This dissertation examines the natural history, potential adaptations, and tradeoffs of fecal cases of Neochlamisus leaf beetles. Fecal cases begin as wrappings around eggs. Each larva maintains and expands its case throughout the juvenile stadia, eventually sealing it for pupation. Once an adult, it cuts a circular cap to escape. In the field, fecal cases are required for larval survival and lab studies showed that fecal material, plant hairs embedded in the external wall of the case, and plant hairs embedded in an internal chamber (i.e., trichome attic) individually reduce predation risk. Normally these plant hairs, or trichomes, are considered adaptations by plants to prevent herbivory. N. platani larvae, which incorporate trichomes into their cases to an extensive degree, can feed on pubescent, denuded, and naturally glabrous leaves equally. Unusually, they also seek out and spend more time on pubescent leaves when given a choice, perhaps co-opting the host's defense for their own protection. I found no evidence that fecal cases moderate the effects of or increase survival in low humidity. Rather, individuals that pupated in low humidity were incapable of escaping the case. This mortality appears to be due to the strengthening of case material when relative humidity was low, becoming too strong for adults to cut and emerge. Strong cases, however, also decrease the ability of predators to penetrate the case. Therefore, an ecological abiotic factor (i.e., relative humidity) causes a plastic response in the animal architecture that imparts a trade-off between normal adult emergence and preventing predation. In addition, I found evidence that fecal cases and larvae may be adapted to prevent overheating. Fecal cases slow the rise in internal temperatures and larvae move to the undersides of leaves and lift the case above the leaf surface when ambient temperatures are high. I also include an observation of a rare 'predation event' of a casebearer, i.e., eggs were incorporated into the bags of the evergreen bagworm. This is by far the most detailed study of this amazing example of animal architecture.

Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase α (pol α), with chromatin. However, the significance of these interactions, and the specific role of Mcm10 during replication initiation is unclear. To address this gap in knowledge, we have begun a structure/function analysis of Xenopus laevis Mcm10 (xMcm10). Chapter 2 describes Mcm10s domain structure which is composed of three structural and functional regions. The amino-terminal domain (NTD) forms a dimerization motif, while the internal (ID) and carboxy-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss)DNA and the catalytic p180 subunit of pol α. Chapter 3 describes the crystal structure determination of xMcm10-ID as well as NMR spectroscopy to map the binary interfaces between xMcm10-ID and ssDNA. In chapter 4, the mechanism by which Mcm10-ID interacts with pol α is investigated using X-ray crystallography, NMR spectroscopy, isothermal titration calorimetry, and fluorescence anisotropy. In addition, the manner in which the ID and CTD operate together to interact with DNA and pol α are investigated. Collectively, the results presented here provide the first mechanistic insight into how Mcm10 might use a hand-off mechanism to load and stabilize pol α within the replication fork. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and pol α, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.

During spinal cord development, precursor cells transition from proliferative divisions to differentiative divisions. Traditionally proliferative divisions, which increase cell numbers, are thought to be symmetric, whereas differentiative divisions are thought to occur both by symmetric and asymmetric divisions. Currently, the mechanisms that control this differentiative cell division fate remain to be defined. However, studies conducted using atypical protein kinase C (aPKC) suggests that aPKC has a conserved function in controlling cell division orientation. In this study, we look at the role aPKC may play in maintaining precursor division in the zebrafish spinal cord. Through time-lapse imaging and loss of function studies we were able to show that aPKC does regulate precursor division in the zebrafish spinal cord, and in its absence excess oligodendrocyte precursor cells (OPCs) are specified at the possible expense of adult precursors.

The yeast respiratory oscillation is a 3 to 5 hour biological rhythm in some strains of Saccharomyces cerevisiae that occurs under a specific range of growth conditions during continuous culture. The cell division cycle, in addition to transcription of many genes, oscillates along with the yeast respiratory oscillation. In this work luciferase reporters were constructed for yeast that provided automated real-time luminescent evidence of cell division synchrony and rhythmic transcriptional regulation in vivo during the yeast respiratory oscillation. This non-invasive, non-destructive luminescent system for monitoring gene activity was used to show an interrelationship between the yeast respiratory oscillation and the cell division cycle. This work also showed that visible light at an intensity of less than one tenth that of full sunlight (primarily in the blue and green wavelengths) noticeably affected the amplitude and period of the yeast respiratory oscillation by interfering with photosensitive substances required for respiration. <p> This dissertation constitutes a series of steps within a larger quest to fully understand the nature of biological rhythms in yeast. In addition to investigating the relationship between respiratory oscillations and cell division, other questions that motivated this research were, Is the respiratory oscillation evidence of an endogenous biological clock? and Does this yeast biological rhythm shown in continuous culture exist in nature? To pursue answers to these and related questions, a number of techniques and investigations involving the production and perception of light were used. This dissertation revolves around the use of light for exploring the biology of yeast; from developing bioluminescent yeast that report gene activity, to studying effects of visible light on yeast respiration and growth, to developing a low cost fluorescent excitation light source for use in microscopy.

Prostaglandin E2 (PGE2) influences many processes in vertebrates, including development, homeostasis, and disease through its GPCRs EP receptors 1-4. PGE2 regulates gastrulation movements during zebrafish embryogenesis, but how it does so was previously unclear, as PGE2 can affect cell adhesion, motility, proliferation, and survival. Our studies reveal that the loss of PGE2 synthesis impairs all gastrulation movements, epiboly, internalization, convergence, and extension, in part due to increased cell adhesion in the embryo. The increase of tight junctions (ZO1) and adherens junctions (E-cadherin) occurs in a germ layer-dependent fashion. In the mesendoderm, PGE2 modulates E-cadherin by stabilizing Snail through the inhibition of Gsk3β by a novel interaction with the Gβγ subunits (in collaboration with K. Jernigan and E. Lee). Moreover, the reduction of PGE2 synthesis results in an endoderm deficiency without significant effect on the mesoderm, possibly due to decreased Nodal signaling. Finally, we present preliminary characterization of a fish harboring a reverse genetics TILLING-generated ep4a nonsense mutation that strongly depletes function of the gene, but manifests no apparent phenotype. In conclusion, our findings suggest that PGE2 signaling can coordinate cell fate specification and movement, in part through its negative regulation of cell adhesion in zebrafish gastrulae.

Dissertation under the direction of Professor Todd R. Graham Drs2 is a type IV P-type ATPase that catalyzes a phospholipid flippase activity in the trans-Golgi network (TGN) in Saccharomyces cerevisiae. Strains harboring a DRS2 null allele (drs2∆) are viable, but exhibit a strong cold-sensitive growth defect. Under this dissertation, a drs2∆ bypass suppressor screen was completed to better understand the mechanism underlying the cold-sensitive growth defect of drs2∆. SDK1 (suppressor of drs2 knockout 1), was cloned and shown to be identical to KES1, an oxysterol binding protein. Genetic studies suggested that Kes1p represses the activity of Drs2p and the closely related P4-ATPases. kes1∆ suppression of the drs2∆ cold-sensitive growth defect requires the closely related Dnf P4-ATPases. Indeed, a flippase assay performed with TGN membranes purified from kes1∆ cells revealed a two-fold increase in Drs2 activity. Addition of recombinant Kes1 repressed Drs2 activity back to wild type levels. Surprisingly, Genetic analyses suggested that Kes1 is hyperactive in drs2∆ cells and is particularly toxic to drs2∆ cells blocked in late stages of ergosterol synthesis. Indeed, the influence of Kes1 on intracellular sterol transport was dramatically enhanced in drs2∆ cells. Thus, the presence of wild-type Drs2 represses Kes1 activity, providing the first evidence for an antagonist relationship between a P4-ATPase and oxysterol binding OSH protein. Drs2 is required for budding of a specific class of exocytic vesicles from the TGN and for AP1/clathrin coated vesicle trafficking between the TGN and early endosome. In contrast, Drs2 and Dnfs act redundantly in transporting proteins from the TGN to the late endosome and vacuole. In the absence of Drs2p, Dnfs can support transport to the late endosome at 30C but not 15C. Deletion of KES1 suppresses the cold-sensitive TGN to late endosome transport defect. These findings lead to a conclusion that the cold-sensitive growth defect of drs2∆ is caused by simultaneous trafficking defects in the TGN to early endosome and TGN to late endosme pathways at low temperature. Moreover, P4-ATPases are a downstream target of the Kes1 repressive effect on vesicular transport.

Type-IV P-type ATPases are putative phospholipid flippases that translocate specific phospholipid substrates from the exofacial to the cytosolic leaflet of membranes to generate phospholipid asymmetry. However, flippase activity has not been reconstituted with any purified type-IV P-type ATPase, and so whether these ATPases directly pump phospholipid across the membrane bilayer is unknown. The major goal of this dissertation project is to test whether Drs2p, a type-IV P-type ATPase from yeast Saccharomyces cerevisiae, is a flippase or not. I show that Drs2p can directly catalyze phospholipid translocation through purification and reconstitution of this ATPase into artificial membranes. This flippase activity is specific to a phosphatidylserine analogue and requires active Drs2p. My data also suggest that only a portion of Drs2p molecules are active after purification and these active molecules may result from partial proteolysis within the carboxyl terminus of Drs2p. This observation is consistent with a model that the carboxyl tail is auto-inhibitory to Drs2p activity. In this study, I demonstrate for the first time the reconstitution of flippase activity with a purified type-IV P-type ATPase.

Human replication protein A (RPA) is a single-stranded DNA (ssDNA) binding protein involved in DNA metabolism. RPA binds ssDNA transiently during initiation of DNA replication. When this dissertation research began, the mechanisms of RPA loading and displacement were not known. Two SV40 T antigen-binding sites on RPA, DNA binding domains A and B of RPA70 (RPA70AB) and C-terminus of RPA32 (RPA32C) have been defined. The origin DNA-binding domain (OBD) of T antigen binds to both sites. Physical interaction between T antigen OBD and RPA70AB was required for the loading of RPA onto ssDNA during initiation of SV40 DNA replication. T antigen formed a ternary complex with RPA and 8-mer ssDNA, but was released from RPA-ssDNA complex when longer ssDNA was available. Thus the ternary complex is a key intermediate for RPA loading. Although RPA32C is not involved in this process, it is crucial for RPA displacement from ssDNA by primosome activity. A charge reversal mutant of RPA32C showed reduced binding affinity for T antigen OBD. The same mutation introduced into intact RPA impaired initiation of replication and primosome activity. Based on these results, a dynamic model of RPA function in the initiation of SV40 DNA replication is proposed, in which successive protein-mediated remodeling of RPA facilitates its binding to ssDNA during origin DNA unwinding and its dissociation from ssDNA upon completion of primer synthesis. Topoisomerase IIβ binding protein 1 (TopBP1), which stimulates SV40 DNA replication, was found to physically interact with T antigen and DNA polymerase α-primase (pol-prim). In this dissertation, Chapter I presents an introduction to SV40 DNA replication as a model system. Chapters II and III are present two publications in which my major research results are included. Chapter IV is a summary and discussion of the functional dynamics of RPA in viral DNA replication. The appendix includes unpublished evidence of TopBP1 interaction with T antigen and pol-prim, which may participate in SV40 DNA replication in infected cells.

DNA damaging agents have been shown to stimulate the localization of human DNA helicase B (HDHB) in nuclear foci, suggesting that HDHB might participate in DNA damage response. In the first part of this dissertation, we found that the chromatin-associated fraction of HDHB increases in cells exposed to a variety of DNA damaging agents. HDHB chromatin accumulation is most prominent in S phase cells exposed to agents that cause replication fork stalling or collapse. Inhibition of checkpoint kinases does not prevent damage-induced accumulation of HDHB on chromatin, suggesting that HDHB associates directly with DNA lesions or with other proteins recruited to lesions. Silencing of HDHB does not affect UV-induced RPA focus formation, but diminishes induction of TopBP1 foci. DNA damage induced CHK1 phosphorylation is impaired in HDHB-depleted cells. These results identify HDHB as a novel factor that associates with damaged chromatin and promotes intra-S phase damage responses. In the second part of this dissertation, we further investigated the possible function of HDHB in homologous recombination. HDHB-depleted cells showed more aphidicolin-induced chromosome breaks than control-depleted cells. HDHB-depleted cells have fewer sister chromatid exchange than control-depleted cells. An in vivo recombination assay showed that HDHB silencing results in impaired homologous recombination. In vitro, recombinant HDHB stimulates Rad51-mediated 5-3 heteroduplex extension. Our studies suggest a function of HDHB in stimulating ssDNA/duplex structure during homologous recombination.

In recent years it has become apparent that microRNAs (miRNAs) are common feature of eukaryotic genomes. The functional products of these non-canonical genes are small, ~22nt RNAs that negatively regulate translation. Targeting of mRNAs occurs when a miRNA base pairs with complementary sequence elements located in the 3 untranslated region of mRNAs. Currently hundreds of miRNA species have been described. However, due to the imprecise pairing between miRNAs and their targets, the functional role of most miRNAs remains unknown. We approached this problem in zebrafish by using a combination of phenotypic analysis and reporter constructs. We determined that miR-214 targets su(fu), a regulator of hedgehog signaling, and that this is critical for appropriate muscle cell specification during somitogenesis. Additionally, we show that the miR-8 family of miRNAs targets a regulator of membrane trafficking, nherf1. This regulation is a critical component of the osmotic stress response. Together, we reveal diverse roles for miRNAs in zebrafish, and demonstrated the feasibility of using this model system to understand miRNA function.