Phosphoinositide Messengers in Cellular Signaling and Trafficking
- Tamás Balla, MD, PhD, Head, Section on Molecular Signal Transduction
- Alejandro Alvarez-Prats, PhD, Staff Scientist
- Yeun Ju Kim, PhD, Staff Scientist
- Gergo Gulyás, MD, PhD, Postdoctoral Fellow
- Alena Koukalova, PhD, Postdoctoral Fellow
- Amrita Mandal, PhD, Postdoctoral Fellow
- Yang Niu, PhD, Postdoctoral Fellow
- Joshua Pemberton, PhD, Postdoctoral Fellow
- Elizabeth Ferrer, BS, Postbaccalaureate Intramural Research Training Award Fellow
- Vijay Joshi, BS, Postbaccalaureate Intramural Research Training Award
- 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, which 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.
Our research 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. However, how cells achieve and maintain the proper lipid composition of their membranes is poorly understood. Cellular processes that affect the 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 can provide new strategies not only to fight various human diseases but also to intercept the life cycle of cellular pathogens, thus offering an alternative to antimicrobial drugs.
The role of ORP3 proteins in cell regulation
Membrane contacts sites (MCSs) between various organelles are emerging as key structural elements where important communication between organelles takes place. MCSs are defined as membrane appositions between membranes of two organelles with a distance of no greater than 30 nm. MCSs have 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, which are the mammalian orthologs of the yeast Osh proteins. The Osh/ORP proteins mediate the transport of specific lipids between cellular membranes, their lipid cargo preference being defined by their lipid transfer ORD domains. One of the salient features of Osh/ORP proteins is that they use a phosphatidylinositol-4-phosphate (PI4P) gradient as a driving force as they counter-transport PI4P in exchange for the specific lipids they move between membranes. Therefore, lipid transport by Osh/ORP proteins is linked to the activity of PI4 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). SOCE is regulated by the ER–luminal Ca2+ concentration, which is sensed by the ER–localized STIM1/STIM2 proteins, which activate the plasma-membrane, Ca2+–selective Orai channels at ER–PM contact sites. In previous studies, we and others showed that PI lipids play important roles in the control of SOCE. We found that PM association of ORP3 is triggered by protein kinase C (PKC) activation, especially when combined with cytoplasmic Ca2+ increases. Once activated by PKC, ORP3–PM association is determined by both the inositol lipids PI(4,5)P2 and PI4P. After activation, ORP3 efficiently extracts PI4P, and to a smaller extent phosphatidic acid, from the PM while it slightly increases PM cholesterol levels. Full activation of ORP3 results in reduced PM PI4P levels, which, in turn, inhibit Ca2+ entry via SOCE. We also identified the C-terminal region of ORP3 that follows the strictly defined lipid transfer ORD domain as critical for the proper localization and function of the protein. The importance of these studies is that they highlight the intimate connection between regulation of PI4P levels and Ca2+ entry at PM–ER contact sites and the 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 control 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 into the role of Golgi in peripheral nerve myelination, we created and characterized a mouse model with genetic inactivation of PI4KB, specifically in SCs. We characterized the phenotype of these mice, focusing on the sciatic nerves. The mice display highly subtle functional defects, which do not show obvious progression with time. Yet, the conduction velocity of the sciatic nerves of mutant animals decreases 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 the nodes of Ranvier and a striking inability of non-myelinating SCs to wrap small diameter fibers in Remak bundles (bundles of a type of unmyelinated nerve fiber). Such changes were linked to Golgi functions affecting cholesterol transport, glycosylation, and to a hitherto unrecognized role of PI4KB in the SC microvilli at the nodes of Ranvier. The studies showed 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.
Additional Funding
- NICHD Scientific Director’s Award (2020–2021)
- NICHD Intramural Research Fellowship for Gergo Gulyás
Publications
- Prinz WA, Toulmay A, Balla T. The functional universe of membrane contact sites. Nat Rev Mol Cell Biol 2020;1:7–24.
- Baba T, Alvarez-Prats A, Kim YJ, Abebe D, Wilson S, Aldworth Z, Stopfer MA, Heuser J, Balla T. Myelination of peripheral nerves is controlled by PI4KB through regulation of Schwann cell Golgi function. Proc Natl Acad Sci USA 2020;117:28102–28113.
- Gulyás G, Sohn M, Kim YJ, Várnai P, Balla T. ORP3 phosphorylation regulates phosphatidylinositol 4-phosphate and Ca 2+ dynamics at plasma membrane-ER contact sites. J Cell Sci 2020;133(6):jcs237388.
- Pemberton JG, Kim YJ, Humpolickova J, Eisenreichova A, Sengupta N, Toth DJ, Boura E, Balla T. Defining the subcellular distribution and metabolic channeling of phosphatidylinositol. J Cell Biol 2020;219:e201906130.
- Balla T, Kim YJ, Alvarez-Prats A, Pemberton J. Lipid dynamics at contact sites between the endoplasmic reticulum and other organelles. Annu Rev Cell Dev Biol 2019;35:85–109.
Collaborators
- Evžen Boura, PhD, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
- John Burke, PhD, University of Victoria, Victoria, Canada
- Martha Cyert, PhD, Stanford University, Stanford, CA
- Juan Marugan, PhD, Division of Pre-Clinical Innovation, NCATS, Bethesda, MD
- Radim Nencka, PhD, Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic
- 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://ballalab.nichd.nih.gov.
