Joseph P Albanesi

University of Texas Southwestern Medical Center, Dallas, Texas, United States

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Publications (78)388.18 Total impact

  • Joseph Albanesi · Hanzhi Wang · Hui-Qiao Sun · Beth Levine · Helen Yin
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    ABSTRACT: For decades, phosphatidylinositol 4-phosphate (PtdIns4P) was considered primarily as a precursor in the synthesis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2). More recently, specific functions for PtdIns4P itself have been identified, particularly in the regulation of intracellular membrane trafficking. PI4K2A/PI4KIIα (phosphatidylinositol 4-kinase type 2 alpha), one of the 4 enzymes that catalyze PtdIns4P production in mammalian cells, promotes vesicle formation from the trans-Golgi network (TGN) and endosomes. We recently identified a novel function for PI4K2A-derived PtdIns4P, as a facilitator of autophagosome-lysosome (A-L) fusion. We further showed that that this function requires the presence of the autophagic adaptor protein GABARAP (GABA[A] receptor-associated protein), which binds to PI4K2A and recruits it to autophagosomes. The mechanism whereby GABARAP-PI4K2A-PtdIns4P promotes A-L fusion remains to be defined. Based on other examples of phosphoinositide involvement in membrane trafficking, we speculate that it acts by recruiting elements of the membrane docking and fusion machinery.
    No preview · Article · Sep 2015 · Autophagy
  • Yan Chen · Jinhui Li · Joachim D. Mueller · Barbara Barylko · Joseph P. Albanesi

    No preview · Article · Aug 2015 · Microscopy and Microanalysis
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    ABSTRACT: The Atg8 autophagy proteins are essential for autophagosome biogenesis and maturation. The γ-aminobutyric acid receptor-associated protein (GABARAP) Atg8 family is much less understood than the LC3 Atg8 family, and the relationship between the GABARAPs' previously identified roles as modulators of transmembrane protein trafficking and autophagy is not known. Here we report that GABARAPs recruit palmitoylated PI4KIIα, a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) and binds GABARAPs, from the perinuclear Golgi region to autophagosomes to generate PI4P in situ. Depletion of either GABARAP or PI4KIIα, or overexpression of a dominant-negative kinase-dead PI4KIIα mutant, decreases autophagy flux by blocking autophagsome:lysosome fusion, resulting in the accumulation of abnormally large autophagosomes. The autophagosome defects are rescued by overexpressing PI4KIIα or by restoring intracellular PI4P through "PI4P shuttling." Importantly, PI4KIIα's role in autophagy is distinct from that of PI4KIIIβ and is independent of subsequent phosphatidylinositol 4,5 biphosphate (PIP2) generation. Thus, GABARAPs recruit PI4KIIα to autophagosomes, and PI4P generation on autophagosomes is critically important for fusion with lysosomes. Our results establish that PI4KIIα and PI4P are essential effectors of the GABARAP interactome's fusion machinery.
    Preview · Article · Jun 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: The Activity-regulated cytoskeleton-associated protein, Arc, is an immediate-early gene product implicated in various forms of synaptic plasticity. Arc promotes endocytosis of AMPA type glutamate receptors and regulates cytoskeletal assembly in neuronal dendrites. Its role in endocytosis may be mediated by its reported interaction with dynamin 2 (Dyn2), a 100 kDa GTPase that polymerizes around the necks of budding vesicles and catalyzes membrane scission. Enzymatic and turbidity assays are used in this study to monitor effects of Arc on dynamin activity and polymerization. Arc oligomerization is measured using a combination of approaches, including size exclusion chromatography, sedimentation analysis, dynamic light scattering, fluorescence correlation spectroscopy, and electron microscopy. We present evidence that bacterially-expressed His6-Arc facilitates the polymerization of Dyn2 and stimulates its GTPase activity under physiologic conditions (37°C and 100 mM NaCl). At lower ionic strength Arc also stabilizes pre-formed Dyn2 polymers against GTP-dependent disassembly, thereby prolonging assembly-dependent GTP hydrolysis catalyzed by Dyn2. Arc also increases the GTPase activity of Dyn3, an isoform of implicated in dendrite remodeling, but does not affect the activity of Dyn1, a neuron-specific isoform involved in synaptic vesicle recycling. We further show in this study that Arc (either His6-tagged or untagged) has a tendency to form large soluble oligomers, which may function as a scaffold for dynamin assembly and activation. The ability of Arc to enhance dynamin polymerization and GTPase activation may provide a mechanism to explain Arc-mediated endocytosis of AMPA receptors and the accompanying effects on synaptic plasticity. This study represents the first detailed characterization of the physical properties of Arc. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Mar 2015 · Biochimica et Biophysica Acta
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    Full-text · Article · Jan 2014 · Biophysical Journal
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    ABSTRACT: Dynamin 2 (Dyn2) is a ~100kDa GTPase that assembles around the necks of nascent endocytic and Golgi vesicles and catalyzes membrane scission. Mutations in Dyn2 that cause Centronuclear Myopathy (CNM) have been shown to stabilize Dyn2 polymers against GTP-dependent disassembly in vitro. Precisely timed regulation of assembly and disassembly is believed to be critical for Dyn2 function in membrane vesiculation, and the CNM mutations interfere with this regulation by shifting the equilibrium toward the assembled state. In this study we use two fluorescence fluctuation spectroscopy (FFS) approaches to show that a CNM mutant form of Dyn2 also has a greater propensity to self-assemble in the cytosol and on the plasma membrane of living cells. Results obtained using brightness analysis indicate that unassembled wild-type Dyn2 is predominantly tetrameric in the cytosol, although different oligomeric species are observed, depending on the concentration of expressed protein. In contrast, an R369W mutant identified in CNM patients forms higher-order oligomers at concentrations above 1μM. Investigation of Dyn2-R369W by Total Internal Reflection Fluorescence (TIRF) FFS reveals that this mutant forms larger and more stable clathrin-containing structures on the plasma membrane than wild-type Dyn2. These observations may explain defects in membrane trafficking reported in CNM patient cells and in heterologous systems expressing CNM-associated Dyn2 mutants.
    Full-text · Article · Sep 2013 · Biochimica et Biophysica Acta
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    ABSTRACT: The mitogen-activated protein kinases (MAPKs) ERK1/2 regulate numerous cellular processes including gene transcription, proliferation, and differentiation. The only known substrates of the MAP2Ks MEK1/2 are ERK1/2; thus, the MEK inhibitors PD98059, U0126, and PD0325901 have been important tools in determining the functions of ERK1/2. By using these inhibitors and genetically manipulating MEK, we find that ERK1/2 activation is neither sufficient nor necessary for regulated insulin secretion from pancreatic beta cells or epinephrine secretion from chromaffin cells. We show that both PD98059 and U0126 reduce agonist-induced calcium entry into cells inde-pendently of their ability to inhibit ERK1/2. Caution should be used when interpreting results from experiments using these compounds.
    No preview · Article · Jul 2013 · Biochemistry
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    Full-text · Article · Jan 2013 · Biophysical Journal
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    Preview · Article · Jan 2013 · Biophysical Journal
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    David M Jameson · Nicholas G James · Joseph P Albanesi
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    ABSTRACT: Communication between cells and their environment, including other cells, is often mediated by cell surface receptors. Fluorescence methodologies are among the most important techniques used to study receptors and their interactions, and in the past decade, fluorescence fluctuation spectroscopy (FFS) approaches have been increasingly utilized. In this overview, we illustrate how diverse FFS approaches have been used to elucidate important aspects of receptor systems, including interactions of receptors with their ligands and receptor oligomerization and clustering. We also describe the most popular methods used to introduce fluorescent moieties into the biological systems. Finally, specific attention will be given to cell maintenance and transfection strategies especially as related to microscopy studies.
    Full-text · Article · Jan 2013 · Methods in enzymology
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    ABSTRACT: Mammalian cells express two classes of phosphatidylinositol 4-kinase (PI4K), designated as Types II and III, that phosphorylate phosphatidylinositol to generate PI4P. A number of studies have indicated that these enzymes are important for Golgi trafficking and both early and late stages of endocytosis. In this study, we focus on PI4KIIβ, a protein that is evenly distributed between membrane and soluble fractions, and is believed to participate in stimulus-dependent phosphoinositide signaling. Using molecular brightness analysis, we found that EGFP-tagged PI4KIIβ exists as two distinct species in the cytoplasm: a soluble monomer and a high-order complex enriched with multiple copies of PI4KIIβ. This observation was confirmed by an autocorrelation analysis that identified two species with distinct mobilities. We further demonstrate that the high-order complex enriched with PI4KIIβ is sensitive to inhibition of palmitoylation, indicating that it is associated with membranes, very likely vesicles. Indeed, we show that the high-order PI4KIIβ complex is sensitive to expression of dynamin 2 (K44A), a dominant-negative inhibitor of endocytosis. Using dual-color heterospecies partition analysis, we directly detected that PI4KIIβ comoves with clathrin light chain on vesicles. This analysis allows us to isolate the comobile species in the presence of strong background contribution from the monomeric pool of PI4KIIβ. Our results strongly suggest that PI4KIIβ is involved in an early stage of endocytosis and is associated with clathrin-coated vesicles. Moreover, we establish molecular brightness as a powerful tool for characterizing cellular cytosolic vesicles that are otherwise difficult to characterize by other techniques.
    Full-text · Article · Oct 2012 · Biophysical Journal
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    ABSTRACT: Type II phosphatidylinositol 4-kinase (PI4KII) produces the lipid phosphatidylinositol 4-phosphate (PI4P), a key regulator of membrane trafficking. Here, we generated genetic models of the sole Drosophila melanogaster PI4KII gene. A specific requirement for PI4KII emerged in larval salivary glands. In PI4KII mutants, mucin-containing glue granules failed to reach normal size, with glue protein aberrantly accumulating in enlarged Rab7-positive late endosomes. Presence of PI4KII at the Golgi and on dynamic tubular endosomes indicated two distinct foci for its function. First, consistent with the established role of PI4P in the Golgi, PI4KII is required for sorting of glue granule cargo and the granule-associated SNARE Snap24. Second, PI4KII also has an unforeseen function in late endosomes, where it is required for normal retromer dynamics and for formation of tubular endosomes that are likely to be involved in retrieving Snap24 and Lysosomal enzyme receptor protein (Lerp) from late endosomes to the trans-Golgi network. Our genetic analysis of PI4KII in flies thus reveals a novel role for PI4KII in regulating the fidelity of granule protein trafficking in secretory tissues.
    Full-text · Article · Jul 2012 · Development
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    ABSTRACT: Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein that contains enzymatically functional GTPase and kinase domains. Several noncoding LRRK2 gene polymorphisms have been associated with susceptibility to Parkinson's disease (PD), Crohn's disease, and leprosy. Many LRRK2 coding polymorphisms have been associated with or causally linked to PD. The G2019S point mutation within the LRRK2 kinase domain is the most common cause of familial PD. The G2019S mutation appears to alter LRRK2 kinase activity. Some but not all studies have reported that LRRK2 kinase activity is dependent upon LRRK2 dimerization and membrane localization. It is important to define the oligomeric state(s) of LRRK2 in living cells, which to date have only been characterized in vitro. Here we use confocal and total internal reflection microscopy coupled with number and brightness analysis to study the oligomeric states of LRRK2 within the cytosol and on the plasma membrane of live CHO-K1 cells. Our results show, for the first time to our knowledge, that LRRK2 is predominantly monomeric throughout the cytosol of living cells, but attains predominately higher oligomeric states in the plasma membrane.
    Full-text · Article · Jun 2012 · Biophysical Journal
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    ABSTRACT: Phosphatidylinositol 4-kinase IIα (PI4KIIα) is predominantly Golgi-localized, and it generates >50% of the phosphatidylinositol 4-phosphate in the Golgi. The lipid kinase activity, Golgi localization, and "integral" membrane binding of PI4KIIα and its association with low buoyant density "raft" domains are critically dependent on palmitoylation of its cysteine-rich (173)CCPCC(177) motif and are also highly cholesterol-dependent. Here, we identified the palmitoyl acyltransferases (Asp-His-His-Cys (DHHC) PATs) that palmitoylate PI4KIIα and show for the first time that palmitoylation is cholesterol-dependent. DHHC3 and DHHC7 PATs, which robustly palmitoylated PI4KIIα and were colocalized with PI4KIIα in the trans-Golgi network (TGN), were characterized in detail. Overexpression of DHHC3 or DHHC7 increased PI4KIIα palmitoylation by >3-fold, whereas overexpression of the dominant-negative PATs or PAT silencing by RNA interference decreased PI4KIIα palmitoylation, "integral" membrane association, and Golgi localization. Wild-type and dominant-negative DHHC3 and DHHC7 co-immunoprecipitated with PI4KIIα, whereas non-candidate DHHC18 and DHHC23 did not. The PI4KIIα (173)CCPCC(177) palmitoylation motif is required for interaction because the palmitoylation-defective SSPSS mutant did not co-immunoprecipitate with DHHC3. Cholesterol depletion and repletion with methyl-β-cyclodextrin reversibly altered PI4KIIα association with these DHHCs as well as PI4KIIα localization at the TGN and "integral" membrane association. Significantly, the Golgi phosphatidylinositol 4-phosphate level was altered in parallel with changes in PI4KIIα behavior. Our study uncovered a novel mechanism for the preferential recruitment and activation of PI4KIIα to the TGN by interaction with Golgi- and raft-localized DHHCs in a cholesterol-dependent manner.
    Preview · Article · Apr 2012 · Journal of Biological Chemistry
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    ABSTRACT: T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. Whereas all the CD3 subunits possess at least one ITAM, the CD3 ε subunit also contains a proline-rich sequence and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. In this study, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3) with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to accumulate stably at the immunological synapse during T cell-APC interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures after T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS-phosphoinositide interactions in supporting T cell activation.
    Full-text · Article · Jun 2011 · The Journal of Immunology
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    Preview · Article · Feb 2011 · Biophysical Journal
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    ABSTRACT: Dynamin 2 is an ubiquitously expressed ∼100 kDa GTPase involved in receptor-mediated endocytosis, Golgi budding, and cytoskeletal reorganization. Dynamin molecules assemble around the necks of budding vesicles and constrict membranes in a GTP-dependent process, resulting in vesicle release. The oligomerization state of dynamin 2 in the membrane is still controversial. We investigated dynamin 2 within the plasma membrane of live cells using total internal reflection microscopy coupled with number and brightness analysis. Our results demonstrate that dynamin 2 is primarily tetrameric throughout the entire cell membrane, aside from punctate structures that may correspond to regions of membrane vesiculation.
    Full-text · Article · Feb 2011 · Biophysical Journal
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    ABSTRACT: Mammalian cells express two isoforms of type II phosphatidylinositol 4-kinase: PI4KIIα and PI4KIIβ. PI4KIIα exists almost exclusively as a constitutively active integral membrane protein because of its palmitoylation (Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Südhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705-7708). In contrast, PI4KIIβ is distributed almost evenly between membranes and cytosol. Whereas the palmitoylated membrane-bound pool is catalytically active, the cytosolic kinase is inactive (Wei, Y. J., Sun, H. Q., Yamamoto, M., Wlodarski, P., Kunii, K., Martinez, M., Barylko, B., Albanesi, J. P., and Yin, H. L. (2002) J. Biol. Chem. 277, 46586-46593; Jung, G., Wang, J., Wlodarski, P., Barylko, B., Binns, D. D., Shu, H., Yin, H. L., and Albanesi, J. P. (2008) Biochem. J. 409, 501-509). In this study, we identify the molecular chaperone Hsp90 as a binding partner of PI4KIIβ, but not of PI4KIIα. Geldanamycin (GA), a specific Hsp90 inhibitor, disrupts the Hsp90-PI4KIIβ interaction and destabilizes PI4KIIβ, reducing its half-life by 40% and increasing its susceptibility to ubiquitylation and proteasomal degradation. Cytosolic PI4KIIβ is much more sensitive to GA treatment than is the integrally membrane-associated species. Exposure to GA induces a partial redistribution of PI4KIIβ from the cytosol to membranes and, with brief GA treatments, a corresponding increase in cellular phosphatidylinositol 4-kinase activity. Stimuli such as PDGF receptor activation that also induce recruitment of the kinase to membranes disrupt the Hsp90-PI4KIIβ interaction to a similar extent as GA treatment. These results support a model wherein Hsp90 interacts predominantly with the cytosolic, inactive pool of PI4KIIβ, shielding it from proteolytic degradation but also sequestering it to the cytosol until an extracellular stimulus triggers its translocation to the Golgi or plasma membrane and subsequent activation.
    Full-text · Article · Feb 2011 · Journal of Biological Chemistry
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    ABSTRACT: Endophilin, which participates in membrane vesiculation during receptor-mediated endocytosis, is a ∼40 kDa SH3 domain-containing protein that binds to the proline/arginine-rich domain of dynamin, a ∼100 kDa GTPase that is essential for endocytic membrane scission. It has been suggested that endophilin is monomeric in the cytoplasm and dimerizes only after it binds to membranes (or perhaps to dimers or tetramers of dynamin). To clarify this issue, we studied the oligomeric state of endophilin both in vitro using analytical ultracentrifugation and fluorescence anisotropy, and in living cells using two-photon fluorescence fluctuation spectroscopy. We analyzed the fluctuation data using the Q-analysis method, which allowed us to determine the intrinsic brightness of the labeled protein complexes and hence its aggregation state in the cytoplasmic regions of the cell. Although a relatively high K(d) (∼5-15 μM) was observed in vitro, the cell measurements indicate that endophilin is dimeric in the cytoplasm, even at submicromolar concentrations. We also demonstrate that endophilin significantly enhances the assembly of dynamin, and that this enhancement is proportional to the fraction of dimeric endophilin that is present. Moreover, there is correlation between the concentrations of endophilin that promote dynamin self-assembly and those that stimulate dynamin GTPase activity. These findings support the view that endophilin-dynamin interactions play an important role in endocytosis.
    Full-text · Article · Feb 2011 · Biophysical Journal
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    Jolene L. Johnson · Yan Chen · Joseph Albanesi · Joachim Mueller

    Full-text · Article · Feb 2011 · Biophysical Journal

Publication Stats

3k Citations
388.18 Total Impact Points

Institutions

  • 1990-2015
    • University of Texas Southwestern Medical Center
      • • Department of Pharmacology
      • • Department of Physiology
      • • Division of Cardiology
      Dallas, Texas, United States
  • 2002-2014
    • University of Texas at Dallas
      • Biochemistry
      Richardson, Texas, United States
  • 1996
    • Children's Medical Center Dallas
      Dallas, Texas, United States
  • 1984-1987
    • National Heart, Lung, and Blood Institute
      베서스다, Maryland, United States