Adam Linstedt - US grants

DESCRIPTION: The aims are focused on studying a recycling mode of Golgi protein localization.

DESCRIPTION (provided by applicant): Understanding the molecular mechanisms that establish and maintain the Golgi during development, cell division, and cellular stress are significant ongoing goals in biomedical research. Our long-term goal is to identify proteins required for Golgi structure and elucidate their mechanism. This work was initiated using an siRNA-based screen targeting candidates, especially golgins, and cell-based Golgi assembly assays followed by rescue after knockdown in conjunction with biochemical, permeabilized-cell, structural, and computational assays. Our screen identified golgin-160 (G160) as a critical requirement for inward motility of Golgi membranes and showed that its absence arrests Golgi assembly at the dispersed ministack stage impairing directed secretion and cell migration during wound healing. This observation raised the possibility that we could answer fundamental questions concerning the unknown identity and regulation of the motor receptor complex that moves secretory cargo and Golgi membranes inward along microtubules. To test the hypothesis that G160 recruits or activates the dynein motor complex we will 1) identify and functionally characterize its interaction with the motor cytoplasmic dynein, 2) identify and functionally characterize its Arf1-dependent interaction with the Golgi membrane, 3) test the acute dependence of the Golgi on G160-based motility by developing a technique to inactivate G160 using light in a time scale of seconds, and 4) elucidate the control mechanism that allows Golgi dispersal and inheritance during cell division. These aims are supported by key preliminary findings. G160 was required for microtubule +tip capture of Golgi membranes. The G160 C-term bound directly to the intermediate chain of the dynein complex. G160 was required for dynein Golgi association and it was also sufficient for functional motor recruitment as G160 targeted to mitochondria recruited dynein and induced mitochondrial inward motility. The G160 N-term mediated its Arf1-dependent membrane association and its binding was regulated such that G160 cycled to the cell periphery and returned inward on microtubules. Photo-inactivation of G160 showed that the Golgi acutely depends on G160-based inward motility because early Golgi enzymes constantly cycle to the cell periphery. Finally, G160 dissociated from the Golgi at mitosis but remained bound to dynein and collected at spindle poles. Thus, our experiments are poised to uncover components in the long-sought Golgi receptor for the dynein motor and elucidate the regulation that uncouples Golgi membranes from microtubule-based motility to allow Golgi partitioning into daughter cells.

DESCRIPTION (provided by applicant): This proposal is focused on basic cell biological mechanisms of a poorly understood membrane trafficking pathway- the endosome-to-Golgi "bypass" pathway- in three human disease-related contexts. The first is cell invasion by bacterial toxins, which co-opt the bypass pathway causing human disease. The second is manganese toxicity, which induces downregulation of a key component of the bypass pathway and neurodegenerative disorders in humans. The third is the generation of a promising early onset serum marker for liver cancer due to alterations in bypass pathway trafficking in hepatocellular carcinoma. Protein and lipid cargo originating at the cell surface or Golgi apparatus gain access to the pathway by moving to early/sorting endosomes. The cargo then "bypasses" the degradative branch of the endocytic pathway involving late endosomes and instead traffics directly to the Golgi apparatus. We are one of the few labs elucidating the mechanisms that govern trafficking in this pathway. Our approach is to identify proteins trafficking in the bypass pathway, map their targeting signals and interactions, and test their functional role using biochemical and morphological assays in conjunction with siRNA-mediated knockdown and gene replacement. Our published (Bachert et al., 2001;Linstedt et al., 1997;Natarajan and Linstedt, 2004;Puri et al., 2002;Puthenveedu et al., 2003) and unpublished work indicates that a) Golgi proteins GPP130 and GP73 cycle in the bypass pathway and their pH-sensitive Golgi targeting depends on this cycling, b) GPP130 and GP73 bypass pathway cycling involves the combined use of Golgi and endosomal determinants present in their lumenal coiled-coil domains, c) GPP130 shows pH-sensitive binding to a large lumenal complex, d) analogous lumenal determinant-mediated sorting of GPP130 occurs in polarized cells and is basolaterally restricted, e) GPP130 is selectively required for bypass pathway trafficking of Shiga toxin and other bypass markers and this functioning depends on both the GPP130 lumenal sorting determinants and the cytoplasmic domain, f) the COG docking complex is also required and acts downstream of GPP130, g) in response to low concentrations of manganese GPP130 trafficking is rapidly and selectively altered leading to GPP130 downregulation, and h) GP73 trafficking in the bypass pathway is altered in HCC such that it is cleaved and its ectodomain is released from cells producing a promising biomarker of the disease. Thus, our major aim is to test the hypothesis that GPP130 and similarly structured bypass pathway components use lumenal, pH-sensitive interactions to be incorporated into a large Golgi retrieval complex that buds from sorting and recycling endosomes. By interacting with this complex, Shiga-like toxins access the bypass pathway. During its formation, the retrieval complex also interacts with transport components essential for efficient bypass pathway trafficking- including the COG docking complex- and the GPP130 cytoplasmic domain plays a critical role in this recruitment. In addition, we will determine the mechanism by which Mn+2 exposure and cellular transformation alter bypass trafficking causing GPP130 downregulation and GP73 ectodomain shedding, respectively. PUBLIC HEALTH RELEVANCE: Defects in membrane trafficking are responsible for many human diseases and our understanding of the molecular basis of these defects is paving the way to future effective therapeutics. An important, but understudied, example is the bypass membrane trafficking pathway from the cell surface to its interior compartments. Our investigation into the mechanism of bypass pathway trafficking is revealing novel and potentially clinically relevant insights into retrieval-based targeting of Golgi proteins, infection of humans by Shiga-like toxins, manganese neurotoxicity (a major problem due to contaminated well water in underdeveloped nations), and production of an early serum biomarker of human liver cancer (the fifth-leading cause of cancer death worldwide). Thus, the work offers possibilities for new modes for drug delivery to the cytosolic compartment, treatment of infectious diseases where bacterial toxins are involved, therapies against manganese-induced motor dysfunction syndrome, and non-invasive early detection of liver cancer.

DESCRIPTION (provided by applicant): This proposal represents a request for funds to purchase an Andor Revolution XD Spinning Disk Confocal System on a Nikon Perfect Focus TIRF Microscope from a group of PHS funded investigators having overlapping imaging needs. The system will be housed in the Department of Biological Sciences'Shared Imaging Facility, which is operated by the Molecular Biosensor and Imaging Center (MBIC) as a multi-user facility in the Mellon College of Science at Carnegie Mellon University. MBIC is currently funded as one of only five National Technology Centers for Networks and Pathways. Its core mission is to develop optical biosensor and imaging informatics technologies for the detection of molecular-level interactions within living cells and biological tissues. The acquisition of the proposed instrument will provide on-site access to state of the art high-speed multi- color confocal and TIRF microscopy instrumentation within the Mellon Institute, which is essential for a variety of live cell and membrane trafficking experiments, as well as for routine analysis and characterization of the fluorescent probe technology developed within MBIC. Instruments that are currently available within MBIC and partner faculty's research laboratories are both aged and unsupported or currently fully utilized. While access to other advanced microscopy instrumentation is possible within the general Pittsburgh area, we have found that this poses a logistical problem for experiments using living cells and developing embryos and is not practical for experiments of this kind. In addition, the capabilities of the proposed system meets a number of current needs that existing instrumentation is incapable of fulfilling. Since MBIC is a multi-disciplinary collaborative center which brings together a diverse collection of Carnegie Mellon faculty spanning several academic departments, colleges and research institutes, the acquired instrument will provide investigators across the university with substantially improved imaging capabilities to integrate into their research programs and will provide new opportunities to address fundamental biological questions using advanced microscopy and imaging methodologies.

DESCRIPTION (provided by applicant): The Golgi apparatus is compartmentalized into cisternae that associate with one another generating stacks. The multiple stacks in mammalian cells move to a peri-centrosomal position where cisternae in neighboring stacks fuse in a homotypic fashion (i.e. cis with cis, etc.) to form a compartmentalized membrane network. The lateral linking of stacks into a ribbon-like network confers optimal processing efficiency and plays a role in cell cycle progression and wound healing. A major goal is to determine the basis for specificity and regulation in the linking reaction. This will provide mechanistic understanding of how membrane networks are formed, compartmentalized, and inherited during cell division. It also promises insight into significant, yet vexing, questions about membrane tethering related to activation of tethering upon membrane binding, promotion of trans interactions, and coordination with SNARE-mediate membrane fusion. Our working model is that GM130 recruits GRASP65 to cis cisternae using a C-terminal PDZ ligand to bind the PDZ2 domain of GRASP65. Then, GRASP65 on adjacent cisternae interact in trans via their PDZ1 grooves, which bind in a homotypic fashion via internal PDZ ligands present in GRASP65. This enhances efficiency and fidelity of bringing the membranes into close proximity for SNARE contacts and membrane fusion. We also hypothesize that a parallel reaction on medial cisternae involves recruitment of GRASP55 by golgin 45 and GRASP55 homotypic interactions. Thus, the specificity of dual PDZ interactions in each GRASP isoform establishes and maintains the compartmentalized membrane network. Finally, evidence suggests that signaling pathways regulate these interactions and we hypothesize that a cascade leads to PLK1-mediated GRASP phosphorylation causing inactivation of the internal tethering ligands to fragment the Golgi ribbon and promote mitotic entry. Many aspects of this model are novel and, if verified, we believe, would be groundbreaking in the field. As a means of providing rigorous and detailed tests we propose to determine the structure of the GRASP domain, which is the conserved part of the two isoforms and contains the two PDZ-like domains. We will then carryout tests of specific hypotheses regarding its structure/function relationship using point mutations and three types of assay: purified protein interactions, in vivo organelle tethering, and live imaging after knockdown and rescue. This work will define the interaction interfaces for tethering and for localizing the GRASP complexes to their respective cisternae, reveal the mechanism and functional role of specificity in tethering distinct Golgi cisternae with distinct GRASP isoforms, and uncover the mechanism of GRASP domain phospho-inhibition. PUBLIC HEALTH RELEVANCE: The goal of this proposal is a structural understanding of the mitotically regulated tethering reaction that links adjacent cisternae to form an intracellular membrane network known as the Golgi ribbon. This work promises important advances in at least two areas of fundamental significance. The first is membrane tethering. Membrane tethering is a fundamental reaction in membrane trafficking that increases fidelity and efficiency of membrane fusion. These reactions form the basis for establishment and maintenance of intracellular compartments. The second concerns how one of these compartments, the Golgi apparatus, is organized and how this supports its function. The Golgi processes newly synthesized proteins and lipids and these reactions are important in preventing and treating human disease. Defects in membrane trafficking are responsible for many human diseases and our understanding of the molecular basis of these defects is paving the way to future effective therapeutics. Further, understanding trafficking and its establishment of secretory compartments is a vital concern in the development of therapeutics targeting the multitude of diseases that arise from defective protein products in which these proteins depend on secretory processes such as protein folding, quality control, glycosylation, proteolytic activation, and localization. Such diseases include cystic fibrosis, prion-related diseases, diabetes, and Alzheimer's disease, to name just a few. Human disease also arises from defects in compartment function itself. For example, disorders of glycan synthesis are a substantial and rapidly growing group and it is becoming increasingly evident that the primary defect can be in the transport and localization of the glycan transferases within the membrane trafficking system comprised by the endoplasmic reticulum and the Golgi apparatus.

SUMMARY Drug-like modulators of the large enzyme family that initiates site-specific O-glycosylation in the Golgi complex (UDP-N-acetyl-?-D-galactosamine polypeptide N-acetyl-galactosaminyltransferases or GalNAc-Ts) hold promise as entirely new therapeutics for major diseases such as osteoporosis, dyslipidemia, heart disease, chronic obstructive lung disease, cancer, and viral outbreaks. Significantly, there are currently no known inhibitors or activators of these initiating enzymes. To address this shortcoming we developed cell-based fluorescent sensors to be used for high-throughput screening to identify isoform-specific, small molecule modulators of GalNAc-T2 and GalNAcT3 mediated O-glycosylation. We will carryout simultaneous screening of compounds with these sensors, which will greatly minimize off-target effects allowing identification of candidates that directly target either enzyme. Preliminary work including a pilot screen has resulted in a few promising candidates arguing that further screening will be successful. Hits will be validated using, among other tests, biochemical assays with purified enzyme preparations. Beyond the scope of this proposal, we plan structural characterization and optimization of any lead compounds as well as initial tests of therapeutic value. Successful identification of isoform-specific modulators promises to be transformative to glycobiology research, and potentially, the clinic.

SUMMARY Glycosylation, which fine-tunes the function of proteins, is the most abundant and diverse posttranslational modification. Despite well documented connections to major diseases such as osteoporosis, dyslipidemia, heart disease, chronic obstructive lung disease, cancer, and viral outbreaks, the enzymes that mediate mucin- type O-glycosylation in the Golgi apparatus have yet to be discovered as druggable targets. The initiating enzymes, a family of 20 ppGalNAc-transferase isozymes (herein termed GalNAc-Ts or T1-T20), determine which substrates are modified and at which sites. Significant questions remain regarding their specificity, regulation, targets and functions and the lack of an in situ activity assay and a pharmacological approach have been critical limitations. To address these shortcomings we are developing a panel of isozyme-specific, cell- based fluorescent sensors for GalNAc-transferase activity. They are being used in high-throughput screening to identify isoform-specific, small molecule modulators of ppGalNAc-T mediated O-glycosylation. We will carryout simultaneous screening of compounds with these sensors, which will greatly minimize off-target effects allowing identification of candidates that directly target the enzymes. Preliminary work has resulted in several isozyme-selective biosensors and a few promising drug-like candidates including a remarkably selective inhibitor of T3 that works in both cells and mice with no apparent toxicity. Hits from the screening will be validated using a battery of assays starting with a multi-well format glycosylation assay using purified enzyme preparations that identifies direct-effect compounds. Beyond the scope of this proposal, we plan structural characterization and optimization of any lead compounds as well as initial tests of therapeutic value. Successful identification of isoform-selective modulators promises to be transformative to this area of glycobiology research, and potentially, the clinic.