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Phosphoinositide Messengers in Cellular Signaling and Trafficking

• Tamás Balla, MD, PhD, Head, Section on Molecular Signal Transduction
• Yeun Ju Kim, PhD, Staff Scientist
• Alejandro Alvarez-Prats, PhD, Postdoctoral Fellow
• Takashi Baba, PhD, Postdoctoral Fellow
• Joshua Pemberton, PhD, Postdoctoral Fellow
• Nivedita Sengupta, PhD, Postdoctoral Fellow
• Mira Sohn, PhD, Postdoctoral Fellow
• Dániel Tóth, MD, PhD, Postdoctoral Fellow
• Marek Korzeniowski, PhD, Special Volunteer
• Ljubisa Vitkovic, PhD, Special Volunteer

Every biochemical process in a eukaryotic cell relies on 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. 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 type 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 cell's normal functions. Better understanding of such processes can not only provide new strategies to fight various human diseases but also intercept the life cycle of cellular pathogens, thus offering an alternative to antimicrobial drugs.

Phospholipid transport controlled by ORP5/8 proteins at plasma membrane–ER contact sites

The project addresses the role of non-vesicular lipid transport at contact sites formed between the plasma membrane (PM) and the 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. The 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 phosphatidylinositol 4-kinase alpha (PI4KA) and elimination of PI4P in the ER by the Sac1 lipid phosphatase enzyme. PI4P is a minor phospholipid, produced by four different PI4P–kinase enzymes, and 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 is also a precursor of PI(4,5)P₂, one of the most important PM phosphoinositide lipids. The transport of PI4P from the PM to the ER by the ORP5/8 proteins represents a divergent pathway, i.e., in competition with 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 that its transport function is switched off when either PI4P or PI(4,5)P₂ levels are lowered in the PM. Such regulation is achieved through the N-terminal pleckstrin homology (PH) domain of ORP5, which provides 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. The mechanism is a safeguard for the cells to maintain PI(4,5)P₂ levels in the PM. We also discovered that ORP8, which under normal conditions is a bystander, is recruited to the PM when PI(4,5)P₂ levels are elevated. Under these conditions, the transport of PI4P from the PM to the ER is increased, thereby reducing the availability of PI4P for PI(4,5)P₂ synthesis and thereby protecting cells from excess PI(4,5)P₂ in the PM. Through this rheostat mechanism, ORP5 and ORP8 play important roles not only in PS transport but also in maintaining a strict control over PM PI(4,5)P₂ levels.

The role of inositol lipids in peripheral nerve myelination

In the second project, we applied our knowledge on the role of PI4KA as a main controller of PM–ER lipid transfer to a whole animal model and studied the importance of these processes in peripheral nerve myelination in mice. 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. Several human diseases present with myelination defects, and a better understanding of the process can help us design strategies to cure or alleviate these pathologic conditions. As pointed out earlier, the PI4KA enzyme is crucial for maintaining the PI4P gradient between the PM and the ER. We genetically inactivated the PI4KA in mice, specifically in Schwann cells, and studied its effect on the myelination process. We found that mice with the PI4KA deletion in Schwann cells (referred to as mutants) show progressive loss of hind-leg function, noticeable from 30 days of age. The mice suffer from a severe myelination defect with substantially reduced myelin thickness and greatly reduced lipid content, most severely affecting phosphatidylserine (PS) and phosphatidylethanolamine (PE). Surprisingly, mouse Schwann cells kept in culture retain their PM PI(4,5)P₂ levels as well as their Akt (anti-apoptotic serine/threonine protein kinase, activator of mTORC1) and mTORC1 (controller of protein synthesis) responses even after prolonged PI4KA inhibition, in spite of the massive reduction in their PM PI4P levels.

The Akt and mTOR responses are a faithful reflection of the signaling initiated by PI(3,4,5)P₃, a lipid that is made by PI 3-kinases from PI(4,5)P₂. When PI(4,5)P₂ levels fall, it would be expected that PI(3,4,5)P₃ signaling would be impaired. So the intact mTOR and Akt responses are further confirmations that there is no impairment of this signaling angle, consistent with the unchanged PI(4,5)P₂ levels. This is important because one would expect that, if plasma membrane PI4P lipid levels fall (as is the case after PI4KA inhibition or inactivation), the lipid PI(4,5)P₂ would also decrease, given that it is made from PI4P.

In contrast, PI4P depletion from the PM causes massive rearrangements of the actin cytoskeleton both in cultured cells and 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.

Additional Funding

• NSERC Postdoctoral Fellowship supporting Dr. Joshua Pemberton
• Natural Sciences & Engineering Research Council of Canada (NSERC) Banting Postdoctoral Fellowship supporting Dr. Joshua Pemberton
• JSPS-NIH Fellowship supporting Dr. Takashi Baba

Publications

1. Sohn M, Korzeniowski M, Zewe JP, Wills RC, Hammond GRV, Humpolickova J, Vrzal L, Chalupska D, Veverka V, Fairn GD, Boura E, Balla T. PI(4,5)P2 controls plasma membrane PI4P and PS levels via ORP5/8 recruitment to ER-PM contact sites. J Cell Biol 2018;217(5):1797-1813.
2. Alvarez-Prats A, Bjelobaba I, Aldworth Z, Baba T, Abebe D, Kim YJ, Stojilkovic SS, Stopfer M, Balla T. Schwann-cell-specific deletion of phosphatidylinositol 4-kinase alpha causes aberrant myelination. Cell Rep 2018;23(10):2881-2890.
3. Balla T. Ca²⁺ and lipid signals hold hands at endoplasmic reticulum-plasma membrane contact sites. J Physiol 2018;596(14):2709-2716.
4. Várnai P, Gulyás G, Tóth DJ, Sohn M, Sengupta N, Balla T. Quantifying lipid changes in various membrane compartments using lipid binding protein domains. Cell Calcium 2017;64:72-82.

Collaborators

• Evžen Boura, PhD, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
• Gerald R. Hammond, PhD, University of Pittsburgh, Pittsburgh, PA
• Stanko Stojilkovic, PhD, Section on Cellular Signaling, NICHD, Bethesda, MD
• Mark Stopfer, PhD, Section on Sensory Coding and Neural Ensembles, NICHD, Bethesda, MD
• Péter Várnai, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary

Contact

For more information, email ballat@mail.nih.gov or visit https://irp.nih.gov/pi/tamas-balla.

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