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GTPases Regulating Membrane Dynamics
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Main description:GTPases that regulate the myriad of membrane fission events that facilitate the assembly and disassembly of COPII (Sar1), COPI (ARF) and clathrin coats (dynamin) involved in exocytic and endocytic trafficking pathways and mitochrondrial organization remain a major challenge for future investigations to understand membrane architecture of eukaryotic cells. This volume of MIE provides a comprehensive set of articles describing the use and application of state-of-the-art methodologies to identify and characterize these GTPases and their rapidly expanding list of regulators and ef...
Main description:
GTPases that regulate the myriad of membrane fission events that facilitate the assembly and disassembly of COPII (Sar1), COPI (ARF) and clathrin coats (dynamin) involved in exocytic and endocytic trafficking pathways and mitochrondrial organization remain a major challenge for future investigations to understand membrane architecture of eukaryotic cells. This volume of MIE provides a comprehensive set of articles describing the use and application of state-of-the-art methodologies to identify and characterize these GTPases and their rapidly expanding list of regulators and effectors. Methodologies focused on biochemical, molecular and advanced imaging techniques provide a wealth of investigational tools for those currently in the field and those entering the field. Many of the methodologies are generally applicable to study of these GTPases in vitro and in vivo to elucidate function in regulation of cell proliferation and signaling in normal tissue and in disease.
- Comprehensive collection of GTPases (ARF, Sar1, Dynamin) GTPases involved in vesicle coat assembly and membrane fission
- Identification and characterization of GTPase GEFs, GAPs and effectors
- General methodologies to study GTPase function in vitro and in vivo
Table of contents:
siRNA cell arrays for high-content screening microscopy; Imaging platforms for measurement of membrane trafficking; Use of the harvestorR data base to annotate the human proteome for GTPase function; Microinjection as a tool to explore Small GTPase function; Visualizing intracellular events in vivo by combined video fluorescence and 3-D electron microscopy; Recombinant production in Baculovirus-infected insect cells and purification of the mammalian Sec13/Sec31 complex; Purification and properties of mammalian Sec23/24 from insect cells; Purification and functional properties of yeast Sec12 GEF; Reconstitution of cargo-dependent COPII coat assembly on proteoliposomes; Assay of COPII vesicle formation using a real-time fluorescence assay; Assay and measurement of phospholipase D activation by Sar1; Use of Acyltransferase Inhibitors to Block Vesicular Traffic Between the ER and Golgi Complex; Tethering assays for COPI Vesicles Mediated by Golgins; Investigating the Role of ADP-Ribosylation Factor 6 in Tumor Cell Invasion and Extracellular Signal-Regulated Kinase Activation; Assay and properties of the Arf GAPs AGAP1, ASAP1 and ArfGAP1; Preparation of myristoylated ARF1 and ARF6; BIG1 and BIG2, Brefeldin A-inhibited Guanine Nucleotide-exchange Factors for ADP-Ribosylation Factors (ARFs); Cytohesin-1: Structure, function, and ARF activation; ADP-Ribosylation Factor Domain Protein 1 (ARD1), a Multifunctional Protein with Ubiquitin E3 Ligase, GAP, and ARF Domains; Expression of BIG2 and Analysis of its Function in Mammalian Cells; Assay and properties of the ARF-GAP, AMAP2, in ARF6 function; Functional assay of EFA6A, a Guanine Nucleotide Exchange Factor for ADP-ribosylation Factor 6 (ARF6), in Dendritic Formation of Hippocampal Neurons; Functional assay of ARNO and ARF6 in neurite elongation and branching; Assay and functional properties of IPCEF1 as regulator of cytohesin GEFs; Assay and properties of the GIT1/p95-APP1 ARFGAP; Preparation and Characterization of Recombinant Golgin Tethers; Purification and functional properties of CtBP/Bars in membrane fission; In vitro Assays of Arf1 interaction with GGA proteins; The role of EFA6, Exhange Factor for Arf6, for tight junction assembly, functions and interaction with actin cytoskeleton; In vitro reconstitution of ARF-regulated cytoskeletal dynamics on Golgi membranes; Assay and properties of Arfaptin 2 binding to Rac1 and ADP-ribosylation Factors (ARFs); Analysis of ARF Interaction with GGAs In Vitro and In Vivo; Arf6 modulates the beta actin-specific regulator, betacap73; Functional assay of effectors of ADP ribosylation factor 6 during clathrin/AP-2 coat recruited of membranes; Assays to study Phospholipase D regulation by inositol phospholipids and ADP-ribosylation factor 6; Assay and functional properties of the tyrosine kinase Pyk2 in regulation of ARF1 through ASAP1 phosphorylation; ARF6 regulation of PI(4,5)P2 synthesis, endocytosis and exocytosis; Interaction of Arl1 GTPase with the GRIP domain of Golgin-245 as assessed by GST (glutathione-S-transferase) pull-down experiments; Functional analysis of Arl1 and Golgin-97 in endosome-to-TGN transport using recombinant Shiga toxin B fragment; Assays used in the analysis of ARL2 and its binding partners;
Assay and functional analysis of the ARL3 effector RP2 involved in X-linked retinitis pigmentosa; Reconstitution of transport to recycling endosomes in vitro; Robust colorimetric assays for dynamin's basal and stimulated GTPase activities; Clathrin-coated vesicle formation from isolated plasma membranes; Assay of biochemical properties of human guanylate-binding protein 1; Stimulation of Dynamin GTPase activity by amphiphysin; Purification and properties of Tuba, a novel dynamin-actin effector; Expression and properties of sorting nexin 9 in dynamin-mediated endocytosis; Rapid purification of native dynamin I and colorimetric GTPase assay;
Assay and functional properties of auxilin-dynamin interactions; Assay and functional analysis DLP1 in peroxisome division; In vitro reconstitution of discrete stages of dynamin-dependent endocytosis; A continuous regenerative coupled GTPase assay for dynamin-related problems; Assay and properties of the mitochondrial dynamin related protein Opa1; Assay and functional analysis of dynamin-like Mx proteins
GTPases that regulate the myriad of membrane fission events that facilitate the assembly and disassembly of COPII (Sar1), COPI (ARF) and clathrin coats (dynamin) involved in exocytic and endocytic trafficking pathways and mitochrondrial organization remain a major challenge for future investigations to understand membrane architecture of eukaryotic cells. This volume of MIE provides a comprehensive set of articles describing the use and application of state-of-the-art methodologies to identify and characterize these GTPases and their rapidly expanding list of regulators and effectors. Methodologies focused on biochemical, molecular and advanced imaging techniques provide a wealth of investigational tools for those currently in the field and those entering the field. Many of the methodologies are generally applicable to study of these GTPases in vitro and in vivo to elucidate function in regulation of cell proliferation and signaling in normal tissue and in disease.
- Comprehensive collection of GTPases (ARF, Sar1, Dynamin) GTPases involved in vesicle coat assembly and membrane fission
- Identification and characterization of GTPase GEFs, GAPs and effectors
- General methodologies to study GTPase function in vitro and in vivo
Table of contents:
siRNA cell arrays for high-content screening microscopy; Imaging platforms for measurement of membrane trafficking; Use of the harvestorR data base to annotate the human proteome for GTPase function; Microinjection as a tool to explore Small GTPase function; Visualizing intracellular events in vivo by combined video fluorescence and 3-D electron microscopy; Recombinant production in Baculovirus-infected insect cells and purification of the mammalian Sec13/Sec31 complex; Purification and properties of mammalian Sec23/24 from insect cells; Purification and functional properties of yeast Sec12 GEF; Reconstitution of cargo-dependent COPII coat assembly on proteoliposomes; Assay of COPII vesicle formation using a real-time fluorescence assay; Assay and measurement of phospholipase D activation by Sar1; Use of Acyltransferase Inhibitors to Block Vesicular Traffic Between the ER and Golgi Complex; Tethering assays for COPI Vesicles Mediated by Golgins; Investigating the Role of ADP-Ribosylation Factor 6 in Tumor Cell Invasion and Extracellular Signal-Regulated Kinase Activation; Assay and properties of the Arf GAPs AGAP1, ASAP1 and ArfGAP1; Preparation of myristoylated ARF1 and ARF6; BIG1 and BIG2, Brefeldin A-inhibited Guanine Nucleotide-exchange Factors for ADP-Ribosylation Factors (ARFs); Cytohesin-1: Structure, function, and ARF activation; ADP-Ribosylation Factor Domain Protein 1 (ARD1), a Multifunctional Protein with Ubiquitin E3 Ligase, GAP, and ARF Domains; Expression of BIG2 and Analysis of its Function in Mammalian Cells; Assay and properties of the ARF-GAP, AMAP2, in ARF6 function; Functional assay of EFA6A, a Guanine Nucleotide Exchange Factor for ADP-ribosylation Factor 6 (ARF6), in Dendritic Formation of Hippocampal Neurons; Functional assay of ARNO and ARF6 in neurite elongation and branching; Assay and functional properties of IPCEF1 as regulator of cytohesin GEFs; Assay and properties of the GIT1/p95-APP1 ARFGAP; Preparation and Characterization of Recombinant Golgin Tethers; Purification and functional properties of CtBP/Bars in membrane fission; In vitro Assays of Arf1 interaction with GGA proteins; The role of EFA6, Exhange Factor for Arf6, for tight junction assembly, functions and interaction with actin cytoskeleton; In vitro reconstitution of ARF-regulated cytoskeletal dynamics on Golgi membranes; Assay and properties of Arfaptin 2 binding to Rac1 and ADP-ribosylation Factors (ARFs); Analysis of ARF Interaction with GGAs In Vitro and In Vivo; Arf6 modulates the beta actin-specific regulator, betacap73; Functional assay of effectors of ADP ribosylation factor 6 during clathrin/AP-2 coat recruited of membranes; Assays to study Phospholipase D regulation by inositol phospholipids and ADP-ribosylation factor 6; Assay and functional properties of the tyrosine kinase Pyk2 in regulation of ARF1 through ASAP1 phosphorylation; ARF6 regulation of PI(4,5)P2 synthesis, endocytosis and exocytosis; Interaction of Arl1 GTPase with the GRIP domain of Golgin-245 as assessed by GST (glutathione-S-transferase) pull-down experiments; Functional analysis of Arl1 and Golgin-97 in endosome-to-TGN transport using recombinant Shiga toxin B fragment; Assays used in the analysis of ARL2 and its binding partners;
Assay and functional analysis of the ARL3 effector RP2 involved in X-linked retinitis pigmentosa; Reconstitution of transport to recycling endosomes in vitro; Robust colorimetric assays for dynamin's basal and stimulated GTPase activities; Clathrin-coated vesicle formation from isolated plasma membranes; Assay of biochemical properties of human guanylate-binding protein 1; Stimulation of Dynamin GTPase activity by amphiphysin; Purification and properties of Tuba, a novel dynamin-actin effector; Expression and properties of sorting nexin 9 in dynamin-mediated endocytosis; Rapid purification of native dynamin I and colorimetric GTPase assay;
Assay and functional properties of auxilin-dynamin interactions; Assay and functional analysis DLP1 in peroxisome division; In vitro reconstitution of discrete stages of dynamin-dependent endocytosis; A continuous regenerative coupled GTPase assay for dynamin-related problems; Assay and properties of the mitochondrial dynamin related protein Opa1; Assay and functional analysis of dynamin-like Mx proteins