The Experts below are selected from a list of 111 Experts worldwide ranked by ideXlab platform

Frances M. Brodsky - One of the best experts on this subject based on the ideXlab platform.

  • Adaptor self-aggregation, adaptor-receptor recognition and binding of Alpha-Adaptin subunits to the plasma membrane contribute to recruitment of adaptor (AP2) components of clathrin-coated pits.
    The EMBO journal, 1993
    Co-Authors: M.p. Chang, William G. Mallet, Keith E. Mostov, Frances M. Brodsky
    Abstract:

    Initiation of receptor-mediated endocytosis by nucleation of clathrin-coated pits involves binding of AP2 adaptor molecules to the plasma membrane. This process was reconstituted in vitro, using plasma membrane fragments, prepared by freeze-thaw lysis of cells, and stripped of their endogenous coat proteins, as targets for binding of purified adaptor molecules and their dissociated subunits. The dissociated Alpha-Adaptin subunit of AP2 bound to plasma membrane fragments, while the dissociated beta-Adaptin subunit did not, suggesting that plasma membrane localization of AP2 adaptors is mediated by Alpha-Adaptin. Membrane binding of intact AP2 adaptor molecules was enhanced by adaptor self-aggregation, which can be modulated by physiological concentrations of inositol phosphates, and may therefore be sensitive to receptor signaling. Adaptor binding was partially inhibited by soluble peptides representing the cytoplasmic domains of the asialoglycoprotein receptor and the polymeric immunoglobulin receptor. These results indicate that direct binding of adaptors to the cytoplasmic domains of receptors contributes to coated pit nucleation but this appears to be a weak interaction, suggesting that an additional recognition signal could be required for high affinity adaptor binding.

  • 100-KD PROTEINS OF GOLGI AND TRANS-GOLGI NETWORK-ASSOCIATED COATED VESICLES HAVE RELATED BUT DISTINCT MEMBRANE-BINDING PROPERTIES
    The Journal of cell biology, 1992
    Co-Authors: D H Wong, Frances M. Brodsky
    Abstract:

    The 100-110-kD proteins (Alpha-, beta-, beta'-, and gamma-Adaptins) of clathrin-coated vesicles and the 110-kD protein (beta-COP) of the nonclathrin-coated vesicles that mediate constitutive transport through the Golgi have homologous protein sequences. To determine whether homologous processes are involved in assembly of the two types of coated vesicles, the membrane binding properties of their coat proteins were compared. After treatment of MDBK cells with the fungal metabolite Brefeldin A (BFA), beta-COP was redistributed to the cytoplasm within 15 s, gamma-Adaptin and clathrin in the trans-Golgi network (TGN) dispersed within 30 s, but the Alpha-Adaptin and clathrin present on coated pits and vesicles derived from the plasma membrane remained membrane associated even after a 15-min exposure to BFA. In PtK1 cells and MDCK cells, BFA did not affect beta-COP binding or Golgi morphology but still induced redistribution of gamma-Adaptin and clathrin from TGN membranes to the cytoplasm. Thus BFA affects the binding of coat proteins to membranes in the Golgi region (Golgi apparatus and TGN) but not plasma membranes. However, the Golgi binding interactions of beta-COP and gamma-Adaptin are distinct and differentially sensitive to BFA. BFA treatment did not release gamma-Adaptin or clathrin from purified clathrin-coated vesicles, suggesting that their distribution to the cytoplasm after BFA treatment of cells was due to interference with their rebinding to TGN membranes after a normal cycle of disassembly. This was confirmed using an in vitro assay in which gamma-Adaptin binding to TGN membranes was blocked by BFA and enhanced by GTP gamma S, similar to the binding of beta-COP to Golgi membranes. These results suggest the involvement of GTP-dependent proteins in the association of the 100-kD coat proteins with membranes in the Golgi region of the cell.

Alan L Schwartz - One of the best experts on this subject based on the ideXlab platform.

  • alterations in the protein composition of maturing phagosomes
    Journal of Clinical Investigation, 1992
    Co-Authors: A Pitt, Luis S. Mayorga, Philip D Stahl, Alan L Schwartz
    Abstract:

    We investigated the protein composition of J774-E clone macrophage phagosomes isolated at different stages of phagolysosome biogenesis. Phagosomes formed by internalizing antibody-coated Staphylococcus aureus for 3 min followed by chase for 0, 4, 9, or 15 min were isolated by density gradient centrifugation. Enrichment and purity of the phagosome preparations were quantitated by radiolabeled ligand recovery, enzyme markers, and electron microscopy. One-dimensional SDS-PAGE analyses of the isolated phagosomes revealed virtually identical protein compositions. However, Western blot analyses with antibodies directed against selected proteins of known itineraries along the endocytic pathway demonstrated distinct differences in phagosome protein compositions. Accumulating within the maturing phagosome were the 31-kD subunit of the vacuolar proton pump, cathepsin D,beta-glucuronidase, the cation dependent mannose 6-phosphate receptor, and LAMP-1. Decreasing within the maturing phagosome were the FcII receptor, the mannose receptor, and Alpha-Adaptin. These results indicate that although the macrophage phagosome's total protein composition changes little during phagolysosome formation, the maturing phagosome both receives and eliminates, possibly by protein recycling, specific membrane and sequestered proteins.

  • Alterations in the protein composition of maturing phagosomes
    1992
    Co-Authors: A Pitt, Luis S. Mayorga, Philip D Stahl, Alan L Schwartz
    Abstract:

    We investigated the protein composition of J774-E clone mac-rophage phagosomes isolated at different stages of phagolyso-some biogenesis. Phagosomes formed by internalizing anti-body-coated Staphylococcus aureus for 3 min followed by chase for 0, 4, 9, or 15 min were isolated by density gradient centrifu-gation. Enrichment and purity of the phagosome preparations were quantitated by radiolabeled ligand recovery, enzyme markers, and electron microscopy. One-dimensional SDS-PAGE analyses of the isolated phagosomes revealed virtually identical protein compositions. However, Western blot analy-ses with antibodies directed against selected proteins of known itineraries along the endocytic pathway demonstrated distinct differences in phagosome protein compositions. Accumulating within the maturing phagosome were the 31-kD subunit of the vacuolar proton pump, cathepsin D, jl-glucuronidase, the cation dependent mannose 6-phosphate receptor, and LAMP-1. De-creasing within the maturing phagosome were the FcII recep-tor, the mannose receptor, and Alpha-Adaptin. These results indicate that although the macrophage phagosome's total pro-tein composition changes little during phagolysosome forma-tion, the maturing phagosome both receives and eliminates, possibly by protein recycling, specific membrane and seques

Juergen A Knoblich - One of the best experts on this subject based on the ideXlab platform.

  • The Endocytic Protein α-Adaptin Is Required for Numb-Mediated Asymmetric Cell Division in Drosophila
    Developmental cell, 2002
    Co-Authors: Daniela Berdnik, Marcos González-gaitán, Tibor Török, Juergen A Knoblich
    Abstract:

    During asymmetric cell division in Drosophila sensory organ precursor cells, the Numb protein localizes asymmetrically and segregates into one daughter cell, where it influences cell fate by repressing signal transduction via the Notch receptor. We show here that Numb acts by polarizing the distribution of Alpha-Adaptin, a protein involved in receptor-mediated endocytosis. Alpha-Adaptin binds to Numb and localizes asymmetrically in a Numb-dependent fashion. Mutant forms of Alpha-Adaptin that no longer bind to Numb fail to localize asymmetrically and cause numb-like defects in asymmetric cell division. Our results suggest a model in which Numb influences cell fate by downregulating Notch through polarized receptor-mediated endocytosis, since Numb also binds to the intracellular domain of Notch.

A Pitt - One of the best experts on this subject based on the ideXlab platform.

  • alterations in the protein composition of maturing phagosomes
    Journal of Clinical Investigation, 1992
    Co-Authors: A Pitt, Luis S. Mayorga, Philip D Stahl, Alan L Schwartz
    Abstract:

    We investigated the protein composition of J774-E clone macrophage phagosomes isolated at different stages of phagolysosome biogenesis. Phagosomes formed by internalizing antibody-coated Staphylococcus aureus for 3 min followed by chase for 0, 4, 9, or 15 min were isolated by density gradient centrifugation. Enrichment and purity of the phagosome preparations were quantitated by radiolabeled ligand recovery, enzyme markers, and electron microscopy. One-dimensional SDS-PAGE analyses of the isolated phagosomes revealed virtually identical protein compositions. However, Western blot analyses with antibodies directed against selected proteins of known itineraries along the endocytic pathway demonstrated distinct differences in phagosome protein compositions. Accumulating within the maturing phagosome were the 31-kD subunit of the vacuolar proton pump, cathepsin D,beta-glucuronidase, the cation dependent mannose 6-phosphate receptor, and LAMP-1. Decreasing within the maturing phagosome were the FcII receptor, the mannose receptor, and Alpha-Adaptin. These results indicate that although the macrophage phagosome's total protein composition changes little during phagolysosome formation, the maturing phagosome both receives and eliminates, possibly by protein recycling, specific membrane and sequestered proteins.

  • Alterations in the protein composition of maturing phagosomes
    1992
    Co-Authors: A Pitt, Luis S. Mayorga, Philip D Stahl, Alan L Schwartz
    Abstract:

    We investigated the protein composition of J774-E clone mac-rophage phagosomes isolated at different stages of phagolyso-some biogenesis. Phagosomes formed by internalizing anti-body-coated Staphylococcus aureus for 3 min followed by chase for 0, 4, 9, or 15 min were isolated by density gradient centrifu-gation. Enrichment and purity of the phagosome preparations were quantitated by radiolabeled ligand recovery, enzyme markers, and electron microscopy. One-dimensional SDS-PAGE analyses of the isolated phagosomes revealed virtually identical protein compositions. However, Western blot analy-ses with antibodies directed against selected proteins of known itineraries along the endocytic pathway demonstrated distinct differences in phagosome protein compositions. Accumulating within the maturing phagosome were the 31-kD subunit of the vacuolar proton pump, cathepsin D, jl-glucuronidase, the cation dependent mannose 6-phosphate receptor, and LAMP-1. De-creasing within the maturing phagosome were the FcII recep-tor, the mannose receptor, and Alpha-Adaptin. These results indicate that although the macrophage phagosome's total pro-tein composition changes little during phagolysosome forma-tion, the maturing phagosome both receives and eliminates, possibly by protein recycling, specific membrane and seques

Antony P Jackson - One of the best experts on this subject based on the ideXlab platform.

  • Alpha-Adaptin, a marker for endocytosis, is expressed in complex patterns during Drosophila development.
    Molecular biology of the cell, 1997
    Co-Authors: S Dornan, Antony P Jackson
    Abstract:

    A Drosophila cDNA encoding a structural homologue of the mammalian coated vesicle component Alpha-Adaptin (AP2 adaptor complex) has been cloned and sequenced. The mammalian and invertebrate sequences are highly conserved, especially within the amino terminal region, a domain that mediates interactions with other components within the AP2 complex and with specific receptors tails. Mammalian Alpha-Adaptins are encoded by two genes; however, Drosophila Alpha-Adaptin has a single gene locus, within polytene bands 21C2-C3 on the left arm of the chromosome 2, closely adjacent to the paired homeobox gene aristaless. There seem to be at least two Drosophila Alpha-Adaptin transcripts expressed, plausibly by alternative splicing. One of the transcripts is more abundant during early embryogenesis and may be of maternal origin. We have studied the distribution of the Alpha-Adaptin protein throughout embryogenesis and at the neuromuscular junction of the third instar larva. During cellularization of the blastoderm embryo, the protein is seen between and ahead of the elongating nuclei, and then redistributes to the cell surface during gastrulation. These observations suggest a role for endocytosis in cellularization and are consistent with the finding that dynamin (the shibire gene product), another component of the endocytic mechanism, is required for cellularization. At later stages of embryogenesis, Alpha-Adaptin is expressed in complex and dynamic patterns. It is strongly induced in elements of the central and peripheral nervous system (e.g., in neuroblasts, the presumptive stomatogastric nervous system, and the lateral chordotonal sense organs), in the Garland cells, the adult midgut precursors, the antenno-maxillary complex, the endoderm, the fat bodies, and the visceral mesoderm. In the larva, Alpha-Adaptin is localized at the plasma membrane in the synaptic boutons of the neuromuscular junctions. The cells expressing high levels of Alpha-Adaptin are known or expected to support high levels of endocytosis; thus, this coated vesicle protein seems to be an excellent marker for endocytic activity. The expression patterns of dynamin, detected in the embryo by in situ hybridization methods, are very similar to those reported here for Alpha-Adaptin reflecting the likely coordinated expression of endocytic components. Taken together with previous evidence, our results suggest that endosomal vesicle trafficking, membrane recycling, and the regulation of endocytosis play critical roles in the wide range of developmental processes.