Cell Biology & Molecular Genetics

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Molecular Mechanisms of the Inhibition of Apoptosis by Mycobacterium tuberculosis
(2009) Miller, Jessica Lynn; Briken, Volker; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persistent intracellular pathogens, such as the human pathogen Mycobacterium tuberculosis (Mtb), to inhibit infection-induced apoptosis of macrophages is important for virulence and to achieve persistence in the host. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis. Here I determine the molecular mechanisms of this host-pathogen interaction. Apoptosis induced by the nuoG deletion mutant (nuoG ) is caspase-8 and TNF-α dependent. This cell death was also reduced in the presence of neutralizers and inhibitors of reactive oxygen species (ROS) and in macrophages derived from NOX2 deficient mice, suggesting that DnuoG induced death is dependent upon NOX2 derived ROS. Correlatively, nuoG infected macrophages also produced more phagosomal ROS than those infected with Mtb, or cells derived from NOX2 deficient mice. NuoG also inhibited apoptosis in human alveolar macrophages in a NOX2 dependant manner. These data suggest that reduction of phagosomal ROS is important for inhibition of apoptosis. Consistent with this hypothesis, Mtb deficient in the ROS neutralizing catalase, KatG, also accumulated ROS in the phagosome and was pro-apoptotic in macrophages. The specific mechanism by which NuoG reduces phagosomal ROS is still unknown. We could not detect secretion of NuoG, so direct neutralization of ROS is unlikely. Interestingly, preliminary data suggests that  nuoG may be defective in secretion of SodA and KatG, enzymes known to be important for neutralizing ROS. In conclusion, these studies revealed that Mtb inhibits macrophage apoptosis by neutralizing phagosomal ROS via the NuoG dependent secretion of SodA and KatG. Furthermore, this research suggests a novel function for NOX2 activity in innate immunity, which is the sensing of persisting intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense for pathogens resistant to the respiratory burst.
EXPLORING THE ROLE OF NFκB HOMOLOGS IN AUTOPHAGIC CELL DEATH IN THE DROSOPHILA SALIVARY GLAND
(2009) Ivory, Adrienne; Wu, Louisa P; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The innate immune response is an ancient, highly conserved means of defense against pathogens. An important mediator of innate immunity is the NF-κB (Nuclear Factor-Kappa B) family of transcription factors. Activation of immune-signaling pathways leads to the nuclear translocation of NFκB proteins which initiate the transcription of antimicrobial peptides (AMPs) that circulate and destroy microbes. In Drosophila, these AMPs are up-regulated during the destruction of larval salivary glands. Salivary gland cells are destroyed via autophagy during metamorphosis. This project sought to determine what, if any, role the NFκB transcription factors have in autophagic cell death. Using the Drosophila model, it was determined that a loss of AMP activity during metamorphosis results in a failure to completely degrade larval salivary glands, and this defect appears to be due to an inability to remove autophagic vacuoles. It is suggested that AMPs may serve to degrade the membranes of autophagic vacuoles.
THE ROLE OF SPERMIDINE IN THE REGULATION OF DEVELOPMENT AND DIFFERENTIATION IN SPERMATIDS OF MARSILEA VESTITA
(2009) Deeb, Faten; Wolniak, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Spermiogenesis in the microspore of the water fern, Marsilea vestita, is a rapid process where a dry microspore containing a single cell undergoes nine successive mitotic divisions to produce 32 spermatids and seven sterile cells. Immediately after the dry microspore is placed into water, cytoplasmic movements precede the first mitotic division; a number of proteins and mRNAs aggregate into zones that later become the spermatogenous initials of the gametophyte. Development is driven by the regulated translation of stored mRNA with little or no new transcription (Hart and Wolniak, 1999). The pattern of translation in the gametophyte is ordered precisely spatially and temporally, which indicates that certain proteins are required in specific locations at specific stages of development. Spermatid differentiation involves the de novo synthesis of 140 basal bodies, the remodeling and condensation of nuclear chromatin and then, nuclear elongation. A complex cytoskeletal structure, the multilayered structure (MLS), is formed at the anterior end of the cell and extends the length of the elongated gamete, and apparently functions in cell and nuclear elongation. This document focuses on the role of kinesin motor proteins and spermidine in the regulation of gametophyte development and spermatid differentiation. Blocking the translation of various kinesin isoforms with RNA interference (RNAi) resulted in arrested development at distinct time points. Centrin and tubulin immunolabeling showed different defects in basal body and microtubule ribbon formation, respectively, with the silencing of specific kinesins. The reduction of spermidine levels in the gametophytes by silencing proteins responsible for its synthesis and transport reveals the involvement of the polyamine in gametophyte cell cycle regulation and in spermatid maturation. In addition, drug inhibition of spermidine synthesis later in development highlighted the importance of its involvement in chromatin remodeling and nuclear elongation. Immunolabeling of spermidine and in situ hybridization assays for its synthesizing enzyme, spermidine synthase, indicated that spermidine levels are controlled in gametophytes by the regulated translation of spermidine synthase. The regulated levels of spermidine in the gametophyte is key to its function as a developmental regulator; spermidine appears to participate in multiple cellular processes in a concentration dependent manner.
THE RELATIONSHIP BETWEEN AUTOPHAGY, CELL SURVIVAL AND CELL DEATH IN A MODEL OF NEURODEGENERATION AND DEVELOPMENT.
(2009) Batlevi, Yakup; Pick, Leslie; Baehrecke, Eric H; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The catabolic degradation of proteins is vital for the proper function and homeostasis of all cells. Autophagy is one of the major catabolic systems, and it is involved in processes that are as diverse as cell survival, cell death, immune reaction, cancer and neurodegeneration. Neurodegenerative diseases often have the pathology of protein accumulation in inclusions, but it is unclear whether these inclusions cause cell toxicity. Here I show that autophagy has protective functions in a model of a polyglutamine neurodegenerative disease in Drosophila. Inhibition of autophagy in this model enhances polyglutmine-induced degeneration, while activation of autophagy suppresses degeneration. Moreover, I observed similar protein aggregates in the larval salivary glands of a Drosophila dynein light chain mutant. This dynein light chain mutant is defective in autophagy, and their salivary glands fail to execute developmentally regulated programmed cell death. Ectopic activation of autophagy is sufficient to suppress the protein accumulation in dynein light chain mutant salivary glands. Both neurons and salivary glands are long-lived post-mitotic cells, and these cells are likely to have unique catabolic needs. Our data indicate that defects in catabolism are responsible for the neurodegenerative and salivary gland cell death defects that I observed, and could explain the association of autophagy with neurodegenerative diseases.
Identification and Characterization of HRG-1 heme transporters in eukaryotes
(2008-11-21) Rajagopal, Abbhirami; Hamza, Iqbal; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Heme is a prosthetic group in proteins that perform diverse biological functions including respiration, gas sensing, xenobiotic detoxification, cell differentiation, circadian clock control and micro RNA processing. In most eukaryotes, heme is synthesized through a multi-step pathway with defined intermediates that are highly conserved through evolution. Despite our extensive knowledge about heme biosynthesis and degradation, the molecules and pathways involved in intracellular heme trafficking are unknown, primarily due to the inability to dissociate the tightly regulated processes of heme biosynthesis and degradation from intracellular trafficking events. Caenorhabditis elegans and related helminths are natural heme auxotrophs that rely solely on exogenous heme for normal development and reproduction. We performed a genome-wide microarray analysis and identified 288 genes that are regulated by heme at the transcriptional level in C. elegans. Here, we characterize two heme-responsive genes, hrg-1 and its paralog hrg-4, that are highly upregulated at low heme concentrations and demonstrate that HRG-1 and HRG-4 are heme transporters. Depletion of hrg-1 and hrg-4 in worms by RNAi results in the disruption of organismal heme homeostasis and abnormal response to heme analogs. HRG-4 traffics to the plasma membrane, and HRG-1 localizes to endo-lysosomal compartments. While hrg-4 appears to be specific to worms, hrg-1 has homologs in vertebrates. Knock-down of hrg-1 in zebrafish results in severe anemia and profound developmental defects, which are fully rescued by worm hrg-1. Human and worm HRG-1 proteins localize together. CeHRG-1, hHRG1 and CeHRG-4 all bind and transport heme. To further understand the in vivo functions of hrg-1 and hrg-4, we characterize the genetic deletions of these genes in C. elegans. Preliminary experiments suggest that the deletion mutants respond abnormally to heme analogs, although these results do not phenocopy the RNAi knock-down studies. We speculate that the deletion strains may have developed compensatory mechanisms in response to the genetic lesions in hrg-1 and hrg-4. Taken together, the studies described herein lay the foundation for identifying the molecular mechanisms for heme transport by the HRG-1 proteins in metazoans and delineating the heme trafficking pathways in C. elegans.
Genetic regulation of autophagic cell death in Drosophila Melanogester
(2008-11-20) Dutta, Sudeshna; Baehrecke, Eric H; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Apoptosis and autophagic cell death are the two most prominent morphological forms of programmed cell death that occur during animal development. While much is known about the mechanisms that regulate apoptosis, relatively little is known about autophagic cell death. The steroid hormone ecdysone coordinates multiple cellular processes during metamorphosis in Drosophila, including cell differentiation, morphogenesis and death. E93 is necessary and sufficient for larval tissue cell death during metamorphosis, including autophagic cell death of salivary glands. Here we characterize new mutant alleles of a dominant wing vein mutation Vein-off (Vno), and provide evidence that E93 and Vno are related. Our data also indicate that E93 functions in steroid regulation of both cell development and death during metamorphosis. E93 encodes a helix-turn-helix DNA binding motif and binds to specific regions of salivary gland polytene chromosomes. We have used genetic and genomic approaches to identify downstream targets of E93. We have identified numerous candidate E93 target genes using DNA microarrays, and have generated transgenic animals to identify downstream target genes of E93 by chromatin immune precipitation. We show that one putative E93 target gene, hippo (hpo), is required for salivary gland cell death. The Wts/Hpo tumor-suppressor pathway is a critical regulator of tissue growth in animals, but it is not clear how this signaling pathway controls cell growth. Our data indicate that salivary gland degradation requires genes in the Wts/Hpo pathway. Wts is required for cell growth arrest and autophagy in dying salivary glands, and regulates the degradation of this tissue in a PI3K-dependent manner.
Live cell imaging to study the assembly and fate of autophagosomes
(2008-10-29) Hailey, Dale Warren; Mather, Ian; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Autophagy (formerly macroautophagy) is a critical process that occurs in all Eukaryotes. Induction of the pathway results in formation of multilamellar membrane-delimited structures that engulf cytosolic proteins, organelles and intracellular pathogens en masse. Capture and catabolism are integral to the diverse roles of autophagy in recycling components, degrading aggregate-prone proteins, removing damaged organelles, depleting cells of organelles and cytosolic mass, and isolating intracellular pathogens. Despite recent attention, fundamental questions about autophagy remain. How do autophagosomes form? What exact roles do they play in non-starvation conditions? To investigate these topics, I developed live-cell imaging approaches to identify substrates and turnover rates of autophagosomes, and to survey intracellular membranes for putative roles in autophagosome formation. The following studies show that autophagosomes utilize lipid derived from the mitochondria during their formation, and that this is a unique aspect of starvation-induced autophagy. During formation, autophagosomal markers transiently localize to punctae on the surface of mitochondria. A tail-anchored outer mitochondrial membrane protein freely diffuses into the newly forming autophagosome until the two organelles dissociate. Starvation-induced autophagosomes produced in this manner engulf cytosolic contents, are 3-MA-sensitive and persist only transiently in cells before fusing with lysosomes. These findings reveal that the outer mitochondrial membrane serves as a major membrane source for autophagosome biogenesis during starvation. Furthermore, the data define a new intracellular pathway from mitochondria to the autophagosomal/lysosomal system. Following, Chapter 1 provides a general survey of autophagy, Chapter 2 discusses advances in live-cell imaging and its use to identify autophagosome substrates, Chapter 3 discusses monitoring autophagosome turnover by photo pulse-labeling in live cells, Chapter 4 presents data implicating the use of mitochondrial membrane in the biogenesis of autophagosomes, and Chapter 5 reviews the implications of the data herein.
The crosstalk between B-cell receptor mediated signaling and the actin cytoskeleton
(2008-08-15) Sharma, Shruti; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Oligomerization of the B-cell receptor (BCR) by antigen leads to both signal transduction and antigen internalization for processing and presentation. Previous studies indicate that these processes intersect at the actin cytoskeleton to coordinate the two cellular processes for the optimal activation of B-cells. The exact mechanism by which signals are transduced via the actin cytoskeleton into the efficient internalization and transport of BCR-antigen complexes is not well delineated. In this thesis, I demonstrate that Bruton's tyrosine kinase (Btk), a Tec kinase in the early signaling pathway of the BCR, is able to transduce signals from the BCR to actin regulatory proteins such as WASP and N-WASP. Upon BCR activation, Btk modulates actin dynamics by increasing the levels of phosphorylated, active WASP and N-WASP in B-cells. Btk regulates the activity of WASP and N-WASP by increasing the levels of PtdIns-4,5-P2 and phosphorylated Vav, both of which are required for WASP and N-WASP activation. Inhibition of Btk activity by a point mutation or a specific inhibitor prevents BCR-induced increases in PtdIns-4,5-P2 as well as in phosphorylated WASP, N-WASP and Vav. Furthermore, Btk deficiency or inhibition leads to a severe reduction in BCR-mediated antigen internalization, processing, and presentation to cognate T-cells. Further studies on the role of WASP show no significant effect of WASP deficiency on BCR internalization, while WASP deficiency affects B-cell development, decreasing the numbers of T1/T2 immature B-cells and marginal zone B-cells. Intriguingly, the protein expression levels of N-WASP and WAVE-2, homologues of WASP, increase in WASP-/- B-cells, implicating a compensatory role for WASP homologues in the absence of WASP. Over-expression of N-WASP's proline-rich domain inhibits BCR-mediated antigen uptake and intracellular transport. All of these data indicate that Btk, which is activated upon BCR binding to antigen, regulates actin dynamics and consequently antigen uptake and transport, by activating WASP and N-WASP via Vav and phosphatidylinositides. This presents a novel mechanism by which BCR-mediated signaling regulates BCR-mediated antigen processing and presentation.
Role of ubiquitination in Caenorhabditis elegans development and transcription regulation during spermatogenesis
(2008-08-12) Kulkarni, Madhura D; Mount, Stephen; Smith, Harold; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Regulation of gene function can be achieved through a variety of mechanisms. In this dissertation, I present the genetic and molecular characterization of two genes involved in two distinct mechanisms of control. Each gene was initially identified by its functional role in sperm development in the model organism Caenorhabditis elegans. The first gene, uba-1, is an essential enzyme involved in protein turnover through ubiquitin-mediated proteolysis. A temperature-sensitive allele, (uba-1)it129, was isolated in a classical genetic screen for mutations that cause sperm-specific sterility. The second gene, spe-44, encodes a putative transcription factor. Its identification by microarray screening for sperm-enriched genes led to the cytological analysis of the deletion allele spe-44(ok1400), by reverse genetics approach. it129 encodes a conditional allele of uba-1, the sole E1 ubiquitin-activating enzyme in C. elegans. E1 functions at the apex of the ubiquitin-mediated conjugation pathway, and its activity is necessary for all subsequent steps in the reaction. Ubiquitin is covalently conjugated to various target proteins. Poly-ubiquitination typically results in target protein degradation, which provides an essential mechanism for the dynamic control of protein levels. Homozygous mutants of uba-1(it129) manifest pleiotropic phenotypes, and include novel roles for ubiquitination in sperm fertility, control of body size, and sex-specific development. We propose a model whereby proteins normally targeted for proteasomal degradation instead persist in uba-1(it129^ts) and impair critical cellular processes. The second gene, spe-44, was identified as a putative sperm gene regulator in C. elegans based on its up-regulated expression during spermatogenesis and its significant sequence homology to the DNA-binding SAND domain. Genetic analysis of a deletion allele of spe-44(1400) has revealed its functional role during sperm development. Cytological analysis of spe-44(ok1400) showed developmental arrest of spermatocytes prior to spermatid differentiation. spe-44 mRNA is expressed in a narrow spatial and temporal window, just prior to spermatocyte differentiation, consistent with its functional role during spermatogenesis. Future study will be directed to find putative targets of spe-44 and the mechanisms that regulate gene expression using microarray analysis and yeast-one hybrid screens. These studies will help to understand transcriptional regulatory aspects of spermatogenesis in C. elegans.
The Role of Mammalian Actin Binding Protein 1 in Coupling BCR Signaling and Antigen Transport Functions
(2008-04-23) Onabajo, Olusegun Okelola; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The B cell receptor (BCR) serves as both signal-transducer and antigen-transporter. Binding of antigens to the BCR induces signaling cascades and antigen-processing and presentation, two essential cellular events for B cell activation. BCR-initiated signaling increases BCR-mediated antigen-processing efficiency by increasing the rate and specificity of antigen transport. Previous studies showed a critical role for the actin cytoskeleton in these two processes. Here I found that actin-binding protein 1 (Abp1/HIP-55/SH3P7) functioned as an actin-binding adaptor protein, coupling BCR signaling and antigen-processing pathways with the actin cytoskeleton. Gene knockout of Abp1 and over-expression of the SH3 domain of Abp1 inhibited BCR-mediated antigen internalization, consequently reducing the rate of antigen transport to processing compartments and the efficiency of BCR-mediated antigen-processing and presentation. BCR activation induced tyrosine phosphorylation of Abp1 and translocation of both Abp1 and dynamin 2 from the cytoplasm to the plasma membrane, where they colocalized with the BCR and cortical F-actin. The inhibitory effect of a dynamin PRD deletion mutant on the recruitment of Abp1 to the plasma membrane and the internalization of the BCR, co-immunoprecipitation of dynamin with Abp1, and co-precipitation of Abp1 with GST fusion of the dynamin PRD, demonstrate the interaction of Abp1 with dynamin 2. In addition to its role in antigen transport and processing, Abp1 is also important for BCR signal transduction. Splenic B cells from Abp1 knockout mice and A20 B cell line with Abp1 knockdown displayed higher levels of protein tyrosine phosphorylation after BCR crosslinking when compared with wild type mice. BCR-triggered ERK phosphorylation in Abp1-deficient splenic B cells occurred sooner and for a much shorter duration than the wild type B cells, while both Abp1 knockout and knockdown significantly reduced BCR-induced phosphorylation of JNK. These results demonstrate that the BCR regulates the function of Abp1 by inducing Abp1 phosphorylation and actin cytoskeleton rearrangement, and that Abp1 facilitates BCR-mediated antigen-processing by simultaneously interacting with dynamin and the actin cytoskeleton. My results further suggest a negative regulatory role for Abp1 in BCR signal transduction.

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