Many fish undergo betanodavirus infection. To study the infection process of dragon grouper nervous necrosis virus (DGNNV), native virus and E. coli-produced virus-like particles (VLPs) were used to analyze the binding and internalization in SSN-1 cells. The binding of DGNNV and VLPs to SSN-1 cells was demonstrated using Western blotting and indirect enzyme-linked immunosorbent assay (ELISA). As estimated by ELISA, the DGNNV particles bound SSN-1 cells in a dose-dependent manner up to 8 ¡Ñ 104 particles per cell. The binding of VLPs was sensitive to neuraminidase and tunicamycin, suggesting that cell-surface sialic acid is involved in binding. The recombinant VLPs block attachment of native virus to the surface of cultured fish nerve cells, blocking infection by the native virus. It is suggesting that the outer shell of DGNNV VLPs is structurally indistinguishable from native virus. The penetration of DGNNV into cells, which was monitored by electron microscopy, appeared mainly to occur via the spherical pit and membrane ruffling pathways. Occasionally, a spherical pit was engulfed by membrane ruffling so as to form a large figure 8-shaped vesicle with an open connection. Our observations suggest that DGNNV utilizes both micro- and macro-pinocytosis pathways to enter SSN-1 cells. Both of nucleotide and amino acid sequences of MGNNV protein A were comparison with all of Nodaviridae members, revealed that MGNNV were most closely related to RGNNV. No correlation of sequences of betanodavirus with geographical habitat was detected. All thirteen nodavirus protein A amino acid sequences contained canonical RNA polymerase motifs in their C-terminal halves and conserved elements of predicted secondary structure throughout. By Phyre web server identification, the BVDV RdRp as the best template for fold recognition of the RdRp domain of MGNNV and allowed the construction of a congruent 3D model.

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Establishment of polarity is a critical process that occurs early during development. In Drosophila melanogaster, axis determination occurs by localization of determinants during oogenesis. Mutations in cappuccino (capu) lead to defects in polarity establishment of both the anterior/posterior (A/P) and dorsal/ventral (D/V) axes during oogenesis. In the oocytes laid by capu mutant females, determinants that define these axes are either mislocalized or are absent. Several lines of evidence suggest that the regulation of cytoskeleton by the gene product encoded by capu is involved in Drosophila oogenesis.Capu, a member of the formin family of proteins, known to be regulators of actin dynamics, interacts both genetically and physically with chickadee (chic) which encodes the actin binding protein Profilin. I show here that mutations in both capu and chic lead to defects in the endocytic uptake of yolk into developing oocytes. I show that mutations in these loci lead to accumulation of abnormally large yolk granules and that this is a post internalization defect in the oocyte of capu and chic females. I also present evidence which indicates that an interaction with capu is necessary for chic regulation of yolk granule biogenesis.This is the first evidence for a formin subfamily of formin proteins to have a role in endocytosis. While this new function identified for the actin associated proteins Capu and Profilin indicates that regulation of actin cytoskeleton plays a role in endocytosis during oogenesis, the mechanism of this regulation and possible actin independent roles played by Capu and Profilin in this process are yet to be determined.Like capu, mutations in spire (spir) also show defects in A/P and D/V axes during oogenesis. Spir is an actin binding protein and like capu, mutations in spir shows defects in cytoskeletal architecture and suggests that capu and spire alter microtubule distribution in the oocyte during oogenesis.To identify molecular partners of capu and spir and their roles during oogenesis, I performed a genome-wide deficiency screen to identify regions of the genome that interact with these genes. I identified regions in the genome that showed interaction with capu and spir. While I was able to narrow down the region of interaction to a smaller cytological interval, gaps in the deficiency coverage and lack of mutants in those regions prevented me from identifying interacting loci in those regions.

During early development, a series of regulated cell movements is required to set up the adult body plan of an organism. Collectively referred to as gastrulation, these coordinated cell movements organize the germ layers and establish the major body axes of the embryo. One such coordinated cell movement, epiboly, describes the thinning and spreading of a multilayered cell sheet to cover the embryo during gastrulation. The zebrafish embryo has emerged as a vital model system to study the cellular and molecular mechanisms that drive epiboly. In the zebrafish, the blastoderm undergoes epiboly to engulf the yolk cell and close the blastopore at the vegetal pole. This is achieved through the coordinated movement of the deep cells, which make up the embryo proper, and two extra-embryonic tissues, the enveloping layer and yolk syncytial layer. Epiboly is essential to the development of most organisms; however, the cellular and molecular mechanisms driving epiboly are poorly understood. Here I report the findings of two distinct projects which addressed the cellular and molecular basis for epiboly in the zebrafish. One cellular mechanism thought to be involved in driving epiboly is the removal of yolk cell membrane ahead of the advancing blastoderm margin. Using a combination of drug- and dominant-negative based approaches to inhibit Dynamin, a key component of the endocytic machinery, I demonstrated that marginal yolk cell endocytosis is dispensable for the successful completion of epiboly. Instead, I found that Dynamin primarily acts in the blastoderm where it maintains integrity of the enveloping layer (EVL) during epiboly. Dynamin maintains EVL integrity through regulation of the Ezrin/Radixin/Moesin (ERM) family of proteins and the activity of the small GTPase Rho A. With the goal of identifying genes involved in regulating epiboly, I characterized the calpain family of calcium-dependent cysteine proteases in the zebrafish and examined the developmental expression patterns of these genes. My study provided insight into the evolution of this large gene family. Furthermore, I found that most members of this family are expressed in the early embryo, suggesting that they may play a role in regulating early developmental processes such as epiboly.

During early development, a series of regulated cell movements is required to set up the adult body plan of an organism. Collectively referred to as gastrulation, these coordinated cell movements organize the germ layers and establish the major body axes of the embryo. One such coordinated cell movement, epiboly, describes the thinning and spreading of a multilayered cell sheet to cover the embryo during gastrulation. The zebrafish embryo has emerged as a vital model system to study the cellular and molecular mechanisms that drive epiboly. In the zebrafish, the blastoderm undergoes epiboly to engulf the yolk cell and close the blastopore at the vegetal pole. This is achieved through the coordinated movement of the deep cells, which make up the embryo proper, and two extra-embryonic tissues, the enveloping layer and yolk syncytial layer. Epiboly is essential to the development of most organisms; however, the cellular and molecular mechanisms driving epiboly are poorly understood. Here I report the findings of two distinct projects which addressed the cellular and molecular basis for epiboly in the zebrafish. One cellular mechanism thought to be involved in driving epiboly is the removal of yolk cell membrane ahead of the advancing blastoderm margin. Using a combination of drug- and dominant-negative based approaches to inhibit Dynamin, a key component of the endocytic machinery, I demonstrated that marginal yolk cell endocytosis is dispensable for the successful completion of epiboly. Instead, I found that Dynamin primarily acts in the blastoderm where it maintains integrity of the enveloping layer (EVL) during epiboly. Dynamin maintains EVL integrity through regulation of the Ezrin/Radixin/Moesin (ERM) family of proteins and the activity of the small GTPase Rho A. With the goal of identifying genes involved in regulating epiboly, I characterized the calpain family of calcium-dependent cysteine proteases in the zebrafish and examined the developmental expression patterns of these genes. My study provided insight into the evolution of this large gene family. Furthermore, I found that most members of this family are expressed in the early embryo, suggesting that they may play a role in regulating early developmental processes such as epiboly.

Amphiphysin (Amph) is a multi-domain protein that has been implicated in synaptic vesicle (SV) endocytosis. In vertebrates, Amph1 associates with SVs and binds to known endocytic proteins, such as dynamin and clathrin. Overexpression of the vertebrate Amph1 SH3 domain is sufficient to inhibit SV endocytosis in the lamprey synapse. However, these in vitro and overexpression studies may not reflect Amph function in vivo. To investigate Amph function in vivo, I used Drosophila melanogaster as a model organism. I discovered that Drosophila Amph was broadly expressed throughout all developmental stages and was also highly expressed in specialized membranes such as the postsynaptic membrane at the larval neuromuscular junction and the t-tubule membranes of muscles. amph mutants were viable and had normal synaptic transmission, results that were inconsistent with a role for Amph in SV endocytosis. However, amph mutants had impaired locomotion, which may reflect a defect in the t-tubule network, a membrane system that is specialized to couple muscle membrane excitation to muscle contraction. To further explore this idea, I undertook a structure-function approach to ask if different Amph functional domains could rescue the t-tubule and locomotory defects observed in amph mutants. Partial rescue was observed for most constructs, suggesting that Amph function was dependent on more than one domain. To further elucidate how Amph functions at the t-tubule network, I used different in vitro methods to investigate novel protein partners for Amph. A GST pull-down approach identified actin as a potential Amph partner, consistent with studies in yeast. However, I could not confirm a direct interaction between Amph and actin in Drosophila. Another candidate partner was the actin-nucleating protein, Wiskott Aldrich Syndrome Protein, WASP. Although WASP and Amph could be coimmunoprecipitated in vitro, WASP was not expressed at the t-tubule membrane, and Wasp mutants had normal t-tubule morphology. Clearly, Amph is essential for normal t-tubule morphology and future work is needed to further define the function of Amph at the t-tubule network.

N-Methyl-D-aspartate receptors (NMDARs) are a principal subtype of excitatory ligandgated ion channel with prominent roles in physiology and disease in the mammalian central nervous system (CNS). Activation of NMDARs requires binding of both glutamate and glycine. Apart from its co-agonist action, glycine can also prime NMDARs for subsequent internalization upon binding of both glutamate and glycine. However, the molecular basis responsible for mediating and regulating glycine priming and NMDAR endocytosis is largely unknown. In my thesis, I discovered the principle that although NMDAR gating and priming share a common requirement for glycine binding, the molecular constraints for gating are distinct from those for priming through two mutations of the glycine binding site in GluN1 subunit of the NMDAR that, while maintaining gating of NMDARs, eliminate glycine priming of the receptors. One of the molecular signatures of glycine priming is recruitment of the endocytic adaptor protein AP-2. I have characterized the two regions in GluN2 subunits required for enhanced AP-2 association. This unexpected result suggests binding of glycine initiates a conformational change transmitted from GluN1 to GluN2 allowing for docking of endocytic machinery. Furthermore, I have discovered that naturally occurring splice variants of GluN1 subunit, containing a 21 amino acid sequence in the N-terminus domain (N1) cassette, abrogate glycine stimulated AP-2 recruitment and glycine-primed NMDAR internalization. These findings imply that there are distinct populations of native NMDARs in the CNS – those lacking N1 that show glycine-primed internalization and those containing N1 that are not primable. Collectively, my thesis work demonstrates a dramatic all-or-none priming effect with splice variants of NMDARs, a highly unexpected discovery providing novel insight into the molecular mechanisms and physiological role of glycine priming. Ultimately, elucidating principles and mechanisms of glycine priming lay the foundation for new types therapeutic approaches for CNS disorders, approaches without the deleterious consequences of directly blocking NMDARs.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 180-186).

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 272-286).

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.