Phosphoinositide-calcium Signaling In Cell Regulation

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Every biochemical process that happens in a 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 a structural framework on which molecular signaling complexes assemble to ensure proper information processing. Lipid composition of cellular membranes is a major determinant of their biophysical properties and is unique to the different cellular organelles. How cells achieve and maintain the proper lipid composition of their membranes is poorly understood. Cellular processes that affect membrane lipid composition of organelles 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. The role of ORP3 proteins in cell regulation: Membrane contacts sites (MCS) between various organelles are emerging as key structural elements where important communication between organelles takes place. MCS is defined as membrane appositions between membranes of two organelles with a distance of no longer than 30 nm. MCS have been primarily featured in non-vesicular lipid transfer and Ca2+ signal propagation, but their importance is likely to reach beyond these two processes. While MCSs can be dynamic, they are stabilized by tethering proteins that also have functional roles. Several proteins have been identified that function in contact sites most of which have been implicated in non-vesicular lipid transfer between the contributing membranes. An important class of molecules that function at MCSs are the ORP (oxysterol-binding protein-related protein) proteins that are the mammalian orthologues of the yeast Osh proteins. The Osh/ORP proteins mediate the transport of specific lipids between cellular membranes, their lipid cargo preference defined by their lipid transfer ORD domains. One of the salient features of Osh/ORP proteins is that they use phosphatidylinositol 4-phosphate (PI4P) gradient as a driving force as they counter-transport PI4P is exchange to the specific lipids they move between membranes. Therefore, lipid transport by Osh/ORP proteins is linked to the activity of PI 4-kinases. In this research period, we investigated the role of ORP3, one of the less-characterized members of this family, in cellular physiology. Specifically, we investigated the role of inositol lipids in the control of ORP3 function and conversely, the possible role of ORP3 in the organization of endoplasmic reticulum (ER)-plasma membrane (PM) contacts with respect to PI4P status and store operated Ca2+ entry (SOCE). We found that PM association of ORP3 is triggered by protein kinase C activation, especially when combined with cytoplasmic Ca2+ increases and is determined by both PM inositol lipids, PI(4,5)P2 and PI4P. After activation, ORP3 efficiently extracts PI4P and to a smaller extent phosphatidic acid from the PM and slightly increases PM cholesterol levels. Full activation of ORP3 results in decreased PM PI4P levels, which, in turn, inhibits Ca2+ entry via SOCE. We also identified the C-terminal region of ORP3 that follows the strictly defined lipid transfer ORD domain, to be critical for the proper localization and function of the protein. The importance of these studies is that they highlighted the intimate connection between regulation of PI4P levels and Ca2+-entry at PM-ER contact sites and a critical role of ORP3 in this process. Notably, defects in SOCE have been found to cause severe human immunodeficiencies, therefore understanding its regulation is of major importance. The role of PI 4-kinase type III beta (PI4KB) in peripheral nerve myelination: Myelination of peripheral nerves is a complex process requiring a coordinated series of molecular events executed by Schwann cells (SCs). Improper myelination and axonal sorting defects cause peripheral neuropathies such as the several forms of Charcot-Marie-Tooth (CMT) disease. Among the genes that are associated with CMT, many controls vesicular trafficking. The fine architecture of myelin exemplified by the delicate structure of the nodes of Ranvier requires communication between the axon and the surrounding SCs and relies upon the proper delivery of molecular cues to their final destinations. The Golgi plays an important role in most of these processes. There is little information about the role of the Golgi in peripheral myelination by SCs. The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P) is a key regulator of Golgi function. It plays a role in defining post-Golgi vesicle exit sites and recruits various adaptors for membrane coats. PI4P also controls delivery of ceramide, glycosyl ceramide and cholesterol from the ER to the Golgi. One of the major regulators of Golgi function is the lipid kinase, PI4KB. Therefore, to gain further insight in the role of Golgi in peripheral nerve myelination, we created and characterized a mice model with genetic inactivation of PI4KB specifically in SC. We have characterized the phenotype of this mice focusing on the sciatic nerves. These mice display very subtle functional defects that do not show obvious progression with time. Yet, the conduction velocity of the sciatic nerves decreased dramatically, and major structural defects were revealed by histochemical and EM analyses. We found that the mutant mice developed a myelination defect characterized by thinner myelin only affecting the large diameter axons, with gross alterations in the structure of nodes of Ranvier and a striking inability of non-myelinating SCs to wrap small diameter fibers in Remak bundles. These changes were linked to Golgi functions affecting cholesterol transport, glycosylation and a hitherto unrecognized role of PI4KB in the SC microvilli at the nodes of Ranvier. These studies have shown that PI4KB is an important component of the myelination process in peripheral nerves, supporting several aspects of Golgi function including sterol and sphingolipid transport, glycosylation and most likely the trafficking of proteins that are important for the process. The unexpected presence of the enzyme in the microvilli of SCs at the nodes of Ranvier together with the defective microvilli in the nodes of mutant mice revealed an important function of PI4KB within the peripheral nervous system, which requires further studies to explore.