Membrane Trafficking

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This is a summary of a presentation our CEO has given at Northwestern University, Chicago - U.S.A. in 2003. Many aspects of protein targeting and membrane trafficking in eukaryotic cells are discussed.
Tags: Gokay-BIOTECH, K.Erden Gokay, Cell Biology, protein sorting, endotubin

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Membrane Trafficking

  1. 1. Principles of Membrane Trafficking in Polarized Epithelia K. Erden GOKAY, M.D.,K. Erden GOKAY, M.D., Ph.D.Ph.D. Ay-GRUP SSA Ltd., Turkey Research Scientist
  2. 2. Classic (old) view:Classic (old) view:  All endosomes eventually get acidified (maturation) and deliver their cargo to the Lysosome.
  3. 3. EGF-R trafficking:EGF-R trafficking:  Ligand triggers endocytosis.  Eventually both the ligand and the receptor are degraded.
  4. 4. LDL-R trafficking:LDL-R trafficking:  Ligand dissociates from the receptor upon delivery to a relatively acidic compartment.  Ligand and receptor are sorted into distinct compartments where ligand is degraded but the receptor is recycled.
  5. 5. Trf-R trafficking:Trf-R trafficking:  Receptor is constitutively internalized.  The ligand is exposed to a relatively acidic environment.  Both the ligand and the receptor are recycled.
  6. 6. PIg-R trafficking:PIg-R trafficking:  Receptor is targeted basolaterally for ligand binding.  Ligand induces internalization.  Ligand bound receptor is delivered apically for apical secretion of the ligand (s-IgA).
  7. 7. Proper membrane trafficking isProper membrane trafficking is required for:required for:  Metabolic regulation: Trf-R, LDL-R, Glut-4  Regulation of signaling pathways: EGF-R, βAR, Furin (TGFβ)  Antigen presentation: MHC-II  Neurotransmitter metabolism: SSV regeneration (Dynamin)  Generation and maintenance of cell polarity: PIg-R (sIgA) {epithelial integrity}
  8. 8. Endosomes are a heterogeneous group ofEndosomes are a heterogeneous group of compartments:compartments:
  9. 9. Principles of Membrane Trafficking:Principles of Membrane Trafficking:  Coat assembly and budding  Fission and transport  Un-coating and priming  Docking and fusion
  10. 10. A common sorting site?A common sorting site? •Transcytotic cargo as well as basolaterally recycling cargo has been shown to meet in an apical compartment (ARE) prior to sorting,
  11. 11. Question:Question: •How does apicalHow does apical endomembraneendomembrane trafficking differ fromtrafficking differ from basolateral trafficking?basolateral trafficking? •What is the nature ofWhat is the nature of apical endosomalapical endosomal targeting cue(s)?targeting cue(s)?
  12. 12. Endotubin:Endotubin: 1195 a.a. single TMD type I glycoprotein with a short (36aa) cytoplasmic domain.
  13. 13. Endotubin expression in the native tissue:Endotubin expression in the native tissue:
  14. 14. A model polarized simple epithelium:A model polarized simple epithelium: • Madin-Darby Canine Kidney (MDCK) cell line. {Renal Collecting tubule like}
  15. 15. Endotubin expression inEndotubin expression in polarized MDCK cells:polarized MDCK cells:
  16. 16. Endotubin-positive structures are apicalEndotubin-positive structures are apical derived early endosomes:derived early endosomes: • 20 min Apical Ricin uptake • 30 min Basolateral Ricin uptake • Anti-LAMP-1
  17. 17. Endotubin-positive endosomes do notEndotubin-positive endosomes do not contain Transferrin:contain Transferrin: • Apical • Perinuclear • Basolateral
  18. 18. Endotubin-positive endosomes do notEndotubin-positive endosomes do not label with Rab-11:label with Rab-11:
  19. 19. Endotubin positive apical endosomalEndotubin positive apical endosomal compartment is distinct from the ARE:compartment is distinct from the ARE:  Red = Transferrin  Green = Endotubin
  20. 20. Coat assembly is a regulated process:Coat assembly is a regulated process: • Arf family of small GTPases regulate Coatomer (Cop-1, Cop-2) assembly onto the donor compartment membrane. • Arf-GEF = ARNO (Cytohesin)
  21. 21. Brefeldin A wash-out:Brefeldin A wash-out: 2-min 10-min
  22. 22. Transferrin Pulse-Chase:Transferrin Pulse-Chase: Transferrin bound to cell surface receptors transiently co localizes with endotubin in non- polarized MDCK cells.
  23. 23. Biogenesis of endotubin-positive apicalBiogenesis of endotubin-positive apical endosomes:endosomes:  Endotubin targets into an apical derived early endosomal compartment distinct from the common (apical) recycling endosome.
  24. 24. Site directed mutagenesis:Site directed mutagenesis:
  25. 25. Targeting of endotubin mutants:Targeting of endotubin mutants: STOP1180 T1186A E1189A I1178A/L1179A • Apical • Perinuclear • Basal
  26. 26. Targeting of endotubin mutants:Targeting of endotubin mutants: E1189A F1180A Apical Ricin uptake for 15 minutes
  27. 27. Domain selective Biotinylation:Domain selective Biotinylation:
  28. 28. TT11861186 is phosphorylated:is phosphorylated: 1: MDCK 2: wt-endotubin 3: STOP1180 4: T1186A
  29. 29. Conclusions:Conclusions:  In polarized epithelia endotubin targets into an apical early endosomal compartment distinct from the ARE.  This targeting process relies on cytoplasmic signals present on intracellular domain of the molecule.  Apical targeting and endosomal targeting (internalization) signals are independent from each other.
  30. 30. Other possible targeting signals:Other possible targeting signals:  Association with glycolipid rich rafts.  Differential glycosylation (N-linked and O- linked) of the ectoplasmic domain.  Other protein-protein interactions (lectin like sorting protein) ?
  31. 31. Endotubin does not partition intoEndotubin does not partition into raft domains:raft domains:
  32. 32. Endotubin - Tac chimeras:Endotubin - Tac chimeras:
  33. 33. Tac vs Tac-ET expression:Tac vs Tac-ET expression: Tac Wildtype Tac-ET C1
  34. 34. Transferrin - BfA:Transferrin - BfA: Tac-ET C1 Tac Wildtype
  35. 35. Tac-ET is present in apical earlyTac-ET is present in apical early endosomes:endosomes: Tac-ET (C1) Apical Ricin 15 min Merge
  36. 36. Targeting of Tac-ET chimeras inTargeting of Tac-ET chimeras in polarized MDCK cells:polarized MDCK cells: Tac-WT C1 C2 C3 • Apical • Perinuclear • Basolateral
  37. 37. Domain selective Biotinylation:Domain selective Biotinylation:
  38. 38. C4 chimera:C4 chimera:
  39. 39. Acknowledgements:Acknowledgements:  * Jean M. WILSON, Ph.D.  Carol GREGORIO, Ph.D.  Paul St. JOHN, Ph.D.  Mani RAMASWAMI, Ph.D.  Tamara Lee COLTON, M.S.  Jennifer SALATA • The University of Arizona, Dept. of Cell Biology and Anatomy. Tucson - ARIZONA
  40. 40. Arf is required for maintenance ofArf is required for maintenance of apical early endosomes:apical early endosomes:  Brefeldin-A treatment causes fusion and tubulation of Endotubin positive endosomes with basolateral early endosomes.  When the drug is washed out, proper polarized sorting resumes.
  41. 41. Membrane trafficking and cell polarity:
  42. 42. Endosomal compartments:Endosomal compartments:  Model polarized epithelial cell.
  43. 43. Glucose transporters:  In the human genome so far there are 11 GLUT isoforms identified (facilitative hexose transporter gene family) but there is only one insulin-responsive transporter is known.  In theory there are 3 ways by which insulin may modulate Glut-4 function: - Altering its transport activity - Upregulating its expression - Altering its endomembrane trafficking
  44. 44. Glut-4 trafficking:  At steady state the transporter targets to endosomes.  Upon initiation of insulin signaling activation of PI-3 kinase, PKCζ and PKB (Akt) cascades result in exocytosis of the transporter.
  45. 45. Furin trafficking:  Furin is an endopeptidase required for cleavage (secretion) of TGF-β from the transmembrane precursor.  Phosphorylation of Furin cytoplasmic domain via CK-II alters its trafficking pathway.
  46. 46. Synaptic vesicle regeneration:  Following neurotransmitter release, rapid endoctosis is required to regenerate SSVs.  Shibire (Dynamin-ts) mutation in the fly is characterized with a temperature sensitive paralysis.
  47. 47. Basic concepts in membrane trafficking:  Despite high degree of regulation and specificity, isolated trafficking events are not error-proof (reliably unreliable).  However, sorting efficiency is boosted up via iterative sorting.  There is high degree of redundancy in membrane trafficking regulators.  Membrane trafficking events are quite plastic and alternate pathways do exist.
  48. 48. Budding and fusion cycle:  Aggregation and coat assembly.  Budding and transport.  Priming (un- coating).  Docking and fusion.
  49. 49. Role players:  Adaptors and coat regulators: Adaptins, Arf, ARNO  Coat proteins: Coatomer, Clathrin, Caveolin  Pinching-off and Priming: Dynamin, Rab, Rabaptin, Rabphylin, Arf-GAF  Docking molecules: t-SNAREs, v-SNAREs  Fusion proteins: NSF, SNAP
  50. 50. Clathrin coated pits:  Clathrin pre-assembles into triskelions composed of 3 heavy and 3 light chains.  Upon cargo binding, adaptins (AP-1, AP-2 and AP-3) mediate clathrin triskelions to polymerize into clathrin cages.
  51. 51. A look to the plasma membrane inner surface:
  52. 52. Dynamin dominant negative mutant:  Dynamin is a GTPase required for vesicle budding.
  53. 53. SNAREs are not sufficient for membrane fusion:  NSF is an ATPase that is requred for membrane fusion.
  54. 54. Endomembrane trafficking pathways are highly conserved: MammalianMammalian Yeast homologYeast homolog  Rab 1a Ypt1p  Arf Sar1p  ARNO Sec7p  NSF Sec18p

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