James D. Jamieson - US grants

The pancreatic acinar cell has been studies in our lab for many years as a model for examining the intracellular transport pathway in a non- neuroendocrine regulated secretor) cell. Recent data from our laboratory indicate that the pancreatic acinar cell contains several immunoreactive members of the SNARE complex of secretory vesicle and target plasma membrane proteins discovered in yeast and neuronal cells. Several of these may be novel mammalian homologues and include isoforms of v-SNAREs (cellubrevin), tSNAREs (syntaxin), and proteins that control membrane fusion (Rab3D, synaptotagmin). This information provides the basis for future studies in which we will examine the role of these proteins in this nonneuroendocrine regulated secretory cell. The Specific Aims are: First, we will characterize membrane proteins involved in regulated exocytosis along the following lines: 1. We will complete the characterization of Rab3D, the acinar cell low Mr GTP binding protein that is granule-specific and likely serves as a molecular switch for regulated exocytosis; 2. We will pursue the molecular characterization of v- and t-SNAREs and associated proteins (synaptotagmin) in the acinar cell and their co-association in compartments along the secretory pathway. This will be carried out by molecular cloning of cDNAs and immunogold immunocytochemistry. 3. We will determine if SNARE proteins relocated in the cell following intense secretagogue stimulation that is associated with amplification of the Golgi: and 4. We plan to assess the function of SNARE proteins in the acinar cell using permeabilized cells which allow entry of probes (antibodies against SNAREs etc. and botulinal toxins). They will be analyzed for effects on the secretory pathway including the function of cellubrevin in maturation of regulated and constitutive secretory vesicles and on exocytosis. Second, we will examine the molecular mechanisms of membrane fusion on the secretory pathway using cell free assays for zymogen granule/plasma membrane fusion and formation of constitutive secretory vesicles. This will allow us to test directly under controlled conditions the effects of antibodies against SNAREs, synaptotagmin, Rab3D and other proteins implicated in membrane targeting and fusion. Third, exocytosis from the acinar cell is associated with massive relocation of secretory granule membranes to the apical surface which is compensated for by membrane retrieval, likely to the Golgi complex. We will examine the route and kinetics of retrieval from stimulated pancreatic lobules using immunogold electron microscopy and antibodies against membrane components of the exocytic and endocytic compartments. Immunoisolation of recycling vesicles from acinar cells stimulated by secretagogues in vitro should allow us to identify associations of membrane proteins during compensatory membrane retrieval. Particular attention will be paid to formation of coated vesicles and the relationship of synaptotagmin and proteins of the SNARE complex in membrane recycling.

Polarized secretory epithelial cells possess several sorting devices to effect vectorial flow of secretory and membrane proteins to their apical and basolateral poles. Our previous studies on pancreatic acinar cells have delineated the kinetics and routes of intracellular transpost of secretory proteins that enter a secretagogue activated (regulated) pathway leading to apical exocytosis while a separate secretory pathway results in basolateral, nonstimulated (constitutive) discharge of basement membrane proteins. Similarly, vectorial delivery of membrane proteins to the apical and basolateral poles of secretory cells is required to establish and maintain structural and functional poolarity. The goal of this project is to define the routes and control mechanisms for constitutive and regulated secretory pathways in pancreatic acinar and other epithelial cells and to characterize the pathways responsible for biogenesis of plasmalemmal polarity. To this end, we will use pancreatic acinar and other cell lines cultured in chambers that allow probing of apical and basolateral secretory compartments. Agents that perturb secretory protein sorting will be tested for their ability to affect differentially apical (e.g. amylase) or basolateral (e.g. basal lamina) secretory pathways. These will include secretagogues, acidothrophic agents, inhibitors of glycosylation and protein synthesis and drugs producing cytoskeletal disassembly. The effects of reduction in temperature and ATP levels will also be examined. Comparable experimental conditions will be used, in conjunction with immnoassays for polarity of apical (gamma-glutamyl transferase) and basolateral (insulin and laminin receptors) membrane protein delivery, to define factors regulating biogenesis of plasmalemmal polarity and to clarify relationships between secretory protein and membrane protein delivery pathways. We will also examine membrane targeting in MDCX cells transfected with cDNAs for gamma-glutamyl transferase, an apical and basolateral membrane protein in pancreatic acinar cells. Finally, we plan to study biogenesis of the regulated secretory pathway in developing pancreas as it acquires secretagogue responsiveness.

Pancreatic cancer continues to present a major cause of cancer mortality in this country in that of the -25,000 new cases diagnosed each year, nearly all will succumb within a few months of detection. This bleak outlook is due to the absence of specific and sensitive diagnostic markers for the early stages of the disease and the lack of decisive means of tumor eradication at the later stages of progression and metastasis. The long range goal of this proposal is to utilize a battery of molecular probes that recognize normal human exocrine pancreatic cell constituents, several of which may be expressed specifically by subsets of pancreatic tumor cells of similar morphology. Poorly- differentiated and moderately-well differentiated pancreatic adenocarcinomas, for example, may be found to resemble several phases of normal tissue differentiation or a number of forms of transformed cells deriving from a particular normal cell type. Several lines of evidence indicate that pancreatic tumors can exhibit phenotypes that correspond to stages of the normal differentiation program. In these studies we plan to use riboprobes for several classes of mRNA: major cell structural components, enzymes involved in metabolism and secretion, hormone receptors, and protein kinases and substrates; antibodies specific for protein kinases and their substrates will be used in consort with gene probes for these molecules. Freshly fixed and snap-frozen pancreatic adenocarcinoma resections and biopsies and normal tissue will be analyzed using the techniques of Northern analysis of tissue extracts and in situ hybridization of tissue sections, as well as light microscopic immunocytochemistry for proteins of interest. Electron microscopy of sections of Epon- embedded fixed tissues will be done for refined morphological analysis. These specimens will be classified pathologically and the patients' clinical course correlated with the data. Sections of pathologic specimens embedded in paraffin from patients with pancreatic adenocarcinoma previously treated in our institution will be similarly examined in order to provide a retrospective clinical-pathologic correlation with new information gained in this study. At the same time, we plan to examine the normal embryonic rat pancreas with the same gene probes and immunologic probes in order to compare the human tumors with an accessible pancreatic developmental system. This analytical scheme should enable us to test the hypothesis that human pancreatic neoplasms reflect aberrant stages in the normal differentiation process and should allow us to identify potential cells of origin and developmental stages that may be the targets of the carcinogenic process in the pancreas.

The overall goal of this Program Project is to understand the molecular mechanisms involved in the control of protein and membrane traffic in normal and transformed cells. It consists of seven individual projects: Project 1: The goal of this project is to characterize Golgi subcompartments and to identify functionally important membrane proteins found in these subcompartments. The trafficking of the lysosomal Man-6-P receptor (46 Kd) through Golgi subcompartments will also be investigated in normal and transformed cells. Project 2: This project takes advantage of the genetic approach and the availability of mutants defective in specific steps along the secretory pathway to identify proteins or regions of proteins that are important in targeting along the exocytic pathway. One such mutant to be investigated has a defective gene that appears to be a member of the ras oncogene family. Project 3: The goal of this project is to investigate the role of protein conformation on signaling transport along the secretory pathway using viral membrane proteins as models. Project 4: This project will investigate the role of the binding protein BiP in post-translational processing and ER to Golgi transit of nascent secretory and membrane proteins in normal and transformed cells. Project 5: The purpose of this project is to determine the mechanisms of sorting and traffic control during endocytosis using closely related Fc receptors in normal and transformed cells expressing this receptor from clone cDNAs. Project 6: The goals of this project are to define the molecular basis for the transient failure of the membrane protein, glycophorin, to be segregated in the ER in transformed Friend Erytholeukemic cells, and to attempt to identify sorter proteins and carrier proteins involved in control of vesicular membrane traffic. Project 7: Hybrid proteins generated by gene fusion and expression technologies have been shown to be blocked at various steps along the biosynthetic pathway. In this project the precise site of the blockage and the molecular features responsible for the blockages will be investigated.

This proposal requests conference grant support for a workshop entitled "Intracellular Protein Sorting in Secretory Cells" to be held in conjunction with the 19th annual meeting of the American Pancreatic Association. This workshop will bring leading researchers in the area of protein sorting and targeting to a meeting that has attracted for the past 18 years outstanding clinical and basic researchers in the pancreas. This workshop is thus timely: rapid advances are being made in a diversity of systems that range from simple eukaryotes (yeast) to more complex mammalian cells. Current information indicates that the governing principles in all these systems are highly conserved in evolutionary time. Thus, new information derived from non-pancreatic systems should be directly relevant to events occurring in the pancreas and the juxtaposition of researchers in both these areas should provide insights into lines of research for the future that may help clarify intracellular and secretory events in the pancreatic acinar cell.

Secretion from mammalian cells is classified as being constitutive (occurring continuously) or regulated (exocytosis occurs in response to neurohormonal stimulation). The pancreatic acinar cell has been a paradigm for studying regulated exocytosis and recent data from our laboratory indicate that the process involves novel members of the rab3 family of small MrGTP-binding proteins based on functional and immunocytochemical studies. Exocytosis from the acinar cell is accompanied by compensatory endocytosis of excess membrane from the apical plasmalemma; the fate of internalized (recycled) membrane is not known. The aim of our project is to examine the biogenesis of regulated secretion in the developing rat pancreas. One to 2 days prior to birth, the following cytodifferentiation, acinar cells do not couple hormonal stimulation with exocytosis but are capable of constitutive secretion. Immediately following birth, stimulus-secretion coupling occurs. Data from our laboratory indicate that maturation of regulated exocytosis may be due to expression of small Mr GTP binding proteins (?rab3-like proteins), their effectors or modulators. This system should allow us to dissect the factors involved in conversion of the acinar cell from a constitutive secreting state (analogous to yeast which possesses only constitutive secretion) to a regulated system. Specific aims are (1) to characterize biochemically the developmental appearance of membrane proteins including members of the rab family that are implicated in regulated exocytosis (rab3) and coupled endocytosis (rab4); (2) to assess biochemically effectors and regulators of G-protein cycles such as GTPases (GAP), GNRP's (guanine nucleotide releasing proteins and GDI's (guanine nucleotide dissociation inhibitors) during maturation of regulated secretion; (3) to use immunocytochemistry to determine the rate of appearance of putative G proteins and other membrane-associated proteins in acquisition of regulated secretion; to use confocal and video microscopy to examine development of coupled exo- and endocytosis in living cells whose exocytic (zymogen granule) and endocytic membranes have been labeled with fluorescent probes and (4) carry out functional studies in which candidate rab proteins, peptides from their effector domains, and antibodies against them and other proteins implicated in coupled exo-endocytosis will be introduced into permeabilized developing acinar cells. The latter approach may complement/bypass the developmental defect in neonatal glands. Future studies will examine pancreatic acinar cell tumors whose secretory refractivity to stimulants may parallel fetal maturation of stimulus-secretion coupling observed in the developing pancreas.

We are requesting support for a Biological Imaging Core that will continue to perform an essential function for the projects associated with this PPG application. Based on the remarkable progress made by members of the PPG during the past grant period, it is clear that the research activities of all of the projects in this proposal are now poised at a point where morphological correlates are essential for placing biochemical and genetic information into a structural context. The Biological Imaging Core's prime purposes are as follow: First, the Core will provide technical support required for all aspects of preparation viewing and interpretation of thin section electron microscopy. A major aspect of EM use will be the availability of techniques for immunocytochemical localization of antigens using pre embedding immunogold labeling and post embedding localizations on frozen thin sections and sections of hydrophilic plastics. Second, the Core will support all aspects of light microscopic immunocytochemistry which is a critical technique for analysis of antigens expressed in cells. The Core will provide practical help with the use of the BioRad MRC 600 laser scanning confocal microscope and will continued to aid in the development of procedures for examination of tagged antigens and other fluorescent probes in live cells. All aspects of image analysis and reconstruction, quantitative analysis of digitized and data display and preparation for publication are available through the Core. Finally, an important aspect of the Core will to provide a focus for interaction amongst investigators in this PPG and to significantly aid in the planning, execution and interpretation of experiments through collaborations. Dr. Jamieson will serve as overall director of this ore; day to day operation of the Core will be carried out by Dr. Paul Webster who is conversant in all aspects of the preparation and analysis of material for light and EM immunocytochemistry. He will oversee the flow of work through the Core, assisted by Ms. Linda Chicione who provides general technical help with specimen preparation and darkroom work. Mr. Philippe Male will provide engineering support and training in the use of the confocal system and electron microscopes. All professionals associated with the Core are available for consultation on experimental approaches, technical advice and for collaborators on most of the projects in the PPG. Finally, a central responsibility of the staff will be training, supervision and quality control of new students and postdoctoral fellows who will participate in PPG projects.

The overall goals of our project are to determine the molecular basis of the biogenesis of regulated secretion in the developing pancreas. Our previous work has shown that 1 to 2 days prior to birth, acinar cells respond to secretagogue stimulation with elevations of intracellular second massagers levels but do not couple this event to exocytosis despite the observation that all of the organelles of the exocytic pathway are present. Regulated exocytosis in response to hormonal stimulation becomes functional approximately 24-48 hours after birth (secretogenesis). Our studies show that secretogenesis is correlated with movement of rab3D (a marker exclusively for zymogen secretogenesis is associated with a dramatic but transient appearance of rab4 (24-48 hour postnatally) in the apical cytoplasm of the cell where it colocalizes with the actin terminal web and not with membranes of the secretory or endocytic pathway. We postulate that the transient expression of rab4 is related to the maturation of an actin "clamp" that may be a terminal event the cascade of events leading to the maturation of regulated exocytosis. We intend to pursue this system further in order to determine the factors responsible for secretogenesis. Specific aims are (1) to characterize the physiological significance of rab3 and rab4 during secretogenesis with regard to state of phosphorylation, interaction with regulatory proteins (e.g., rab GDI and proteins that may be involved in actin binding), and to evaluate function in cell line, AR42J whose regulated secretory pathway can be hormonally induced and in which specific mRNAs for rab proteins are eliminated with anti-sense nucleotides; (2) to examine the expression of other small GTP-binding proteins and members of the SNARE family that may be developmentally regulated during secretogenesis; (3) to study secretogenesis in organ culture under defined conditions in order to determine the effect of candidate hormones on maturation of regulated secretion; and (4) to examine an acinar cell tumor and a derived inducible cell line, AR42J, that exhibits many of the properties of normal prenatal pancreas for expression and distribution of rab proteins involved in normal secretogenesis. Comparison between the developing gland and the acinar cell tumor may provide information on the potential defects in secretion that characterize the morphology of this rat tumor and several types of human pancreatic cancer.