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. 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. Two projects have been completed this year, both dealing with the role of non-vesicular lipid transport at contact sites formed between plasma membrane (PM) and endoplasmic reticulum (ER) in maintaining proper lipid composition and signaling at the PM. In the first series of studies, we characterized the roles of two lipid-transport proteins, oxysterol binding protein like protein 5 and -8 (ORP5 and ORP8) in the transport of phosphatidylserine (PS) from the ER to the PM. These proteins use a phosphatidylinositol 4-phosphate (PI4P) gradient between the PM (high PI4P) and the ER (low PI4P) to support the transport of PS from the ER to the PM. The PI4P gradient between the PM and ER is maintained by the production of PI4P in the PM by the lipid kinase, PI4KA and the elimination of PI4P in the ER by the Sac1 phosphatase enzyme. PI4P is a minor phospholipid produced by four different PI 4-kinase enzymes that has important functions in the cells as it recruits and organizes protein complexes in endocytic membranes such as in the Golgi and the late endosomes, but it also is a precursor of one of the most important PM phosphoinositide lipid, PI(4,5)P2. The fact that PI4P can be transported from the PM to the ER by the ORP5/8 proteins represents a competing pathway diverting away from the canonical route of PI4P conversion to PI(4,5)P2. In our studies we found that PI4P transport by the ORP5/8 proteins from the PM to ER is, in fact, regulated by the PI(4,5)P2 content of the PM. We showed that under normal conditions ORP5 is the major component of this PI4P transport process but its transport function is switched off when either PI4P or PI(4,5)P2 levels are reduced in the PM. This regulation is achieved through the N-terminal pleckstrin homology (PH) domain of ORP5, which provides the contact with the PM and which requires both PI4P and PI(4,5)P2 for proper PM interaction. Without this interaction, ORP5 is unable to transport lipids between the PM and the ER. This mechanism is a safeguard for the cells to maintain PM PI(4,5)P2 levels. We also discovered that ORP8, which under normal conditions is a bystander, is recruited to the PM when PI(4,5)P2 levels are increased. Under these conditions the transport of PI4P from the PM to the ER is increased thereby decreasing the availability of PI4P for PI(4,5)P2 synthesis thereby protecting cells from too much PI(4,5)P2 in the PM. Through this rheostat mechanism, ORP5 and ORP8 play important roles not only in PS transport but also keeping PM PI(4,5)P2 levels under strict control. In the second projects, we applied this knowledge to the whole animal and studied the importance of these processes in peripheral nerve myelination. Myelination is a process in which long neuronal processes are wrapped in a series of PM sheets provided by Schwann cells in the peripheral nerves. There are a number of human diseases that present with myelination defects and better understanding of the process can help us design strategies to cure or alleviate these pathologic conditions. As pointed out earlier, PI4KA is the enzyme that is crucial for maintaining the PI4P gradient between the PM and the ER. We have genetically inactivated the PI4KA in mice specifically in Schwann cells and studied its effect on the myelination process. We found that mice with PI4KA deletion in Schwann cells (referred to as mutants) show progressive loss of hindleg function noticeable from age of 30 days. These mice suffer from severe myelination defect with substantially reduced myelin thickness and greatly reduced lipid content most severely affecting phosphatidylserine (PS) and phosphatidylethanolamine (PE). Surprisingly, mouse SCs kept in culture retain their PM PI(4,5)P2 levels as well as their Akt and mTORC1 responses even after prolonged PI4KA inhibition in spite of the massive reduction in their PM PI4P levels. In contrast, PI4P depletion from the PM causes massive rearrangements of the actin cytoskeleton both in cultured cells an in the nerves of affected animals. Our studies highlight the central role of PI4KA in the myelination process and show that the role of the enzyme is more closely linked to actin dynamics and PS metabolism than to PI(3,4,5)P3-mediated signaling cascades.
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