Intersections of distinct biological pathways in cells allow for nodes of metabolic regulation. This work describes the discovery of the intersection of two pathways in yeast mitochondria: RNA processing and fatty acid synthesis and attachment. Analysis of the components of the pathways is presented here along with a model illustrating the connection as a potential mode of regulation of mitochondrial gene expression.A genome-wide screen of respiratory-deficient <italic>Saccharomyces cerevisiae</italic> deletion strains for defects in mitochondrial RNA processing revealed that two novel genes affect processing of mitochondrial tRNAs by RNase P. One gene encodes Htd2, an enzyme in the type II mitochondrial fatty acid synthesis pathway (FAS II). The other gene is described here as encoding Lip3, an enzyme involved in the synthesis and attachment of the co-factor lipoic acid, which is synthesized from a product of the FAS II pathway.RPM1 is the mitochondrial-encoded RNA subunit of mitochondrial RNase P. The multigenic transcription unit containing RPM1 also contains tRNA<super>pro</super>. Maturation of RPM1 necessitates processing of the tRNA by RNase P. Thus, RNase P is required for maturation of its own RNA component, constituting a positive feedback cycle. The present work demonstrates that a product of the FAS II pathway is necessary for the assembly or activity of RNase P, as deletion of any gene encoding an FAS II enzyme results in inefficient processing of tRNApro from the transcript.Analysis of the enzymes involved in the synthesis and attachment of lipoic acid to target proteins is also described here. Disruption of any of these enzymes affects protein lipoylation and tRNA processing. Gcv3, a target of lipoylation, was found to be required for lipoylation as well as for efficient tRNA processing.A second feedback cycle controlling pyruvate dehydrogenase activity and fatty acid synthesis may be functional under certain conditions. Pyruvate dehydrogenase, which provides acetyl-CoA for the FAS II pathway, requires lipoic acid for its activity. It is hypothesized that the two feedback cycles and the role of Gcv3 may provide switch-like regulation of mitochondrial gene expression in response to the nutritional state of the cell.

The experiments reported here describe mechanisms involved in the establishment of the dorsal-ventral axis in the mud snail, Ilyanassa obsoleta. Ilyanassa and other spiralians utilize an embryonic organizer to induce dorsal identity, and thus establish the bilateral axis. The D macromere embryonic organizer in Ilyanassa is specified at the four-cell stage by the inheritance of the polar lobe, but does not function as an inductive center until the 24-cell stage. Previously it was assumed that the D macromere of Ilyanassa functioned autonomously through its inheritance of the polar lobe. I have found this is not the case. Rather, I describe the role that the micromeres play in the activation of the D macromere organizer. Specifically, I have found that micromeres of the first and second quartet are necessary for at least three known characteristics of the D macromere: the activation of MAPK in the D macromere, the division of the D macromere, and the inductive capacity of the D macromere. Thus, while the polar lobe is necessary for D macromere function, its inheritance does not provide the D macromere with functional autonomy.The localized activation of MAPK was the first molecular component of dorsal-ventral patterning to be identified in Ilyanassa and other spiralians. In addition to being activated in the D macromere organizer, MAPK is also activated in the micromeres that are induced by the D macromere. I undertook a pharmacological screen to identify other components involved in dorsal-ventral patterning. I have found that a member of the Protein Kinase C (PKC) family is also involved in the establishment of the dorsal-ventral axis in Ilyanassa. Inhibition of PKC disrupts patterning, resulting in a radialized animal. In addition, I have found that PKC functions in the same path as MAPK. PKC is necessary for the proper activation of MAPK in the D macromere organizer and the micromeres. These results suggest that either the same transduction pathway is used repeatedly in the establishment of the dorsal-ventral axis or that patterning is the result of one global signal. These results drastically change our view of dorsal-ventral patterning during spiralian development.

The polyamines positively charged molecules that are able to affect nucleic acid structure. Investigation of polyamine and RNA interaction shows that the two are able to form aggregates and these aggregates may be responsible for the inhibition of RNA decapping that is seen in the presence of polyamines. The polyamines are also responsible for a post-translational modification of a protein, eukaryotic initiation factor 5a (eIF5A). This protein is known to affect translation and RNA decay. This investigation shows that eIF5A is able to affect the formation of foci by Dhh1 but does not affect DCP1 or DCP2 foci formation. This investigation also shows that eIF5A mutation leads to a depression of polysome profiles and 35S-met incorporation and therefore eIF5A may truly be a translation initiation factor. This study also describes unique interactions of eIF5A with Dhh1p and eIF5A-independent effects of the polyamines on gene expression. The inhibition of eIF5A's hypusine modification leads to an increase in phoshorylation of eIF2&amp;#945; and this may contribute the induction of apoptosis and inhibition of protein synthesis associated with inhibition of eIF5A's hypusine modification.

Long-distance signaling is an important process in the development of Arabidopsis thaliana. A leucine-rich repeat receptor-like kinase (LRR-RLK), XYLEM INTERMIXED WITH PHLOEM1 a.k.a. C-TERMINALLY ENCODED PEPTIDE RECEPTOR 1 (XIP1/CEPR1), functions in vascular development and has recently been implicated in nitrogen sensing and response. Previous results indicate that XIP1/CEPR1 also interacts with multiple proteins involved in sugar metabolism and transport as well as other metabolic proteins, which indicates a possible role for XIP1/CEPR1 in mediating sugar transport. xip1-1 seeds, which grow slowly in the cold in comparison to Columbia wild-type plants, were previously EMS mutagenized and screened for suppressors of the cold-sensitive phenotype. One of these suppressors, 9-12, maps to the lower region of chromosome V and several possible causative EMS-like mutations have been identified that may link XIP1/CEPR1 to a more general vascular transport role.

Each step of vascular development needs to be carefully regulated; endothelial precursors must be specified, these cells then proliferate and coalesce to form vascular cords, and finally they lumenate, undergo angiogenic branching and remodeling, and recruit smooth muscle cells to establish a mature vessel. An aberration at any of these steps during embryonic development is incompatible with life, and vascular pathologies in the adult are associated with numerous diseases including stroke, arteriosclerosis, diabetic retinopathies and cancer progression. My work has aimed to understand how endothelial precursors are specified, and more precisely the cell-signaling pathways and transcriptional networks that guide their fate. This work leads us to conclude the following: (1) blood island precursor cells in the Xenopus embryo can give rise to either blood or endothelial cells, and it is BMP-mediated activation of the erythroid transcriptional program that regulates cell fate, (2) endothelial specification requires the Ets transcription factor Etv2. Persistence of Etv2 expression in blood/endothelial cell precursors allows these cells to develop into endothelium, and overexpression of Etv2 in any of the three germ layers causes activation of every endothelial marker examined. Along the way we have characterized a number of small-molecule inhibitors that should be useful to the Xenopus community and applicable to other model systems.

The eyespot of the single-celled alga Chlamydomonas aids the cell in detecting the direction of light in the environment. The complex assembly and asymmetric placement of the eyespot provides a model to ask questions about assembly and asymmetric placement of organelles. Understanding the mechanisms that underlie assembly and asymmetric placement of the eyespot can be applied more broadly to their functions in other eukaryotic organisms. This study sought to understand the role of a key protein in those processes, Channelrhodopsin-1 (ChR1). ChR1 was found to localize along the entire length of the D4 rootlet from the region around the daughter basal body to the eyespot. ChR1 was found to primarily localize to the plasma membrane side of the D4, suggesting that ChR1 was being pulled through the plasma membrane from the region around the basal bodies to the eyespot. Further, ChR1 was found to be able to localize to the eyespot even with the truncation of the large cytoplasmic C-terminal domain, suggesting that ChR1 is able to complex with another protein that is being trafficked to the eyespot. One such protein was thought to be ChR2, the other light-activated ion channel localized to the eyespot. Efforts to isolate a mutation in ChR2 were unsuccessful. Initial efforts were made in this dissertation to perform proteomic studies of ChR1 and identify its interacting partners. ChR1 is not the master regulator of either placement or assembly of the eyespot, but work in this study lays the groundwork to further investigate transport of ChR1 and interacting proteins to the eyespot and their role in assembly of the eyespot.

Lysosomes are membrane-bound organelles that have diverse functions in eukaryotic cells. Malfunctions in lysosomes result in a range of diseases known as Lysosomal Storage Disorders. After fusing with late endosomes to form hybrid organelles, lysosomes bud off and are reformed in a poorly characterized process known as lysosome formation or reformation. Only one mammalian regulator of lysosome formation has been identified, the non-selective cation channel TRPML1. In the highly similar process of Autophagic Lysosome Reformation (ALR), three known regulators have also been identified, the vesicle-coating protein clathrin and two phosphatidylinositol kinases that catalyze the formation of the membrane phospholipid PI(4,5)P₂. Here, we use an inhibitor screen coupled with a live imaging assay to identify the actin microfilament as a novel regulator of lysosome formation.

Microtubule-based mitochondrial transport into dendrites and axons is vital for sustaining neuronal function. Transport along microtubules proceeds in a series of plus- and minus-end directed movements facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective long distance transport remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites. To directly examine Miro's significance for kinesin- and/or dynein-mediated mitochondrial motility, we live imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of either antero- or retrograde mitochondrial transport by selectively impairing kinesin- or dynein-mediated movements, depending on the direction of net transport. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We concluded that dMiro promotes effective antero- and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrion's programmed direction of transport. To determine how Miro achieves this control mechanistically, we introduced point mutations that render each GTPase either constitutively active or inactive. Expression of either first GTPase mutant impaired antero- (inactive) or retrograde motor movements (active) in a direction dependent manner. The active state of the second GTPase domain up-regulated the number of consecutive kinesin motions during anterograde transport but impaired kinesin transport biases while the inactive second GTPase state impaired transport in either direction. Together, these data suggest that Miro's first GTPase domain is major factor that controls the execution of either the antero- or retrograde directional program while Miro's second GTPase may provide a signal that supports or disfavors transport. In addition, the active state of the first and the second GTPase domain increased the length of stationary mitochondria but only the first GTPase domain modified motile mitochondrial lengths. Overexpression of these mutations generated opposing effects. We conclude that both domains control antero- and retrograde transport in a switch-like manner.

Receptor-like kinases (RLKs) are encoded for by one of the largest gene families in Arabidopsis and represent the predominant form of cell surface receptors in plants. RLKs mediate signal transduction in diverse processes including steroid-mediated growth pathways, pathogen-triggered innate immune responses. Here I present characterization of mutant phenotypes, expression patterns, and genetic interactions for the BAK1 INTERACTING RECEPTOR (BIR) family of Leucine-rich Repeat-RLKs, three members of which have had no previous characterization. Furthermore, I show that cell death, aerial growth, and lateral root development defects in bir1-1 are suppressed by mutations of the LRR-RLK co-receptor BRI1-ASSOCIATED KINASE 1 (BAK1); I identify a novel primary root growth phenotype in bir1-1 mutants, as well as a lateral root development phenotype for bir3 mutants; and primary root growth and aerial defects in bir3.bir4;bak1 triple mutants. Using an allelic series of bak1 mutations I show that bir phenotypes are dependent upon particular functions of BAK1, and propose that the BIR family exhibits a novel function, previously undescribed for LRR-RLKs, as regulators of co-receptor/ligand-binding receptor complex specificity.

During Arabidopsis embryogenesis, cell-cell signaling plays an essential role in establishing an organized body plan centered around two major axes of development: apical-basal and radial. Two topics of great interest are how the layered structure is initiated and maintained during and after embryogenesis and how communication between layers is achieved to allow for coordinated development. Recent research involving Receptor-Like Kinases (RLKs) in plants suggests that their roles in integrating various signals are important in many aspects of development, including embryonic and post-embryonic patterning. The research presented here describes the roles of two pairs of RLKs with independent roles in two different signaling environments. The first RLK pair, GSO1 and GSO2, function in root development at the transition to photoautotrophic nutrition to integrate sugar signals and regulate root growth. GSO1 and GSO2 regulate root epidermal cell identity by controlling the pattern of cell division of stem cells. The second pair of RLKs, RPK1 and TOAD2, function to control root development by regulation of meristem proliferation and a coordinated response to signaling molecules of the CLE family. The response of wild-type roots to treatment with CLE peptides (A-type) is meristem growth arrest, resulting in short roots. toad2 mutants are insensitive to the effect of CLE peptides in reducing meristem size and TOAD2 also regulates RPK1 upon CLE stimulation. Although responding to different signals, the two pairs of RLK share a common output of regulating cell proliferation in and around the root meristem, especially in the epidermis of the root.