N-linked glycans serve as quality control tags in the eukaryotic secretory pathway. The endoplasmic reticulum (ER) protein UDP-glucose: glycoprotein glucosyltransferase 1 (GT1) is the main enzyme that modifies carbohydrate tags based upon the folding state of the maturing substrate. GT1 adds glucoses to non-glucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate as well as its ability to differentiate between native or aberrant secretory cargo is poorly understood. Additionally, due to inherent difficulties in studying GT1 activity in the cell, identification of endogenous substrates and the necessity of reglucosylation remain unknown. Here, we analyzed the role of GT1 in glycoprotein maturation in the intact mammalian ER. GT1 post-translationally reglucosylates N-linked glycans in slow-folding regions of substrate glycoproteins. Maturation mutants that disrupt oxidation or oligomerization also support regio-specific reglucosylation by GT1. Our studies have also revealed an abundant endogenous substrate of GT1, identified as prosaposin. GT1 is critical for the maturation of endogenous prosaposin. In the absence of GT1, the endogenous protein is mislocalized to large intracellular juxtanuclear aggregates. Together, these results propose that GT1 acts as an ER quality control sensor by post-translationally targeting glycans on slow folding or non-native domains to recruit chaperones specifically to critical unstable regions. GT1 plays a vital role in endogenous protein folding and trafficking, since in its absence misfolded proteins accumulate intracellularly. This investigation provides new insight into the integral role of GT1 in glycoprotein maturation.

A pollen tube is the cell that navigates sperm cells through the style and delivers them to the ovum, in a rapid and highly-polarized fashion. Underlying the process of pollen tube growth are a dynamic actin cytoskeleton and oscillating gradients and fluxes of ions. The work presented in this dissertation focuses on visualzing the actin cytoskeleton and gauging calcium dynamics in growing pollen tubes. Three different GFP-labeled actin-binding domains are used to probe the actin cytoskeleton of growing Lilium and Nicotiana pollen tubes. Each marker highlights different aspects of the actin cytoskeleton, whereas no single marker reveals the entire spectrum of actin present. Thus, GFP-ADF and GFP-talin reveal elements of the cortical fringe of actin, whereas GFP-fimbrin generously labels actin filaments in the shank of the pollen tube. Because high levels of expression inhibit pollen tube growth and cause structural aberrations of the actin cytoskeleton, I urge caution in their use. A steep tip-focused gradient of calcium that oscillates with the same periodicity as growth rate, is a consistent feature of growing pollen tubes. Conventional methods for monitoring calcium in growing pollen tubes rely on microinjection of fluorescent dyes, a procedure that is both invasive and technically challenging in the small pollen tubes of the model species Nicotiana tabacum and Arabidopsis thalaiana . Here, two transformable calcium indicators, yellow cameleon YC2.1 and ratiometric pericam, are introduced as effective indicators of cytoplasmic calcium in growing pollen tubes. Finally, mag-fura 2 is employed to measure the calcium concentration in the lumen of the endoplasmic reticulum ([Ca 2+]L) of growing Lilium formosanum pollen tubes. I provide evidence that calcium ions are periodically withdrawn from the apex and funneled into the endoplasmic reticulum. I thus support the hypothesis that the endoplasmic reticulum sequesters calcium in the clear zone and thereby confines the tip-focused gradient to the apex. The endoplasmic reticulum therefore emerges as a potential key player in the process of pollen tube growth.

Programmed cell death is essential for normal development and adult tissue homeostasis in almost all multicelluar organisms. Acheron gene was first isolated from the intersegmental muscles (ISMs) in Manduca sexta as a death-associated gene. Subsequently, we cloned human and mouse homolog of Acheron. Acheron encodes a novel protein that has not been previously characterized. Protein structure analysis revealed that Acheron proteins are structurally related to La proteins, but define a novel subfamily. Tissue expression analysis showed that mAcheron is widely expressed in most tissues at both the RNA and protein levels, with brain and heart displaying the highest levels. In mouse C2C12 cells, endogenous Acheron is constitutively expressed in cycling myoblasts and myotubes. Despite the presence of a putative nuclear localization site, the protein is localized predominantly in the cytoplasm. Analyses of the different Acheron transfected C2C12 cells suggested that Acheron is implicated in mediating differentiation and apoptosis in C2C12 cells by differentially regulating the expression of MyoD, Myf5 and Bcl-2. Acheron expression allows C2 C12 cells to up-regulate MyoD and differentiate into myotubes when the cells are induced to undergo differentiation. However, it does not support the myoblast self-renewal by specifically inhibiting the expression of Bcl-2, a key survival factor for ‘reserve’ cells in DM. Inhibition of Acheron activity by tAch (a putative dominant negative regulatory factor of Acheron) or antisense Acheron results in greatly increased ‘reserve’ cell population and decreased differentiation under differentiation condition. The mediation of differentiation and survival by Acheron may be achieved through its regulation on integrin—FAK signaling. To help determine how Acheron functions, we performed a yeast 2-hybrid screen with Acheron as the bait. A clone that contains partial cDNA of Ariadne was isolated from the screen. Ariadne contains RING finger domain and is known to bind to ubiquitin E2 conjugase. In vitro ubiquitination assay revealed that Ariadne has ubiquitin E3 ligase activity. We speculate that Ariadne may function as an E3 to target Acheron for ubiquitination and subsequent proteasome-dependent degradation.

Human lysosomal α-N-acetylgalactosaminidase (α-NAGAL) is responsible for the break down of glycolipids and glycopeptides that contain a terminal α-linked N-acetylgalactosamine residues. Deficiency of α-NAGAL results in Schindler and Kanzaki diseases. α-NAGAL is closely related to another lysosomal enzyme, α-galactosidase (α-GAL), which breaks down glycolipids and glycopeptides with a terminal α-linked galactose residues. Fabry disease results from a deficiency of α-galactosidase activity. We studied the reaction mechanism of both enzymes using biochemistry and X-ray crystallography, and found that α-GAL and α-NAGAL use an identical reaction mechanism, and differ only in substrate specificity. We solved the first structure of human α-NAGAL, allowing us to examine the disease-causing patient mutations in the context of a high-resolution 3D atomic structure, moving Schindler and Kanzaki disease into the realm of personalized molecular medicine. We then developed the first ever proof-of-principle treatment of Schindler and Kanzaki disease, by developing and characterizing 2 pharmacological chaperones that show promise to treat Schindler and Kanzaki diseases, which currently have no treatment options.

Comparative cytological and molecular phylogenetic data suggest that amitochondriate trichomonads (Archaeprotista: Parabasalia) are an early branching lineage of eukaryotes. Prominent in their cells is the karyomastigont, a cytoskeletal organellar system that includes a nucleus, 4–5 kinetosomes, an axostyle, a parabasal body (Golgi complex), and associated fibers. The karyomastigont is an ancestral character of eukaryotes. Genera of the trichomonad family Calonymphidae (Calonympha, Coronympha, Metacoronympha, Stephanonympha and Snyderella) are polymastigont: multinucleate cells with many karyo- or akaryomastigonts (karyomastigonts that lack nuclei). Termite hindgut trichomonads were examined for their susceptibility to antibiotics, pattern of cell division, and to test the hypothesis that DNA is associated with centriole-kinetosomes. Several types of bacterial symbionts are harbored on the surface membranes, in the cytoplasm and nuclei of these anaerobic protists rendering them susceptible to antibacterial antibiotics. Caduceia sp., a devescovinid trichomonad with four types of bacterial symbionts, was removed within 14 days from the Cryptotermes cavifrons (Florida) gut when penicillin and streptomycin were fed to the termite. Snyderella tabogae was far less susceptible to the antibiotic treatment, which also enhanced formation of calcium-rich crystals in the intestine. Metacoronympha divides asymmetrically. M. senta from Incisitermes nr. incisus (Trinidad) also showed a bimodal distribution of cell size. Approximately one third of the population averaged 100 μm in length with as many as 1000 karyomastigonts, unlike the previously described 50 μm mean length with up to 350 karyomastigonts. The cytoskeleton of Snyderella tabogae, the only calonymphid whose complete set of nuclei are not attached to centriole-kinetosomes, consists solely of 500 or more akaryomastigonts (>2000 undulipodia and their kinetosomes). In Snyderella tabogae, groups of 20–50 akaryomastigonts simultaneously beat and form locally organized regions of the cortex. These polymastigont cells are ideal to seek centriole-kinetosome-associated DNA. Fluorescent cytological (DAPI, SYTOX, acridine orange, ethidium bromide, propidium iodide) as well as Feulgen stains were negative: no DNA was present in the centriole-kinetosomes of S. tabogae. However, DAPI accumulates in the parabasal bodies (Golgi complex).

Viral infection of cells often requires many host cell factors. Despite the identification of numerous viral receptor molecules relatively little is known about the roles of these molecules in viral entry, and intracellular targeting. Major histocompatibility complex (MHC) class I proteins are components of the Simian Virus 40 (SV40) cell surface receptor. This interaction between virus and host cell is of interest since MHC class I proteins have typically been studied for their importance in cellular immune responses. MHC class I proteins acquire antigenic peptides in the endoplasmic reticulum (ER). SV40 is unique in its ability to target the ER from the cell surface. Thus, the possibility exists that surface MHC class I proteins may recycle and target the ER. A major question is do MHC class I proteins internalize and target SV40 to the ER. The goal of this thesis was to better characterize SV40 entry, and determine if MHC class I proteins are directly involved in viral internalization. Basic studies were therefore conducted to determine the fate of MHC class I proteins on fibroblasts. SV40 entry into cells was found to occur rather slowly. The majority of preadsorbed virions remain at the cell surface for up to 2.5 hours. Furthermore, three hours were required for preadsorbed SV40 to internalize and escape antiserum neutralization. This suggests that SV40 infectious entry is relatively slow. Cytosol acidification greatly reduced clathrin-dependent endocytosis, but had no inhibitory effect on SV40 infectious entry. Therefore, SV40 infectious entry does not occur by receptor-mediated endocytosis. Treatment of cells with PMA, nystatin, or filipin prevents internalization via caveolae. All three treatments prevented SV40 infectious entry. Thus, caveolae may be the sites of SV40 penetration into cells. To evaluate the role of MHC class I proteins in SV40 endocytosis, cell surface proteins were labeled with $\sp{125}$I. Protease treatment of cells was used to distinguish proteins that have internalized from those remaining at the cell surface. Protease resistant $\sp{125}$I-MHC class I proteins were not detected in the absence, or presence of SV40. Furthermore, a $\sp{125}$I-MHC class I protein-specific monoclonal antibody did not internalize into cells. These results suggest that surface MNC class I proteins are not internalized into cells, and SV40 does not induce their internalization. Truncated $\sp{125}$I-MHC class I proteins were detected in the media. 1,10-phenanthroline prevented accumulation of $\sp{125}$I-MHC class I proteins in the media. Thus, metalloprotease activity is involved in shedding of surface MHC class I proteins from CV-1 cells. The loss of the B$\sb2$-microglobulin subunit from the MHC class I heavy chain was found to precede metalloprotease cleavage of the heavy chain. Collectively, these results suggest that the fate of MHC class I proteins on fibroblast is shedding into the media, and these molecules are probably not directly involved in SV40 entry.

Programmed cell death (PCD) is an essential developmental process in all multicellular organisms. It serves multiple functions including selected removal of unneeded and/or deleterious cells, and regulation of cell numbers (reviewed in Milligan and Schwartz, 1996). The intersegmental muscles (ISMs) of the hawkmoth Manduca sexta provide an ideal model for studying PCD (reviewed in Schwartz, 1992). In response to a decline in the circulating titer of the steroid hormone 20-hydroxyecdysone (20-HE), these cells initiate a death program which includes both the up- and down-regulation of specific genes. Following eclosion, the ISMs undergo PCD that results in the complete destruction of the muscles during the subsequent 30 hours (Finlayson, 1956). This dissertation examines in detail the repression of actin and myosin heavy chain expression that occurs when the muscles become committed to die. At the protein level, actin expression was reduced by 84% at the time the muscles were committed to die, which presumably plays a role in the rapid dissolution of the muscles. When the ISMs became committed to die, there were dramatic increases in proteolytic activity that are correlated with an approximately eightfold increase in the absolute amounts of multicatalytic proteinase (MCP). At the time of commitment, four new MCP subunits were observed to be associated with the complex. Correlated with the addition of these new subunits was a dramatic increase in the levels of immunodetectable MCP throughout the cytoplasm and within the nuclei of dying muscles. These changes in MCP were regulated by the same hormonal signals that mediate cell death. Cells dying by PCD often display a characteristic set of features termed apoptosis. These features include chromatin condensation, DNA fragmentation, membrane blebbing and phagocytic removal of the dying cells. However, dying ISMs display few characteristics of apoptosis. Interestingly, apoptotic cell death does occur in Manduca embryogenesis. Evidently Manduca possesses the necessary biochemical machinery to undergo apoptosis and does so in specific developmental circumstances. These data suggest that more than one cell death mechanism is used during development.

During muscle differentiation in vertebrates, myoblasts initially form in somites and then migrate to proper locations in the trunk and limbs. Once there, these cells are faced with one of three choices: differentiate into myotubes, arrest as satellite cells, or initiate apoptosis and die. The molecular mechanisms that regulate the decision of myoblasts to die are poorly understood. To gain insight into this process, we have cloned death-associated genes from the intersegmental muscles of the moth Manduca sexta, a model system for developmentally regulated muscle cell death. One of the genes isolated in this screen was DALP (Death Associated LIM Only Protein), a protein that shares 52% similarity at the protein level with mammalian Hic-5. Ectopic expression of DALP in the skeletal muscles of the fruit fly Drosophila caused atrophy and disorganization of the contractile apparatus. To determine the role of DALP/Hic-5 in mammalian myogenesis, we took advantage of the mouse myoblast cell line C2C12. Ectopic expression of either DALP or Hic-5 blocked the ability of myoblasts to differentiate following serum withdrawal. These cells failed to express muscle differentiation markers such as MyoD or myosin heavy chain. In addition, these cultures displayed greatly enhanced rates of cell death. Hic-5 expression is restricted to mononucleated and apoptotic C2C12 cells in serum-depleted medium. The effects of ectopic DALP or Hic-5 expression could be prevented by contact with wild type C2C12 cells or by ectopic expression of MyoD. Gene profiling experiment demonstrated that the ectopic expression of Hic-5 results in enhanced expression of pro-apoptotic Bcl-2 family members and of polyubiquitin. Taken together, these data suggest that Hic-5 acts upstream of MyoD and functions as a negative-regulator of myoblast differentiation and may facilitate the initiation of apoptosis. In separate studies, the functional roles of another death-associated molecule, m56, were studied in Ratl fibroblasts. Our data strongly support the hypothesis that m56 is a proteasome subunit and misexpressing m56 can sensitize Ratl cells to apoptotic stimuli.

The pituitary gland is the major endocrine gland in the forebrain. The hormones secreted from this gland regulate vital processes such as reproduction, growth and stress response. Distinct endocrine cells arise from pituitary precursors cells. The endocrine cells are spatially organized along the anterior-posterior axis within the anterior lobe of the pituitary, the adenohypophysis. Several signaling molecules have been shown to play roles in the development of this endocrine gland. Previous work in our lab showed that Sonic Hedgehog (Shh) is required for induction and patterning of the adenohypophysis. Hedgehog (Hh) signaling is involved in many developmental processes including induction, patterning and differentiation of many tissues. In addition, independent studies show that Fibroblast growth factor (Fgf) signaling also plays a role in the development of zebrafish adenohypophysis. ^ One of the main aims of my dissertation was to determine how the Hh and Fgf signaling pathways specify the functional patterning of the adenohypophysis. Using small molecule inhibitors I show that high levels of Hh signaling are required for the formation of the anterior adenohypophysis, the pars distalis (PD) and high levels of Fgf signaling are required for the formation of the posterior adenohypophysis, the pars intermedia (PI). My dissertation work also shows that high Hh levels are required for differentiation of the endocrine cells in the PD, and in contrast high Fgf levels are required for differentiation of the endocrine cells in the PI. Using live-imaging of a transgenic zebrafish line, I show that the PD and PI originate from distinct regions. My analyses has revealed that graded Hh and Fgf signaling help pattern the adenohypophysis along anterior-posterior axis by guiding endocrine cell differentiation in a dose dependent manner. ^ A related aim of my research was to analyze the role of Hh signaling in zebrafish neural tube patterning. The transcriptional response to varying Hh levels is well characterized in chick and mouse neural tube, and this transcriptional response has been partially described in zebrafish. The analysis of the Hh transcriptional response in wild type, Hh mutant and Hh over-expressing embryos show that there is a conserved transcriptional response to Hh signaling in the zebrafish neural tube. My comprehensive analyses of the Hh transcriptional response in the zebrafish neural tube provides a useful tool for the characterization of Hh signaling in zebrafish. ^

A-kinase anchoring protein AKAP95 is implicated in mitotic chromosome condensation by recruiting the condensin complex. Here, we report a differential regulation of condensation of maternal and paternal chromosomes mediated by AKAP95 and chromatin composition in mitotic mouse zygotes. AKAP95 is synthesized upon oocyte activation, targeted to the female pronucleus and specifically associates with maternal chromosomes at mitosis. Peptide competition and rescue experiments show that AKAP95 is required for recruitment of the mCAP-D2 condensin subunit to, and condensation of, maternal chromosomes. In contrast, AKAP95 is dispensable for mCAP-D2 targeting and condensation of paternal chromosomes. In vitro nuclear reconstitution and disassembly assays indicate that human hCAP-D2 targets protamine-containing chromatin independently of AKAP95, but requires AKAP95 for association with histone-containing chromosomes. We propose a concept whereby (1) recruitment of condensins to chromatin is affected by chromatin composition and (2) AKAP95 renders histone-containing chromatin permissive to condensin targeting.