Mrr cmb journal_club_20111115
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Mrr cmb journal_club_20111115

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FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network - Mol. Biol. Cell. 2010 21(17): 3041 – 3053

FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network - Mol. Biol. Cell. 2010 21(17): 3041 – 3053

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  • STEC, HUS, assemble in ER, apoptosis
  • Different cargo, different golgins

Mrr cmb journal_club_20111115 Mrr cmb journal_club_20111115 Presentation Transcript

  • FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport fromRecycling Endosomes to the trans-Golgi Network Mol. Biol. Cell. 2010 21(17): 3041 – 3053 Presented by Mariam Rizkallah Cell and Molecular Biology Course, The American University in Cairo November 15, 2011 Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • OutlineMariam Rizkallah - BIOT502 - Nov 15, 2011
  • Introduction: Vesicular TrafficImage credit: Molecular Cell Biology, Fifth Edition © 2003 W.H. Freeman and Company Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Introduction: Vesicular Traffic Nascent Peptides Fate From ER Enzymes and structural Transport vesicles proteins remain in ER bud to cis-Golgi Vesicle membrane proteins Cisternal maturation recycled or missorted through Golgi bud back to ER Golgi-resident proteins Secretion Lysosome membrane retrotransport to Golgi (regulated or soluble proteins or constitutive) bud to late endosome Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Introduction: Where to Go?• Soluble NSF Attachment Protein REceptor (SNAREs) crucial for cargo sorting• Membrane budding from donor organelle, and fusion with target organelle• Vesicle internal membrane proteins (v-SNAREs) join with target membrane proteins (t-SNAREs) Image credit: Molecular Cell Biology, Fifth Mariam Rizkallah - BIOT502 - Nov 15, 2011 Edition © 2003 W.H. Freeman and Company
  • Introduction: Where to Go?• Rab (GTPase superfamily), and Rab-mediated docking• VAMP (vesicle-associated membrane protein) as type of v-SNAREs• Syntaxin (integral) and SNAP-25 (attached) as types of t-SNAREs• SNARE coiled-coil complexes• NSF-catalyzed hydrolysis of ATP and dissociation of the SNARE complexesImage credit: Molecular Cell Biology, Fifth Edition © 2003 W.H. Freeman and Company Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • The Trans-Golgi Network (TGN)•Extension of the trans-Golgi•Sorting station (proteins and lipids)•Golgins: regulate transport and maintain structure•Cargo is either:- Transported to lysosomes (mannose 6-phosphate receptor)- Packaged for secretion The intracellular structures known at the- Reside TNG (Golgins) Golgi complex are involved in protein•Problem: Which Golgin regulates processing and secretion. what transport? Image credits: Golgi - Biology Encyclopedia Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Model for FIP1/RCP Function in Retrograde TransportImage credits: http://adoptamicrobe.blogspot.com/2006/06/escherichia-coli.html Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Retrograde Transport of TNG38, STxB and VAMP4• Major players: - Rab11: Retrograde transport regulation - Golgin-97: TNG’s t-SNAREs (GRIP domain and FIP1/RCP-binding/coiled-coil domain) - FIP1/RCP: RE’s v-SNAREs Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Who Did It?• Identifying the major players through: - Knockdown of Rab11a/b or its interacting protein FIP1/RCP to prove that EE/RE –and not EE/LE— to TNG transport is Rab11-mediated [Control: ] - Investigation of FIP1/RCP direct binding to Golgin- 97 in vitro and in vivo to prove that binding is required for the retrograde transport from EE/RE – and not EE/LE— to the TGN [Control: CI-MPR6] Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Results and Conclusions1. Rab11 Is Required for the Localization of TGN38 to TGNRationale: Investigation of the role of Rab11Methods:HeLa cells-Tac-TNG38 transfected with siRNA againstRab11a and/or bKnockdown of both a/b resulted in: - Retention of TNG38 - Redistribution of FIP1/RCP to cytoplasm - Decrease in FIPs levels (FIP1/RCP and FIP5/Rip11) Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Results: Rab11 Is Required for the Localization of TGN38 to TGNImmunoblottingLocalization of FIP1/RCP Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Results: Rab11 Is Required for the Localization of TGN38 to TGN E-G: Mock Staining With Antibodies H-J: FIP1/RCP Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Results and Conclusions2. FIP1/RCP Is Required for Retrograde TGN38 TransportRationale:Investigation of role of different FIPsMethods:HeLa cells-Tac-TNG38 transfected with siRNA againstKnockdown of FIP5/Rip11, FIP1/RCP, and FIP3 resulted in: - Redistribution of Tac-TGN38 from TGN toperinuclear punctuate organelles Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Results: FIP1/RCP Is Required for Retrograde TGN38 Transport Immunoblotting Localization of FIP1/RCP Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Results: FIP1/RCP Is Required for Retrograde TGN38 Transport C-E: Mock F-H: FIP1/RCP KD I-M: Higher magnification Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Results: FIP1/RCP Is Required for Retrograde TGN38 Transport Slides from 17-29 were not prepared HeLa cells treated with either mock siRNA or FIP1/RCP siRNA Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • Conclusion1. Identified Golgin-97 as binding partner for FIP1/RCP (Immunoppt and yeast two-hybrid and glutathione bead pulldown assays)2. Knockdown results in accumulation of TGN38 in endocytic organelles containing the retromer complex (SNX1), EpsinR, and transferrin receptor.3. Interaction between Golgin-97 and FIP1/RCP suggests that FIP1/RCP may act as a tethering factor, which mediates EE/RE-derived retrograde carrier docking and subsequent fusion at the TGN. Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • References• Jing, J., Junutula, J. R., Wu, C., Burden, J., Matern, H., Peden, A. A., & Prekeris, R. (2010). FIP1/RCP binding to Golgin-97 regulates retrograde transport from recycling endosomes to the trans-Golgi network. Molecular biology of the cell, 21(17), 3041-53. doi:10.1091/mbc.E10-04-0313• Lee, M.-S., Cherla, R. P., & Tesh, V. L. (2010). Shiga Toxins: Intracellular Trafficking to the ER Leading to Activation of Host Cell Stress Responses. Toxins, 2(6), 1515-35. Molecular Diversity Preservation International. doi:10.3390/toxins2061515 Mariam Rizkallah - BIOT502 - Nov 15, 2011
  • QuestionsMariam Rizkallah - BIOT502 - Nov 15, 2011
  • Mariam Rizkallah - BIOT502 - Nov 15, 2011