. When ion-paired with a transportable anion it rotates across the barrier and releases the anion on the other side of the membrane. A similar process returns it to its original position. This proposed mechanism, based on the three-dimensional model, can account for the passive, electroneutral, anion exchange observed for band 3. Dianions can be transported through a similar mechanism with the additional participation of a histidine residue. Both residues are located on M10.

Cell-targeted regulation of gene expression through synthetic RNA devices

The ability to interface with and program cellular function remains a challenging research frontier in biotechnology. Although the emerging field of synthetic biology has recently generated a variety of gene-regulatory strategies based on synthetic RNA molecules, few strategies exist through which to control such regulatory effects in response to specific exogenous or endogenous molecular signals. Here, we present the development of an engineered RNA-based device platform to detect and act on endogenous protein signals, linking these signals to the regulation of genes and thus cellular function.

We describe efforts to develop an RNA-based device framework for regulating endogenous genes in human cells. Previously developed RNA control devices have demonstrated programmable ligand-responsive genetic regulation in diverse cell types, and we attempted to adapt this class of cis-acting control elements to function in trans. We divided the device into two strands that reconstitute activity upon hybridization. Device function was optimized using an in vivo model system, and we found that device sequence is not as flexible as previously reported. After verifying the in vitro activity of our optimized design, we attempted to establish gene regulation in a human cell line using additional elements to direct device stability, structure, and localization. The significant limitations of our platform prevented endogenous gene regulation.

We next describe the development of a protein-responsive RNA-based regulatory platform. Employing various design strategies, we demonstrated functional devices that both up- and downregulate gene expression in response to a heterologous protein in a human cell line. The activity of our platform exceeded that of a similar, small-molecule-responsive platform. We demonstrated the ability of our devices to respond to both cytoplasmic- and nuclear-localized protein, providing insight into the mechanism of action and distinguishing our platform from previously described devices with more restrictive ligand localization requirements. Finally, we demonstrated the versatility of our device platform by developing a regulatory device that responds to an endogenous signaling protein.

The foundational tool we present here possesses unique advantages over previously described RNA-based gene-regulatory platforms. This genetically encoded technology may find future applications in the development of more effective diagnostic tools and targeted molecular therapy strategies.

Effects of TI-299423 on Neuronal Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric, ligand-gated, cation channels found throughout the central and peripheral nervous system, whose endogenous ligand is acetylcholine, but which can also be acted upon by nicotine. The subunit compositions of nAChR determine their physiological and pharmacological properties, with different subunits expressed in different combinations or areas throughout the brain. The behavioral and physiological effects of nicotine are elicited by its agonistic and desensitizing actions selectively on neuronal nAChRs. The midbrain is of particular interest due to its population of nAChRs expressed on dopaminergic neurons, which are important for reward and reinforcement, and possibly contribute to nicotine dependence. The α6-subunit is found on dopaminergic neurons but very few other regions of the brain, making it an interesting drug target. We assayed a novel nicotinic agonist, called TI-299423 or TC299, for its possible selectivity for α6-containing nAChRs. Our goal was to isolate the role of α6-containing nAChRs in nicotine reward and reinforcement, and provide insight into the search for more effective smoking cessation compounds. This was done using a variety of in vitro and behavioral assays, aimed dually at understanding TI-299423’s exact mechanism of action and its downstream effects. Additionally, we looked at the effects of another compound, menthol, on nicotine reward. Understanding how reward is generated in the cholinergic system and how that is modulated by other compounds contributes to a better understand of our complex neural circuitry and provides insight for the future development of therapeutics.

Organization and function of the phage Lambda chromosome

The process of prophage integration by phage λ and the function and structure of the chromosomal elements required for λ integration have been studied with the use of λ deletion mutants. Since att^φ, the substrate of the integration enzymes, is not essential for λ growth, and since att^φ resides in a portion of the λ chromosome which is not necessary for vegetative growth, viable λ deletion mutants were isolated and examined to dissect the structure of att^φ.

Deletion mutants were selected from wild type populations by treating the phage under conditions where phage are inactivated at a rate dependent on the DNA content of the particles. A number of deletion mutants were obtained in this way, and many of these mutants proved to have defects in integration. These defects were defined by analyzing the properties of Int-promoted recombination in these att mutants.

The types of mutants found and their properties indicated that att^φ has three components: a cross-over point which is bordered on either side by recognition elements whose sequence is specifically required for normal integration. The interactions of the recognition elements in Int-promoted recombination between att mutants was examined and proved to be quite complex. In general, however, it appears that the λ integration system can function with a diverse array of mutant att sites.

The structure of att^φ was examined by comparing the genetic properties of various att mutants with their location in the λ chromosome. To map these mutants, the techniques of heteroduplex DNA formation and electron microscopy were employed. It was found that integration cross-overs occur at only one point in att^φ and that the recognition sequences that direct the integration enzymes to their site of action are quite small, less than 2000 nucleotides each. Furthermore, no base pair homology was detected between att^φ and its bacterial analog, att^B. This result clearly demonstrates that λ integration can occur between chromosomes which have little, if any, homology. In this respect, λ integration is unique as a system of recombination since most forms of generalized recombination require extensive base pair homology.

An additional study on the genetic and physical distances in the left arm of the λ genome was described. Here, a large number of conditional lethal nonsense mutants were isolated and mapped, and a genetic map of the entire left arm, comprising a total of 18 genes, was constructed. Four of these genes were discovered in this study. A series of λdg transducing phages was mapped by heteroduplex electron microscopy and the relationship between physical and genetic distances in the left arm was determined. The results indicate that recombination frequency in the left arm is an accurate reflection of physical distances, and moreover, there do not appear to be any undiscovered genes in this segment of the genome.

The binding and catalytic properties of lysozyme

The binding and catalytic properties of hen's egg white lysozyme have been studied by a variety of techniques. These studies show that the enzyme has three contiguous binding subsites, A, B, and C. The application of nuclear magnetic resonance (NMR) spectroscopy to probe the binding environment of several saccharides to lysozyme has demonstrated that the reducing end sugar rings of chitotriose, chitobiose and the β-form of N-acetylglucosamine all bind in subsite C. The central sugar ring of chitotriose and the sugar ring at the nonreducing end of chitobiose were found to bind in subsite B, while the nonreducing end sugar residue of chitotriose occupied subsite A. The dynamics of the binding process has also been investigated by NMR. The formation rate constant of chitobiose--and chitotriose-enzyme complexes were found to be about 4 X 10^-6 M^-1 sec^-1 with small activation energies.

The stereochemical path of the lysozyme catalyzed hydrolysis of glycosidic bonds has been shown to proceed with at least 99.7% retention of configuration at C-1 of the sugar. The lysozyme catalyzed hydrolysis of glucosidic bonds has been shown to be largely carbonium ion in character by virtue of the α-deuterium kinetic isotope effect (k_H/k_D = 1.11) observed for the reaction. It is probable that the mechanism of action of the enzyme involves a carbonium ion intermediate which is stereospecifically quenched by solvent. However, acetamido group participation cannot be ruled out for natural substrates.

Circadian clock mutants of Drosophila melanogaster

Three mutants of Drosophila melanogaster have been isolated in which the free-running period of the circadian eclosion rhythm and the adult locomotor activity rhythm is affected. One mutant is arrhythmic, another has a short period of 19 hours, and the third has a long period of 28 hours. The mutants retain their phenotypes over the temperature range 18° to 25° C. All three mutants map near the tip of the X chromosome (distal to the centromere). By deficiency mapping, the short-period mutation has been localized to the 3B1-2 region. Complementation tests show that all three mutations affect the same functional gene.

Analysis of activity rhythms of individual mosaic flies indicates that the site of action of the short-period mutation is probably located in the head of the fly. A few activity patterns of split-head and mixed-head mosaics appear to possess both mutant and heterozygous components, suggesting that the fly head may contain two complete clocks capable of maintaining their periodicities independently.

The short-period mutation affects both the duration of the light-insensitive part of the oscillation and the degree to which the clock can be reset during the light-sensitive part of the oscillation.

Both the short-period and long-period mutant eclosion rhythms can be entrained to a period of 24 hours by a 12:12 light-dark cycle having a light intensity at least two orders of magnitude greater than that required to entrain the normal rhythm. The arrhythmic mutant does not entrain under these conditions. In the presence of a temperature cycle, however, the arrhythmic mutant does entrain, but its rhythm damps out when the temperature cycle is removed.

Evidence is presented that Pittendrigh's two-oscillator model for the clock in D. pseudoobscura applies to D. melanogaster as well. The three clock mutations primarily affect the light- sensitive driving oscillator. The arrhythmic mutation appears to have eliminated the driving oscillator while leaving the temperature-sensitive driven oscillator relatively intact.