Every biochemical process that happens in an eukaryotic cell relies upon a molecular information flow that leads from receptors that inform the cell about its environment all the way to the molecular effectors that determine the appropriate cellular response. A proper information transmission requires a high degree of organization where the molecular players are organized into different cellular compartments so that the specificity of the cellular response can be properly maintained. Breakdown of this organization is the ultimate cause of all human diseases even if the affected molecular pathways differ according to the kind of disease, such as cancer, diabetes or neurodegenerative diseases just to name a few. Research described in this report has focused on the question of how cells organize their internal membranes to provide with the structural framework on which molecular signaling complexes assemble to ensure proper information processing. As detailed below, these cellular processes are often targeted by cellular pathogens such as viruses to force the cells to produce the pathogen instead of performing the cells normal functions. Better understanding of these processes not only can provide new strategies to fight various human diseases but also to intercept the life cycle of cellular pathogens offering an alternative to antimicrobial drugs. In one set of experiments in collaboration with Dr. Hajnoczkys group at Jefferson University in Philadelphia, PA, we aimed at understanding of how mitochondria (Mito) communicate with the endoplasmic reticulum (ER) in special membrane areas where the two organelles are juxtaposed. It has been known for a period of time that Ca2+ ions stored in the ER can be released into the cytosol during cellular activation and very effectively taken up by Mito. This efficient Ca2+ transfer is possible because of the juxtaposition of Mito to some areas of the ER forming a compartment where local Ca2+ concentrations can reach significantly higher levels than in the cytosol. Ca2+ taken up by mitochondria regulates many normal Mito functions such as biological oxidation but also can trigger processes that lead to cell death. In these experiments we designed a novel strategy to determine in living mammalian cells whether such juxtapositioned Mito-ER membrane areas exist and can be visualized by fluorescence microscopy and also intended to measure the Ca2+ concentration within this special membrane domain. For this we used a drug-inducible heterodimerization system that is based upon the rapamycin-mediated heterodimerization of the FKBP12 protein and the small FRB fragment of mTOR. These protein modules were targeted respectively to the surface of the ER and Mito and tagged with the fluorescent proteins CFP and mRFP. Addition of rapamycin clustered these respective targeted proteins in the areas where the Mito and the ER are juxtaposed allowing assessment of the membrane areas that form these contact zones. In further experiments we targeted genetically encoded Ca2+ indicators that have been modified to lower their Ca2+ affinities to these ER-Mito contact zones using the same strategy and showed that the Ca2+ increases in these microdomains can reach several folds higher levels than those reported in the cytoplasm. These results for the first time gave reliable estimatesof the Ca2+ concentrations in these ER-Mito contact zones and will allow us and other researchers to investigate the functionality of these Ca2+ compartments in various disease states. In another set of experiments we studied the role of phosphatidylinositol 4-phosphate (PI4P) in Golgi function. PI4P is a regulatory lipid that has been implicated in a number of cellular processes such as maintenance of organelle morphology and function, vesicular trafficking and lipid transport. PI4P has its highest levels in the Golgi and it has been postulated that PI4P is critical for the attachment of specific protein factors to the Golgi membranes. These factors determine the destination of the membranes that bud off and the rate at which this happens in the Golgi compartment. Since there are four different PI 4-kinase enzymes that can make PI4P in the Golgi and it is not possible to completely eliminate the activity of all four enzymes without causing cell death, it is difficult to assess the role of PI4P in any specific biological process. Therefore, we designed a strategy to acutely and completely eliminate PI4P from the Golgi. This was again based on the drug-inducible heterodimerization system described above. The FRB domain was targeted to the surface of the Golgi using the Tgn38 protein and the cytoplasmic version of the Sac1 phosphatase enzyme that is able to remove the phosphate from PI4P was fused to FKBP12. Rapamycin addition causes the cytoplasmic Sac1 enzyme to bind to the Golgi, where its membrane-bound substrate, PI4P, is rapidly dephosphorylated. We were able to demonstrate that, indeed, this approach has worked leading to the rapid and complete elimination of Golgi PI4P in selected cells. We then determined that PI4P depletion prevented the exit of any Golgi derived membranes leading to a complete blockade of the secretion process. We also showed that both clathrin and several clathrin adapter proteins, such as GGA1 and -2 but not GGA3 lost its localization from the Golgi after PI4P depletion, but the small GTP-binding protein, Arf1 was not affected. These experiments also clarified that Golgi made little contribution to the PI4P supply of the plasma membrane an important process in ensuring proper transmembrane signaling. The importance of PI 4-kinases in the maintenance of intracellular membrane architecture has surfaced in unexpected ways, making these enzymes relevant targets in potential antiviral therapies. Last year several groups have reported that one of the PI4Ks (PI4KIIIalpha) is a critical host factor necessary for hepatitis C virus (HCV) replication in liver. In a separate research project, the group of Alton-Bonnet in Rutgers University in collaboration with our group showed that another PI4K enzyme (PI4KIIIbeta) is upregulated during infection with several small RNA viruses such as coxackie and polio. PI4KIIIbeta not only got upregulated but was also part of the reorganization of the ER membrane that the viral proteins initiated. Inhibition of this enzyme either by pharmacological or genetic means greatly diminished the ability of these viruses to replicate in their host cells. These studies identified PI4Ks as important host factors for RNA virus replication offering new strategies for fighting these viruses. Several pharmaceutical companies have initiated programs to explore whether targeting PI4Ks is a sensible way of fighting diseases caused by these RNA viruses. These developments exemplify how basic research can yield unforeseeable benefits with potentially high impact in public health.
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