Tamas Balla

Eunice Kennedy Shriver National Institute of Child Health and Human Development, Роквилл, Maryland, United States

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Publications (188)1128.47 Total impact

  • Y. J. Kim · M. L. Guzman-Hernandez · E. Wisniewski · N. Echeverria · T. Balla

    No preview · Article · Feb 2016 · Biochemical Society Transactions
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    ABSTRACT: Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics, as well as changes in cytoplasmic Ins(1,4,5)P3 levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.
    No preview · Article · Dec 2015 · Biochimica et Biophysica Acta
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    ABSTRACT: The spatial organization of vascular endothelial growth factor (VEGF) signaling is a key determinant of vascular patterning during development and tissue repair. How VEGF signaling becomes spatially restricted and the role of VEGF secreting astrocytes in this process remains poorly understood. Using a VEGF-GFP fusion protein and confocal time-lapse microscopy, we observed the intracellular routing, secretion and immobilization of VEGF in scratch-activated living astrocytes. We found VEGF to be directly transported to cell-extracellular matrix attachments where it is incorporated into fibronectin fibrils. VEGF accumulated at β1 integrin containing fibrillar adhesions and was translocated along the cell surface prior to internalization and degradation. We also found that only the astrocyte-derived, matrix-bound, and not soluble VEGF decreases β1 integrin turnover in fibrillar adhesions. We suggest that polarized VEGF release and ECM remodeling by VEGF secreting cells is key to control the local concentration and signaling of VEGF. Our findings highlight the importance of astrocytes in directing VEGF functions and identify these mechanisms as promising target for angiogenic approaches. GLIA 2015.
    Full-text · Article · Nov 2015 · Glia
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    ABSTRACT: Phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P2) is a quantitatively minor phospholipid in eukaryotic cells that plays a fundamental role in regulating endocytic membrane traffic. Despite its clear importance for cellular function and organism physiology, mechanistic details of its biology have so far not been fully elucidated. In part, this is due to a lack of experimental tools that specifically probe for PtdIns(3,5)P2 in cells to unambiguously identify its dynamics and site(s) of action. In this study, we have evaluated a recently reported PtdIns(3,5)P2 biosensor, GFP-ML1Nx2, for its veracity as such a probe. We report that, in live cells, the localization of this biosensor to sub-cellular compartments is largely independent of PtdIns(3,5)P2, as assessed after pharmacological, chemical genetic or genomic interventions that block the lipid’s synthesis. We therefore conclude that it is unwise to interpret the localization of ML1Nx2 as a true and unbiased biosensor for PtdIns(3,5)P2.
    Preview · Article · Oct 2015 · PLoS ONE

  • No preview · Conference Paper · Jul 2015
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    ABSTRACT: Sustained agonist-induced production of the second messengers InsP3 and diacylglycerol requires steady delivery of phosphatidylinositol (PtdIns) from its site of synthesis in the ER to the plasma membrane (PM) to maintain PtdIns(4,5)P2 levels. Similarly, phosphatidic acid (PtdOH), generated from diacylglycerol in the PM, has to reach the ER for PtdIns resynthesis. Here, we show that the Drosophila RdgB homolog, Nir2, a presumed PtdIns transfer protein, not only transfers PtdIns from the ER to the PM but also transfers PtdOH to the opposite direction at ER-PM contact sites. PtdOH delivery to the ER is impaired in Nir2-depleted cells, leading to limited PtdIns synthesis and ultimately to loss of signaling from phospholipase C-coupled receptors. These studies reveal a unique feature of Nir2, namely its ability to serve as a highly localized lipid exchanger that ensures that PtdIns synthesis is matched with PtdIns(4,5)P2 utilization so that cells maintain their signaling competence. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · May 2015 · Developmental Cell
  • Gerald R.V. Hammond · Tamas Balla

    No preview · Article · May 2015 · Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
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    ABSTRACT: Improved versions of inositol-1,4,5-trisphosphate (InsP3) sensors were created to follow intracellular InsP3 changes in single living cells and in cell populations. Similar to previous InsP3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP3 receptor (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP3 binding affinity. Tagging the InsP3R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of Ins P3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP3 and Ca2+ levels in BRET experiments, the Cameleon D3 Ca2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P2 resulted in the fall of InsP3 level, followed by the decrease of the Ca2+-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca2+-signal preceded the fall of InsP3 level indicating an InsP3-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP3 sensor can be used to monitor both the increase and decrease of InsP3 levels in live cells suitable for high-throughput BRET applications.
    Full-text · Article · May 2015 · PLoS ONE
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    ABSTRACT: Polymicrogyria is a structural brain abnormality involving the cerebral cortex that results from impaired neuronal migration and although several genes have been implicated, many cases remain unsolved. In this study, exome sequencing in a family where 3 fetuses had all been diagnosed with polymicrogyria and cerebellar hypoplasia allowed us to identify regions of the genome for which both chromosomes were shared identical-by-descent, reducing the search space for causative variants to 8.6% of the genome. In these regions, the only plausibly pathogenic mutations were compound heterozygous variants in PI4KA, which Sanger sequencing confirmed segregated consistent with autosomal recessive inheritance. The paternally-transmitted variant predicted a premature stop mutation (c.2386C>T; p.R796X) whilst the maternally-transmitted variant predicted a missense substitution (c.5560G>A; p.D1854N) at a conserved residue within the catalytic domain. Functional studies using expressed wild-type or mutant PI4KA enzyme confirmed the importance of p.D1854 for kinase activity. Our results emphasize the importance of phosphoinositide signalling in early brain development. © The Author 2015. Published by Oxford University Press.
    Full-text · Article · Apr 2015 · Human Molecular Genetics
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    ABSTRACT: Professional phagocytic cells ingest microbial intruders by engulfing them into phagosomes, which subsequently mature into microbicidal phagolysosomes. Phagosome maturation requires sequential fusion of the phagosome with early endosomes, late endosomes, and lysosomes. Although various phosphoinositides (PIPs) have been detected on phagosomes, it remained unclear which PIPs actually govern phagosome maturation. Here, we analyzed the involvement of PIPs in fusion of phagosomes with various endocytic compartments and identified phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 3-phosphate [PI(3)P], and the lipid kinases that generate these PIPs, as mediators of phagosome-lysosome fusion. Phagosome-early endosome fusion required PI(3)P, yet did not depend on PI(4)P. Thus, PI(3)P regulates phagosome maturation at early and late stages, whereas PI(4)P is selectively required late in the pathway.
    No preview · Article · Mar 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Biased agonism on the type I angiotensin receptor (AT1-R) can achieve different outcomes, via activation of G protein-dependent and -independent cellular responses. In this study, we investigated whether the biased activation of the AT1-R can lead to different regulation and intracellular processing of the receptor. We analyzed β-arrestin binding, endocytosis and subsequent trafficking steps such as early and late phases of recycling of the AT1-R in HEK293 cells expressing wild type or biased mutant receptors in response to different ligands. We used Renilla luciferase tagged receptors and yellow fluorescent protein (YFP) tagged β-arrestin2, Rab5, Rab7 and Rab11 proteins in bioluminescence resonance energy transfer (BRET) measurements to follow the fate of the receptor after stimulation. We found that not only is the signaling of the receptor different upon using selective ligands, but the fate within the cells is also determined by the type of the stimulation. β-arrestin binding and the internalization kinetics of the angiotensin II (AngII)-stimulated AT1-R differed from those stimulated by the biased agonists. Similarly, AngII-stimulated wild type AT1-R showed differences compared to a biased mutant AT1-R (DRY/AAY AT1-R) regarding β-arrestin binding and endocytosis. We suggest that the differences in the internalization kinetics of the receptor in response to biased agonist stimulation are due to the differences in plasma membrane PtdIns(4,5)P2 depletion. Moreover, the stability of the β-arrestin binding is a major determinant of the later fate of the internalized AT1-R receptor. The American Society for Pharmacology and Experimental Therapeutics.
    No preview · Article · Mar 2015 · Molecular pharmacology
  • Gerald R V Hammond · Tamas Balla
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    ABSTRACT: Polyphosphoinositides (PPIn) are an important family of phospholipids located on the cytoplasmic leaflet of eukaryotic cell membranes. Collectively, they are critical for the regulation of many aspects of membrane homeostasis and signaling, with notable relevance to human physiology and disease. This regulation is achieved through the selective interaction of these lipids with hundreds of cellular proteins, and thus the capability to study these localized interactions is crucial to understanding their functions. In this review, we discuss current knowledge of the principle types of PPIn-protein interactions, focusing on specific lipid-binding domains. We then discuss how these domains have been re-tasked by biologists as molecular probes for these lipids in living cells. Finally, we describe how the knowledge gained with these probes, when combined with other techniques, has led to the current view of the lipids' localization and function in eukaryotes, focusing mainly on animal cells. This article is part of a Special Issue entitled Phosphoinositides. Copyright © 2015. Published by Elsevier B.V.
    No preview · Article · Feb 2015 · Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
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    ABSTRACT: The yeast Efr3p protein is a major regulator of the Stt4p phosphatidylinositol 4-kinase at ER-PM contact sites. Its mutant fly homologue, Rbo displays diminishing light responses attributed to progressively impaired PLC signaling. Here we find that Efr3s play a role in maintaining responsiveness to angiotensin II (AngII) receptors. RNAi-mediated depletion of EFR3A and EFR3B impaired the sustained phase of cytosolic Ca(2+) response to high concentration of AngII in HEK293 cells expressing the wild type but not a truncated AT1a receptor, missing the phosphorylation sites. Efr3 depletion had minimal effect on the recovery of plasma membrane phosphoinositides during stimulation, and AT1 receptors still underwent ligand-induced internalization. A higher level of basal receptor phosphorylation and a larger response was observed after stimulation. Moreover, Gq activation more rapidly desensitized after AngII stimulation in Efr3 downregulated cells. Similar but smaller effect of EFR3 depletion was observed on the desensitization of the cAMP response after isoproterenol stimulation. These data suggest that mammalian Efr3s contribute to the control of the phosphorylation state and hence desensitization of AT1a receptors and could affect GPCR responsiveness in higher eukaryotes.
    Preview · Article · Nov 2014 · Journal of Cell Science
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    ABSTRACT: CDP-diacylglycerol Synthases (CDS)1 are critical enzymes that catalyze the formation of CDP-diacylglycerol (CDP-DAG) from phosphatidic acid (PA). Here we show in vitro that the two isoforms of human CDS, CDS1 and CDS2, show different acyl chain specificities for its lipid substrate. CDS2 is selective for the acyl chains at the sn-1 and sn-2 positions, the most preferred species being 1-stearoyl-2-arachidonoyl-sn-phosphatidic acid. CDS1, conversely, shows no particular substrate specificity, displaying similar activities for almost all substrates tested. Additionally, we show that inhibition of CDS2 by phosphatidylinositol is also acyl chain dependent, with the greatest inhibition seen with the 1-stearoyl-2-arachidonoyl species. CDS1 shows no acyl chain dependent inhibition. Both CDS1 and CDS2 are inhibited by their anionic phospholipid end products, with phosphatidylinositol-(4,5)-bisphosphate showing the greatest inhibition. Our results indicate that CDS1 and CDS2 could create different CDP-DAG pools that may serve to enrich different phospholipid species with specific acyl chains.
    Full-text · Article · Nov 2014 · Biochemistry
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    ABSTRACT: Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4-kinase type IIα (PI4K IIα), in complex with ATP solved by X-ray crystallography at 2.8 Å resolution. The structure revealed a non-typical kinase fold that could be divided into N- and C-lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C-lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane.
    Full-text · Article · Oct 2014 · EMBO Reports
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    Gerald R V Hammond · Tamas Balla
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    ABSTRACT: The inositol lipids play many essential roles in eukaryotic physiology, although the action has usually focused on the special properties of their headgroup. Now, a study by Clark et al (2014) re-focuses attention on the hydrophobic lipid tails, showing that these too exhibit unique biochemical properties—and are also likely to play a fundamental role in the biology of the lipid.
    Preview · Article · Sep 2014 · The EMBO Journal
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    ABSTRACT: Specificity of membrane fusion in vesicular trafficking is dependent on proper subcellular distribution of soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs). Although SNARE complexes are fairly promiscuous in vitro, significant specificity is achieved in cells due to spatial segregation and shielding of SNARE motifs prior to association with cognate Q-SNAREs. In this study we identified phosphatidylinositol 4-kinase IIα (PI4K2A) as a binding partner of vesicle-associated membrane protein 3 (VAMP3), a small R-SNARE involved in recycling and retrograde transport, and found that the two proteins co-reside on tubulo-vesicular endosomes. PI4K2A knockdown inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3-mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly decreased association of VAMP3 with its cognate Q-SNARE, Vti1a. Although binding of VAMP3 to PI4K2A did not require kinase activity, acute depletion of PtdIns4P on endosomes significantly delayed VAMP3 trafficking. Phospholipid modulation of SNARE function has been proposed based on in vitro studies and our study provides mechanistic evidence in support of these claims by identifying PI4K2A and PtdIns4P as regulators of an R-SNARE in intact cells.
    Full-text · Article · Jul 2014 · Journal of Cell Science
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    ABSTRACT: Polyphosphoinositides are an important class of lipid that recruit specific effector proteins to organelle membranes. One member, phosphatidylinositol 4-phosphate (PtdIns4P) has been localized to Golgi membranes based on the distribution of lipid binding modules from PtdIns4P effector proteins. However, these probes may be biased by additional interactions with other Golgi-specific determinants. In this paper, we derive a new PtdIns4P biosensor using the PtdIns4P binding of SidM (P4M) domain of the secreted effector protein SidM from the bacterial pathogen Legionella pneumophila. PtdIns4P was necessary and sufficient for localization of P4M, which revealed pools of the lipid associated not only with the Golgi but also with the plasma membrane and Rab7-positive late endosomes/lysosomes. PtdIns4P distribution was determined by the localization and activities of both its anabolic and catabolic enzymes. Therefore, P4M reports a wider cellular distribution of PtdIns4P than previous probes and therefore will be valuable for dissecting the biological functions of PtdIns4P in its assorted membrane compartments.
    Preview · Article · Apr 2014 · The Journal of Cell Biology
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    ABSTRACT: Vascular endothelial growth factor (VEGF) is a critical regulator of endothelial cell differentiation and vasculogenesis during both development and tumor vascularization. VEGF-165 is a major form that is secreted from the cells via a poorly characterized pathway. Here we used GFP and epitope-tagged VEGF-165 and found that its early trafficking between the ER and the Golgi requires the small GTP binding proteins, Sar1 and Arf1- and that its glycosylation in the Golgi compartment is necessary for efficient post Golgi transport and secretion from the cells. The relative temperature insensitivity of VEGF secretion and its Sar1 and Arf1 inhibitory profiles distinguished it from other cargoes using the "constitutive" secretory pathway. Prominent features of VEGF secretion were: the retention of the protein on the outer surface of the plasma membrane and the stimulation of its secretion by Ca(2+) and PKC. Importantly, shedding of VEGF-165 from the cell surface together with other membrane components appears to be a unique feature by which some of VEGF is delivered to the surroundings to exert its known biological actions. Understanding VEGF trafficking can reveal additional means by which tumor vascularization can be inhibited by pharmacological interventions.
    Full-text · Article · Feb 2014 · Molecular biology of the cell
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    ABSTRACT: Phosphatidylinositol 4-kinase type IIIα (PI4KA) is a host factor essential for hepatitis C virus replication and hence is a target for drug development. PI4KA has also been linked to endoplasmic reticulum exit sites and generation of plasma membrane phosphoinositides. Here, we developed highly specific and potent inhibitors of PI4KA and conditional knock-out mice to study the importance of this enzyme in vitro and in vivo. Our studies showed that PI4KA is essential for the maintenance of plasma membrane phosphatidylinositol 4,5-bisphosphate pools but only during strong stimulation of receptors coupled to phospholipase C activation. Pharmacological blockade of PI4KA in adult animals leads to sudden death closely correlating with the drug's ability to induce phosphatidylinositol 4,5-bisphosphate depletion after agonist stimulation. Genetic inactivation of PI4KA also leads to death; however, the cause in this case is due to severe intestinal necrosis. These studies highlight the risks of targeting PI4KA as an anti-hepatitis C virus strategy and also point to important distinctions between genetic and pharmacological studies when selecting host factors as putative therapeutic targets.
    Preview · Article · Jan 2014 · Journal of Biological Chemistry

Publication Stats

12k Citations
1,128.47 Total Impact Points

Institutions

  • 2008-2015
    • Eunice Kennedy Shriver National Institute of Child Health and Human Development
      Роквилл, Maryland, United States
  • 1987-2015
    • National Institutes of Health
      • • Section on Molecular Signal Transduction
      • • Program in Developmental Neuroscience
      • • Section on Reproductive Endocrinology
      베서스다, Maryland, United States
  • 1987-2011
    • National Institute of Child Health and Human Development
      베서스다, Maryland, United States
  • 1981-2008
    • Semmelweis University
      • Department of Physiology
      Budapeŝto, Budapest, Hungary
  • 2007
    • National Heart, Lung, and Blood Institute
      Maryland, United States
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2005
    • National Eye Institute
      Maryland, United States
  • 2000
    • University of California, San Francisco
      • Department of Cellular and Molecular Pharmacology
      San Francisco, California, United States
  • 1983
    • Semmelweis University
      Budapeŝto, Budapest, Hungary