Eric Baehrecke - US grants

A Fluorescence Image Deconvolution-Reconstruction Microscope will be used for a variety of different applications with living cells. Use will be restricted to observations of living cells that are present singly, or in thin specimens. This microscope generates a stack of fluorescent images at different focal planes, and then employs a set of sophisticated algorithms to determine point-spread functions for sources of fluorescence in the specimen. Out-of focus noise is subtracted from the signals in the image slices, and the slices are restacked to generate a three-dimensional reconstruction of the object at high resolution. Since small, bright objects placed against a dark background are detected as spots, it is possible to image fluorescence sources that are smaller than the theoretical limit of resolution for the microscope.

During the last twenty years, developments in the design of novel indicator fluorescent probes have enabled biologists to attack formerly intractable problems in cell biology and cell physiology. The cellular processes that have been amenable to this kind of analysis include photoreception, neuronal transmission, animal development, hormonal signaling, triggered gene expression, mitotic regulation, and chemotaxis. The developments in fluorescent dye design have moved in parallel with improvements in our ability to visualize and measure low light intensity signals from small numbers of molecules in living cells, with newly-designed optical microscopes and large pixel array CCD camera detectors. It is reasonable to expect that a significant expansion of analysis of processes in living cells will result from combined developments of reporter molecules and digital imaging technologies. It seems clear that the convergence of developments in photochemistry, biochemistry, cell biology, physiology and microscopy are all about to intersect within the living cell, and when accurate assessments of changing abundance and activity of a variety of molecules can be made in vivo and through time.

This microscope will be placed in an imaging facility that provides faculty, postdocs, graduate and undergraduate students with access to several high performance microscopes equipped with the capacity to view small quantities of fluorescent reporter molecules in living cells.

The long term goal of this SGER Proposal is to investigate restrictions to protein synthesis in cells and to develop enhanced heterologous protein production systems. This will be accomplished by studying the protein output of different transfected cell types in living fruit flies. Green fluorescent protein (GFP) will be the construct marker and the production will be directly observed by insect organ fluorescence. IL-2, the product protein, will be fused to the GFP using a histidine tag to facilitate protein isolation, and an enzymatic cleavage site incorporated in the system along with an appropriate promoter. Various promoters will be used to drive expression in different tissues. The work should lead to high protein-producing cell lines and/or select producing strains of Drosophila and information on the factors controlling protein production, e.g., promoters, chaperones and foldases, etc.

DESCRIPTION (provided by investigator): Programmed cell death plays an important role during animal development, and defects in this process result in a variety of human disorders including cancer and autoimmunity. Apoptosis and autophagic cell death are the two most prominent morphological forms of programmed cell death that occur during development. The regulation of apoptosis is relatively well understood, but little is known about the mechanisms that mediate autophagic programmed cell death. We are studying autophagic programmed cell death during development of the fruit fly Drosophila melanogaster using larval salivary gland cell death as a model. An increase in steroid triggers a genetic hierarchy that activates nearly synchronous cell death in salivary glands. These developmentally-regulated cell deaths utilize apoptosis genes, including caspase proteases, but inhibition of caspases and caspase mutations only partly inhibit salivary gland degradation. Salivary glands possess the morphology of cells that die by autophagic cell death, and autophagy is required for their complete degradation. While much is known about the function and regulation of autophagy in yeast, less is known about the mechanisms that regulate this fundamental process in animal cells, and little is known about the function of autophagy in programmed cell death. Our hypothesis is that the cell-specific use of autophagy in multicellular organisms involves specific regulatory mechanisms that integrate with core autophagy pathways that are conserved from yeast to humans. We have made the surprising discovery that engulfment genes are induced before autophagic cell death, and that the engulfment receptor drpr is required for tissue autonomous autophagy and degradation of salivary glands. Our goal is to characterize the engulfment factors that regulate autophagy, and investigate how their regulation is coordinated in the context of autophagy that participates in cell death during development. Here we propose to: (1) determine the function of engulfment genes in the regulation of autophagy and death of salivary glands, (2) investigate the regulation and function of engulfment receptors during autophagy, and (3) investigate the signaling downstream of Drpr and determine how Drpr regulates autophagy. The recent association of autophagy with neurodegenerative disorders and cancer indicates the importance of investigating the understudied process of autophagic programmed cell death.

DESCRIPTION (provided by applicant): Autophagy is a conserved catabolic process that is used to deliver cytoplasmic material to the lysosome for degradation, and has been implicated in cancer and other disorders. Molecular alterations in the autophagy gene beclin1 are associated with human cancers, and studies in mice have shown that decreased beclin1 function causes dramatic increase in epithelial and hematopoietic malignancies. Beclin1 (Atg6 in flies) is a core component of the evolutionarily conserved Vps34/class III phosphatidylinositol 3 (PI3) kinase complex that regulates the formation of PI3 phosphate (PI3P) lipids. Although the Vps34 complex regulates autophagy, the function of PI3P in multiple vesicle compartments indicates that the tumor suppressor function of beclin1/Atg6 may be more complex than through the regulation of autophagy alone. Similar to beclin1 mutant mice, our data indicate that animals with null mutations in Drosophila beclin1 (Atg6) possess increased hematopoietic cells resulting in blood cell tumors. Significantly, Atg6 mutant clones of eye and ovarian follicle epithelial cells possess a growth advantage over wild- type cell neighbors that is not shared by cells with mutations in either Vps34 or the essential autophagy gene Atg1. These results indicate that significant differences exist between Atg6 and Vps34 mutant cells even though these genes are thought to encode core components of all PI3P regulatory complexes. Our data also suggest that the interpretation of beclin1 phenotypes likely over-simplify the function of this tumor suppressor. Therefore, our hypothesis is that Atg6 phenotypes are caused by alteration of more than autophagy alone. Our goal is to use the strength of Drosophila genetics to determine how Atg6 influences cell and tissue growth. Here we propose to: (1) determine Atg6 mutant cellular defects, (2) investigate the genetic relationship between Atg6, Ref(2)P/p62, NF-kB and tissue overgrowth, and (3) characterize novel factors and pathways that are involved in Atg6-regulated tissue overgrowth. The importance of Atg6/Beclin1 and the Vps34 regulatory complex in all normal cells and in cancer illustrate the significance of these studies.

Project Summary/Abstract Innate immunity is an ancient defense response that evolved with the earliest metazoan creatures, and is the first line of defense against microbial infection. These responses rely on the recognition of microbes by germline-encoded receptors, and drive the production of numerous chemical, biological, and cellular defense responses. In the face of constant microbial assault, innate immunity is essential for the survival of nearly all multicellular organisms. On the other hand, over-exuberant or inappropriate innate immune responses are the underlying cause of morbidity and mortality associated with many infectious and autoimmune diseases. The endocrine system, through steroids as well as sex hormones and vitamin D, has profound pro- or anti- inflammatory effects on the innate immune response. This crosstalk between the innate immune and endocrine systems is found throughout the animal kingdom, and likely evolved with some of the earliest animals. This proposal uses the fruit fly Drosophila melanogaster as a model for the study of these interactions. Flies offer many advantages for these studies, including experimental tractability with arguably the most robust genetic system for in vivo studies, extensive knowledge of steroid hormone regulatory networks, and an innate immune system without the complexity of the adaptive immune response. Furthermore, many aspects of the innate immune responses are highly conserved with mammals, and discoveries made in flies can be translated into paradigm shifting findings in mammals. Particularly relevant for this proposal are the conserved NF-?B signaling pathways, which drive the immediate response to infection, and the modulation of these signaling pathways by steroid hormones. A thorough mechanistic analysis of how the innate immune response is regulated by steroid hormones in the Drosophila model system will provide a deeper understanding of these ancient regulatory interactions, and are likely to identify new avenues for manipulating these interactions in vector insects and/or mammals. Preliminary data demonstrate that the insect steroid hormone 20-hydroxyecdysone has a profound enhancing effect on NF-?B dependent innate immune responses, through regulating the expression of the bacterial sensing receptor PGRP-LC. This regulatory network enables the ecdysone to prime the innate immune response, creating more effective immune defenses in times of stress. The experiments outlined in Aim 1 are designed to elucidate the molecular mechanisms underlying this hormonal control of immunity, while Aim 2 will probe the molecular and cellular mechanisms by which the steroid ecdysone responds to stress and primes immune defenses. In Aim 3, we will probe the role steroid-regulated immune signaling in driving developmentally programmed autophagic cell death and tissue degradation. All three of these Aims build upon, and extend in exciting new directions, the findings from our previous cycle of support for this grant.

? DESCRIPTION (provided by applicant): Programmed cell death plays an important role during animal development, and defects in this process result in a variety of human disorders including cancer, neurodegeneration and autoimmunity. Apoptosis and autophagic cell death are the two most prominent morphological forms of programmed cell death that occur during development. The regulation of apoptosis is relatively well understood, but little is known about the mechanisms that mediate autophagic programmed cell death. We are studying steroid-activated autophagic cell death in Drosophila, and are using the midgut of the larval intestine as a model. An increase in steroid triggers a genetic program that activates midgut cell death. These developmentally-regulated cell deaths do not depend on apoptosis genes, including caspase proteases, and they possess the morphology of cells that die by autophagic cell death. Significantly, autophagy (Atg) genes are required for midgut degradation where they regulate programmed cell size reduction. While much is known about the function and regulation of macro-autophagy (autophagy) in yeast, less is known about the mechanisms that regulate this process in animal cells in vivo, and little is known about the function of autophagy during cell death. It has been assumed that the mechanisms controlling autophagy are identical between yeast and humans. Our hypothesis is that the cell-specific use of autophagy in multicellular organisms involves previously unrecognized regulatory mechanisms that integrate with core autophagy pathways. In support of this hypothesis, we have discovered that the conserved E1 and E2 enzymes encoded by Atg7and Atg3 are not required for autophagy and degradation of the fly midgut, while these genes are required for starvation-triggered autophagy in flies. By contrast, autophagy in midgut cells depends on Uba1, the E1 used for ubiquitination. These and other data indicate that we have discovered a novel mechanism by which ubiquitin regulates Atg7 and Atg3-independent autophagy, and our goal is to characterize molecular mechanisms that control autophagy during midgut cell death. Here we propose to: (1) investigate the role of Atg8 in midgut autophagy and cell size reduction, (2) determine the role of ubiquitin-binding proteins and Parkin substrates in autophagy, and (3) characterize new genes that are required for clearance of mitochondria and autophagy. The recent association of autophagy with neurodegenerative disorders and cancer indicates the importance of investigating the understudied role of autophagy during programmed cell death.

ABSTRACT Autophagy is used by all cells to deliver cytoplasmic material to the lysosome for degradation. Significantly, autophagy has been implicated in several human diseases, including inflammatory disorders, cancer and neurodegeneration. Most of what we know about the regulation of autophagy is based on pioneering studies in yeast that defined the core autophagy machinery, but recent studies in animals have revealed that autophagy can possess different regulatory mechanisms in distinct cell types. Our research program aims to understand how autophagy is regulated in 2 cell types during development of Drosophila. This system possesses several advantages for these studies, including robust genetic, genomic and cell biological tools that enable sophisticated cellular analyses at single cell resolution. We have focused on studying autophagy in dying larval salivary gland cells and midgut enterocyte cells of the intestine as models. Both salivary gland and midgut cells require autophagy for proper death and degradation, but use entirely different mechanisms for the activation of autophagy. Salivary gland autophagy is regulated by an ancient inflammatory signaling pathway that includes the complement factor Mcr and the engulfment receptor Draper, but this pathway is not required for autophagy in either fatbody cells following nutrient deprivation or midgut cells of the intestine during development. By contrast, midgut cells of the intestine require a ubiquitin-dependent autophagy program that interfaces with mitochondrial dynamics through the novel Vps13D protein. Significantly, Vps13D is not required for autophagy in either fatbody or salivary gland cells. Our future research program contains 4 projects that will address key questions in the autophagy field. What is the role of inflammatory signaling in developmental autophagy? What is the role of mitochondrial dynamics in autophagy? What is the role of ubiquitin in autophagy? What is the role of previously undiscovered pathways in context-specific regulation of autophagy? These proposed studies will address a critical gap in our knowledge about the cell context-specific mechanisms that regulate autophagy within an animal. Given the strong conservation of autophagy mechanisms between Drosophila and mammals, we expect that what we discover will provide insight into the diversity of mechanisms that control autophagy in humans, and how alterations in autophagy in different cell contexts may lead to disease. Furthermore, an understanding of the diversity of mechanisms that control autophagy in animals is essential knowledge for the design of rationale strategies to target autophagy for disease therapies.

ABSTRACT Autophagy is used by all cells to deliver cytoplasmic material to the lysosome for degradation. Significantly, autophagy has been implicated in several human diseases, including inflammatory disorders, cancer and neurodegeneration. Most of what we know about the regulation of autophagy is based on pioneering studies in yeast that defined the core autophagy machinery, but recent studies have revealed that autophagy has unique regulatory mechanisms in higher animals. Our hypothesis is that the regulation of autophagy in multicellular animals involves unknown mechanisms that integrate with known autophagy pathways. In support of our hypothesis, we recently identified previously uncharacterized genes encoding members of the solute transporter (SLC) family that are required for autophagy during salivary gland developmentally programmed cell death, including CG11665/hermes and CG5805. Significantly, hermes encodes a pyruvate transporter that is required for autophagy during salivary gland degradation. hermes mutant salivary glands have elevated mTOR signaling, and decreased mTOR function suppresses the hermes salivary gland phenotype. CG5805 encodes a putative mitochondrial amino acid transporter that is also required for autophagy in salivary glands. Our data suggests that the CG5805 and hermes salivary gland phenotypes are related, possibly through nutrient sensing mechanisms. In addition, hermes mutants have phenotypes suggesting the that these transporters may function in the regulation of autophagy in adult intestine stem cells. Our goal is to characterize the role of these SLCs in autophagy, cell health and death during development and adulthood. Here we propose to: (1) determine how Hermes regulates autophagy during development, (2) investigate CG5805 and its relationship to hermes and autophagy, and (3) characterize the role of transporters and autophagy in adult stem cell and intestine health. The association of autophagy with age-associated disorders illustrates the importance of investigating the relationship between autophagy, cell and animal health.