The ability of SV40 to activate the DNA damage response raises the question of whether the virus also induces DNA damage. Broken nuclear DNA in SV40-infected BSC40 cells could be detected at 5 hours post infection (hpi) using Comet and TUNEL assays. DNA breaks were not detected during infections of non-permissive cells. Agents that inhibit intracellular trafficking of SV40 to the endoplasmic reticulum (ER), or redox processes required for uncoating in the ER, prevented DNA breaks. An increase in phosphorylated histone H2AX (γH2AX), a marker of DNA damage signaling, was detected at 5 hpi and the number of γH2AX-positive cells continued to increase during the infection. A greater percentage of γH2AX positive cells were found with increasing viral multiplicity of infection. To test if viral gene expression is required for DNA breaks, SV40 was UV inactivated to prevent early gene expression, without affecting virus uptake or uncoating. Cells infected with UV-inactivated SV40 displayed γH2AX and DNA breaks at a level comparable to that induced by mock-irradiated virus. Because SV40 is uncoated in the ER during entry, we hypothesized that ER stress, disruption of Ca+2 homeostasis, and reactive oxygen species (ROS) could lead to DNA breaks. Consistent with this notion, the antioxidant N-acetyl cysteine (NAC) prevented nuclear DNA fragmentation, but not DNA damage signaling. Future work will clarify how SV40 entry generates host DNA breaks.

Dendritic spines are small actin-rich structures that receive the majority of excitatory synaptic input in the brain. The actin-based dynamics of spines are thought to mediate synaptic plasticity, which underlies cognitive processes such as learning and memory. However, little is known about the molecular mechanisms that regulate actin dynamics in spines and synapses. In this study, we show that the multifunctional actin-binding protein VASP regulates the density, size, and morphology of dendritic spines by inducing actin assembly in these structures. Knockdown of endogenous VASP by siRNA led to a significant decrease in the density of spines and synapses, while expression of siRNA-resistant VASP rescued this defect. The ability of VASP to modulate spine and synapse formation, maturation, and spine head enlargement is dependent on its actin-binding Ena/VASP homology 2 (EVH2) domain and its EVH1 domain, which contributes to VASP localization to actin-rich structures. Moreover, VASP increases the amount of PSD scaffolding proteins and the number of surface GluR1-containing a-amino-3-hydroxy-5-methyl-4- isoxazole propionic acid (AMPA) receptors in spines. VASP knockdown results in a reduction in surface AMPA receptor density, suggesting a role for this protein in regulating synaptic strength. Consistent with this, VASP significantly enhances the retention of GluR1 in spines as determined by fluorescence recovery after photobleaching (FRAP) and increases AMPA receptors-mediated synaptic transmission. Collectively, our results suggest that actin polymerization and bundling by VASP are critical for spine formation, spine expansion, and the modulation of synaptic strength.

BIOLOGICAL SCIENCES KCTD12 PROTEINS REGULATE ULK2 TO CONTROL THE DEVELOPMENT OF ASYMMETRIC HABENULAR NEUROPIL ROBERT W. TAYLOR Dissertation under the direction of Professor Joshua T. Gamse The habenular nuclei (Hb) are part of an ancient conduction pathway that controls diverse behaviors. In zebrafish, the Hb develop robust asymmetries including connectivity, morphology, and gene expression patterns. One striking example of asymmetric gene expression is the predominantly left-sided expression of kctd12.1 and the right-sided kctd12.2. Though used extensively as markers for Hb sidedness, there is a paucity of information relating to the function of Kctd12 proteins during zebrafish Hb development. We have used a cross-species yeast 2-hybrid approach to identify Unc-51-like Kinase 2 (Ulk2) as a novel Kctd12.1 interactor. We then asses the consequences of genetic manipulation of both Ulk2 and Kctd12 proteins on Hb development. We have discovered a system by which Ulk2 activity promotes the elaboration of Hb neuropil, and this activity is negatively regulated by Kctd12 proteins, leading to proper neuropil development, and perhaps shaping the asymmetric morphology of the zebrafish Hb.

The work presented in this dissertation focuses on a how the telomerase complex assembles both in vivo and in vitro and begins to explore how one member of the telomerase enzyme in the budding yeast, Saccharomyces cerevisiae, termed Est3p, functions within the complex. I have discovered that Est1 protein levels are lower in G1 phase of the cell cycle due to regulated degradation by the ubiquitin-proteasome system. Additionally, I have elucidated a direct protein-protein interaction between the TEN domain of the Est2 protein and a charged region of the Est3 protein. Finally, I have begun to uncover a potential function of the Est3 protein in stimulating or activating telomerase activity in vitro. This activity appears to be dependent upon stable interaction with the Est2 TEN domain.

This project explores the synthesis of heterogeneous G1-3T yeast telomere sequences by the enzyme telomerase. I demonstrate that a mutant in the essential N-terminal TEN domain of Est2p (telomerase reverse transciptase), glutamic acid 76 to lysine (est2-LTE76K), restricted possible alignments between the DNA primer and the RNA template and increased in vivo processivity. Within the context of telomerase, the Est2p TEN domain is thought to contribute to enzyme processivity by mediating an anchor-site interaction with the DNA primer. I showed that binding of the purified TEN domain (residues 1-161) to telomeric DNA is enhanced by the E76K mutation. These results suggest a novel role for the anchor-site in mediating primer/template alignment within the active site of yeast telomerase.

Hemi-gap-junction (HGJ) channels composed of connexin (Cx) proteins are proposed as the key component to mediate the negative feedback pathway from horizontal cells (HCs) to photoreceptors in the outer retina. The goal of the research undertaken in this dissertation was to test whether HGJ channels in retinal HCs could serve their proposed roles in the negative feedback pathway. First, the biophysical properties of HGJ channels and their modulation by the neuromodulator zinc using electrophysiological methods were characterized. Both outward and inward hemichannel currents were elicited in cultured solitary zebrafish retinal HCs. In particular, inward hemichannel currents elicited at negative potentials persisted under physiological conditions and satisfy the requirement of ephaptic communication in the feedback pathway. In addition, to uncover the molecular basis of the HGJ currents, the function and properties of various Cx proteins were studied using morpholino-based gene knockdown and a cx55.5 mutant zebrafish. The data suggest that inward hemichannel currents are solely dependent on the expression of Cx55.5, while outward hemichannel currents are dependent by both Cx55.5 and Cx52.6. In summary, this study expands our understanding in the properties and function of HGJ channels and the mechanisms underlying neuronal network adaptation in retinal circuitry.

BIOLOGICAL SCIENCES THE RHO FAMILY GEF ASEF2 REGULATES ADHESION DYNAMICS AND THEREFORE CELL MIGRATION BY MODULATING RAC AND RHO ACTIVITY JEANNE MALLOY BRISTOW Dissertation under the direction of Assistant Professor Donna J. Webb The Rho family of small GTPases, including Rac, Cdc42, and Rho, are key regulators of cell migration and its underlying processes. By modulating the actin cytoskeleton, these GTPases control cell polarization, leading edge protrusion, and cell-matrix adhesion turnover. GTPases act as molecular switches whose activity is tightly regulated by their bound guanine nucleotide state. Guanine nucleotide exchange factors (GEFs) tightly regulate GTPase activity by facilitating the exchange of bound GDP for GTP. Asef2 is a recently identified Rho family GEF that has been shown to activate Rac and Cdc42. However, its function as a regulator of cell migration and adhesion dynamics is poorly understood. Immunofluorescence microscopy revealed that Asef2 localizes with actin and Akt at the leading edge of migrating HT-1080 human fibrosarcoma cells. Our data indicate Asef2 activates Cdc42 and Rac and indirectly inhibits Rho through a Rac-mediated pathway. We have shown that Asef2 promotes migration and rapid adhesion turnover in a Rac-dependent manner. Further, Asef2-mediated random migration and adhesion turnover effects are dependent on a previously unknown mechanism requiring Asef2-mediated Rac, PI3K, and Akt activation leading to Rho inhibition. Together, these data indicate a role for Asef2 as an important regulator of cell migration and adhesion dynamics as it coordinately regulates the activity of Rho family GTPases.

Ecological speciation is defined as the process by which reproductive isolation arises between populations as a result of ecologically based divergent selection between environments. Selection is ecological when individual survival and reproduction are dependent on interactions with its environment. Selection is divergent when it works in contrasting directions on specific traits between populations or individuals. I used populations of the leaf beetle <i>Neochlamisus bebbianae</i> associated with Bebbs willow (<i>Salix bebbianae</i>) and red maple (<i>Acer rubrum</i>) that are currently undergoing ecologically-driven divergence and speciation to address the role that ecology plays in generating reproductive isolation and speciation. These two host forms offer an excellent study system to answers questions regarding ecological speciation due to their geographic overlap and their divergence in habitat preferences, host-associated fitness, and host-associated assortative mating. This thesis involves two complimentary projects aimed at (a.) understanding the genetic architecture of traits associated with ecological adaptation to different hosts that contribute to reproductive isolation and (b.) examining the unknown nature of postmating isolation that has evolved between these ecologically differentiated populations. To address these projects I have used a single experimental design that employs controlled genetic crosses within and among populations of <i>N. bebbianae</i> adapted to each host plant over multiple geographic localities, multiple years and within one year, over multiple generations. In doing so, I was able to uncover and contrast the genetic basis for host use on each plant and the role of different postmating barriers that arise during the speciation process.

Telomeres are protective protein/DNA complexes that cap the ends of linear chromosomes. The repetitive, TG-rich telomeric sequences are elongated by the telomerase ribonucleoprotein. In Saccharomyces cerevisiae, the catalytic core of telomerase consists of the reverse transcriptase Est2p and the TLC1 RNA, which contains the template for nucleotide addition. The Est1 and Est3 proteins serve regulatory roles in vivo but are dispensable for in vitro telomerase activity. The mechanism through which Est3p assembles with the holoenzyme is debated within the field, specifically concerning Est1ps role in Est3p recruitment. Based on evidence demonstrating that Est1p is not absolutely required to recruit Est3p, I hypothesize that Est1p stimulates Est3p recruitment. To identify residues of the Est1 protein responsible for this function, I used a combination of in vivo and in vitro experiments and two genetic screens. Using an in vivo assembly assay, I determined that a putative Est3p recruitment domain lies between amino acids 499 and 563 of Est1p and optimized the protocol for analyzing point mutations within this region. Additionally, I have designed two genetic screens to isolate alleles of EST1 specifically disrupted for stimulating Est3p recruitment. The first uses an Est2-Est3 fusion protein predicted to bypass only the Est3p recruitment function of Est1p. The second utilizes a galactose-inducible allele of EST3 to screen for Est1p mutants rescued by over-expression of Est3p. Identification of separation-of-function alleles of EST1 with these tools will clarify the precise mechanisms of yeast telomerase complex assembly and potentially provide insight into the interactions of human telomerase components.

Cell migration is a complex process that requires the coordination of signaling events that take place in distinct locations within the cell. Adaptor proteins are emerging as key modulators of spatially integrated processes because of their ability to localize to different subcellular compartments and bring together important signaling proteins at these sites. However, the role that adaptor proteins play in regulating cell migration is not well understood. Here, we show a novel function for the adaptor protein containing a pleckstrin homology (PH), phosphotyrosine binding (PTB), and leucine zipper motif 1 (APPL1) in modulating cell migration. APPL1 impairs the turnover of adhesions at the leading edge of cells thereby inhibiting their migration. The ability of APPL1 to impair migration is dependent on its PTB domain, which interacts with Akt, suggesting the interaction of APPL1 with Akt is important for its effect on migration. Interestingly, APPL1 decreases the amount of active Akt in cells. We show that APPL1 modulates migration and adhesion dynamics via a mechanism that involves regulation of Akt function. Src has been shown to regulate Akt function through the phosphorylation of two Akt tyrosine residues, and intriguingly, we have found that APPL1 reduces Src-mediated tyrosine phosphorylation of Akt. Therefore, we propose a model in which APPL1 regulates adhesion dynamics and cell migration by altering Src-mediated tyrosine phosphorylation of Akt. Our results further underscore the importance of adaptor proteins in modulating the flow of information through signaling pathways by demonstrating a critical new function for APPL1 in regulating cell migration and adhesion turnover. In addition, we provide the first comprehensive phosphorylation map of APPL1. Using mass spectrometry, we identify 13 phosphorylated residues within APPL1. Four of these sites are located in important functional domains; one within the BAR domain, two cluster near the edge of the PH domain, and one within the PTB domain. These phosphorylation sites may control APPL1 function by regulating the ability of APPL1 domains to interact with other proteins and membranes.