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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2020 Annual Report of the Division of Intramural Research

Phosphoinositide Messengers in Cellular Signaling and Trafficking

Tamas Balla
  • Tamás Balla, MD, PhD, Head, Section on Molecular Signal Transduction
  • Alejandro Alvarez-Prats, PhD, Staff Scientist
  • Yeun Ju Kim, PhD, Staff Scientist
  • Gergo Gulyas, 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
  • 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.

Research described in this report 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, cellular processes that 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.

Intracellular distribution of phosphatidylinositol in living mammalian cells

Phosphorylated inositol phospholipids (PPIn) are a class of phospholipids that are present in tiny amounts but have very important regulatory functions, as they organize protein signaling complexes on specific membrane compartments. They are produced by phosphoinositide kinases that can phosphorylate specifically one of three positions of the inositol ring of one of the major classes of phospholipids, phosphatidylinositol (PI). Although the cellular localization of most PPIn has been thoroughly studied and mapped, the cellular distribution of PI, their more abundant precursor, has not been determined. In this review period, we developed new molecular tools to gain information on PI distribution in intact live mammalian cells without disrupting their membrane integrity. Using structural information available for bacterial PI–specific phospholipase C enzymes (PI-PLCs), we selected the highly active enzyme from Bacillus cereus to use it as a platform for protein engineering. Bacillus cereus (Bc)PI-PLC shows remarkable specificity for PI and does not display catalytic activity towards phosphorylated PPIn species or other phospholipids. Capitalizing on such features and to mark the intracellular distribution of PI, we generated mutant forms of the enzyme targeting residues within the conserved catalytic domain that would abolish enzymatic activity but maintain substrate coordination within the active site. Fusion of this mutant BcPI-PLC to the green fluorescent protein (GFP) and expression in mammalian cells allowed visualization of the distribution of the protein by confocal microscopy in living cells. We then designed BcPI-PLC constructs with minimal interfacial binding, and therefore low basal catalytic activity from the cytosol. Using the rapamycin-inducible heterodimerization system, we modified the enzymes, which were capable of rapidly hydrolyzing PI when recruited in the proximity of membrane-embedded substrate. Acute targeting of this recruitable BcPI-PLC to various organelles, together with detection of diacylglycerol (DAG), the product of PI hydrolysis, could serve as a proxy to assess the PI content of that membrane. Our findings using these tools showed that PI is localized to the endoplasmic reticulum (ER), the site of its synthesis, but is also enriched in the cytosolic leaflets of the Golgi complex, peroxisomes, and mitochondria. Strikingly, we did not find significant amounts of PI within the plasma membrane (PM) or in endosomal compartments in any of the mammalian cell types examined.

As part of these studies, we also developed bioluminescence energy-transfer (BRET)–based biosensors to monitor organelle-specific PI, DAG, and PPIn dynamics at the level of cell populations and combined the method with the use of the recruitable BcPI-PLC construct to characterize the role of PI availability and supply for the generation of PPIn species within distinct membrane compartments of live cells. The studies reveal the explicit need for the sustained delivery of PI from the ER, rather than its absolute steady-state content, to maintain monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments. Overall, our findings mapped for the first time the PI distribution in mammalian cells and revealed a suspected, yet never formally proven, role for PI transfer and substrate channeling in the spatial control of PPIn metabolism. The importance of the studies is that, using these new molecular tools, researchers will be able to characterize the biochemical processes and their molecular machinery that are responsible for the transport of PI from the ER and how these molecules contribute to establish the proper lipid composition of organelle membranes.

Role of the phosphatidylinositol 4-kinase beta–interacting c10orf76 protein in viral replication

As mentioned in above, viral replication requires host factors that many viruses hijack to establish their optimal membrane niche for their replication. Targeting such processes would be an effective means of combating virus infections and propagation. In a separate set of studies, we collaborated with the groups of John Burke and Frank van Kuppeveld to understand the role of the enigmatic c10orf76 protein in viral replication with respect to its interaction with the lipid kinase phosphatidylinositol 4-kinase B (PI4KB). PI4KB is a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P), a critical regulatory lipid in the Golgi complex. PI4KB has been identified as one of the essential host factors necessary for replication of several small picornaviruses in mammalian cells. PI4KB can interact with many protein-binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB–associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. In this collaborative effort, the Burke lab used hydrogen-deuterium exchange mass spectrometry to characterize the c10orf76–PI4KB complex. The studies revealed that c10orf76 and PI4KB directly interact, that binding is mediated by the kinase linker region of PI4KB, and that formation of the heterodimeric complex is modulated by protein kinase A (PKA)–dependent phosphorylation. Using mutant proteins that interrupt the association between PI4KB and c10orf76, we found that PI4KB is required for the recruitment of c10orf76 to the Golgi, but that PI4KB will still bind to the Golgi, without c10orf76 protein. van Kuppeveld's group showed that, while all enteroviruses require PI4KB for replication, replication of c10orf76–dependent enteroviruses requires intact c10orf76–PI4KB interaction, whereas viruses whose replication is independent of c10orf76 still require PI4KB but can replicate with PI4KB mutants that are unable to recruit c10-rf76. The studies also revealed that c10orf76 controls the small GTP–binding protein Arf1 and that the regulation is important to maintain the level of PI4P in the Golgi complex. The studies' significance is that they characterized important protein components of human cells that are used by some viruses for their replication, knowledge that will be used to assess the host protein requirement for the replication of any future viruses that pose a health risk to the public.

Identification of a new membrane sub-compartment in secretory cells

Our group was involved in a multicenter study that identified ribosome-associated vesicles as a dynamic sub-compartment of the ER in secretory cells. Using a combination of live-cell microscopy with in situ cryo-electron tomography, ER dynamics in several secretory cell types, including pancreatic β-cells and neurons, were directly visualized under near-native conditions, imaging approaches that identified a novel, mobile vesicular compartment of the ER, characterized by the presence of ribosomes. The ribosome-associated vesicles (RAVs) were found primarily at the cell periphery, observed across different cell types and species. The studies showed that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture and that they also interact with mitochondria via direct intermembrane contacts. The findings described a new ER sub-compartment within cells, the function of which is still under investigation.

Additional Funding

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


  1. Prinz WA, Toulmay A, Balla T. The functional universe of membrane contact sites. Nat Rev Mol Cell Biol 2020;1:7-24.
  2. 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.
  3. McPhail JA, Lyoo H, Pemberton JG, Hoffmann RM, van Elst W, Strating JR, Jenkins ML, Stariha JT, Powell CJ, Boulanger MJ, Balla T, van Kuppeveld FJ, Burke JE. Characterization of the c10orf76-PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication. EMBO Rep 2019;21:e48441.
  4. Carter SD, Hampton CM, Langlois R, Melero R, Farino ZJ, Calderon MJ, Li W, Wallace CT, Tran NH, Grassucci RA, Siegmund SE, Pemberton J, Morgenstern TJ, Eisenman L, Aguilar JI, Greenberg NL, Levy ES, Yi E, Mitchell WG, Rice WJ, Wigge C, Pilli J, George EW, Aslanoglou D, Courel M, Freyberg RJ, Javitch JA, Wills ZP, Area-Gomez E, Shiva S, Bartolini F, Volchuk A, Murray SA, Aridor M, Fish KN, Walter P, Balla T, Fass D, Wolf SG, Watkins SC, Carazo JM, Jensen GJ, Frank J, Freyberg Z. Ribosome-associated vesicles: a dynamic subcompartment of the endoplasmic reticulum in secretory cells. Sci Adv 2020;6:eaay9572.
  5. 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.


  • 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
  • Zachary Freyberg, MD, PhD, University of Pittsburgh, Pittsburgh, PA
  • 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
  • Frank van Kuppeveld, PhD, Utrecht University, Utrecht, The Netherlands
  • Péter Várnai, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary


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