Clathrin-mediated Endocytosis

1. Clathrin-mediated Endocytosis Friday Jan. 13, 2006 Sandra Schmid slschmid@scripps.edu Phone 784-2311

2. The plasma membrane is a communication device

3. Endocytosis is critical for all aspects of cell physiology - ensures efficient uptake of nutrients - attenuates signaling from receptor tyrosine kinases and G-protein coupled receptors - mediates immune responses by bulk or specific sampling of external environment - remodels PM constituents to change how cells respond to their environment (e.g. for acute alterations in expression of signaling receptors, membrane transporters, cell adhesion molecules) - cleans up cell debris at sites of inflammation, repair and/or during development - provides a pathway for invasion by pathogens and toxins

4. Multiple portals of entry into the mammalian cell control how cells interact with their environment Emerging concept: Endocytosis is cargo driven Recent review: Conner and Schmid (2003) Nature 422:37-42

5. Clathrin-mediated endocytosis

6. Clathrin-mediated endocytosis: • Ubiquitous and most active endocytic pathway. May account for bulk of ‘fluid-phase’ endocytosis. • Endocytic machinery is well-defined (i.e. clathrin, adaptors, dynamin, numerous accessory proteins). • Clathrin assembly/clathrin coats drive membrane invagination and cargo selection. • Sorting signals on cytoplasmic tails of receptors are well-defined (Y-based, diLeu, ubiquitin?). • Multiple cargo-selective adaptors (a growing list) decode endocytic sorting signals. Some are cell type and/or receptor specific (ARH, -arrestins, grb2)

7. Methods to study clathrin-mediated endocytosis: • In vivo: • follow ligand internalization by resistance to surface stripping, surface quenching • assay by cell associated radioactivity, fluorescence (FACS, microscopy), or streptavidin/biotin • Live cell microscopy to follow GFP-clathrin directly • (confocal microscopy or TIR-FM/epi) • In vitro: • Ligand uptake in perforated cells or from purified PM • Coat assembly and fission on model liposomes

8. In vitro assay for clathrin-mediated endocytosis using perforated cells Quench external with avidin Transferrin (T) biotinylated with a cleavable disulfide bond Reduce disulfide with MesNa or TCEP

9. Rat liver PM sheets (stored @ -80°C) Incubate @ 37°C with Cytosol, ATP, GTP Centrifuge 20 k•g x 1 min @ 4°C Centrifuge 110 k•g x 10 min @ 4°C LSP HSP Endocytic CCV formation from purified PM LSS

10. LSP HSP buffer only 20% 10% complete 4°C LRP TfnR ASGPR SR-B1 Cargo Receptors are Selectively Incorporated into the High Speed Pellet Fraction

11. Intensity Distance ~1500 nm Clathrin Coated Vesicle Formation in living cells Visualized by Near Simultaneous TIR-FM/WF-epi TIR-FM WF-epi WF epi-fluorescence WFandTIR-FM TIR-FM

12. Membrane fission/endocytosis in living cells expressing pH-sensitive GFP-transferrin-R (TfnR-phl) TfnR-phlourin 1st frame pH 7.4 Subsequent pH 5.5 Merrifield et al., 2005 Cell 121:593

14. Clathrin, AP2 and dynamin are necessary but not sufficient for CME Miwako and Schmid (2006)

15. A growing list of endocytic accessory proteins Clathrin assembly Cargo Recognition Curvature sensing/ generating Actin regulatory Coat disassembly Dual role for Dynamin SNX9 Slepnev and DeCamilli (2000)Nat. Rev. Neuro. 1:165

16. Clathrin assembly Cargo recognition A growing list of endocytic accessory proteins Clathrin assembly Cargo Recognition Curvature sensing/ generating Actin regulatory Coat disassembly Dual role for Dynamin SNX9 Slepnev and DeCamilli (2000)Nat. Rev. Neuro. 1:165

17. Clathrin triskelions self-assemble to form a closed polygonal lattice Fotin, Harrison, et al. Nature 2004 432:573-579

18. The multifunctional AP2 adaptor complex plays a central role in clathrin-mediated endocytosis D.J. Owen, B.M. Collins, P.R. Evans ­ ADAPTORS FOR CLATHRIN COATS: Structure and Function Annual Review of Cell and Developmental Biology Vol. 20: 153-191rett M. Collins1, and ­ Philip R. Evans2

19. 863 628 312 NPF DLL DPF AAK1 is activated by assembled clathrin and couples cargo recruitment to coat assembly Kinase Q/P/A-rich AID Conner and Schmid, JCB 2002, 2003

20. …and resistant to inhibition by DN-AAK1 Conner and Schmid 2003 JCB 162:773 Clathrin-mediated endocytosis of some cargo receptors is AP2 independent! Motley et al. 2003 JCB 162:909 Transferrin EGF CD8-LDLR Internalized ligand (% of surface bound)

21. A growing number of cargo-selective adaptor molecules: Endocytosis is cargo driven Review by Traub 2003 JCB 163:203

22. A growing list of endocytic accessory proteins Clathrin assembly Cargo Recognition Curvature sensing/ generating Actin regulatory Coat disassembly Dual role for Dynamin SNX9 Slepnev and DeCamilli (2000)Nat. Rev. Neuro. 1:165

23. Vesicle formation requires curvature generating activities Curvature-sensing activities can establish distinct functional domains on an emerging bud

24. Alternate mechanisms for generating and/or sensing curvature e.g. LPAAT activity PI-kinases/phosphatases Phospholipase D, A2 e.g. ANTH, ENTH, ALPS Domain-containing proteins e.g. AP2/clathrin lattices (via PIP2-binding) Dynamin BAR domain-containing proteins

25. and can generate curvature at high concentrations BAR domain dimers sense curvature (amphiphysin, endophilin, SNX9)

26. PH GED PRD GTPase Middle Dynamin self-assembly drives tubulation Zhang and Hinshaw 2001 NCB 3:922

27. Stahelin, R. V. et al. J. Biol. Chem. 2003;278:28993-28999 ANTH and ENTH domains can sense or generate curvature with amphiphatic helices (AP180, epsin)

28. Curvature-sensitive activities can establish functionally distinct domains on emerging buds Positive curvature Negative curvature Antonny, B. Nature, 2003; EMBO J 2006,

29. Actin regulatory A growing list of endocytic accessory proteins Clathrin assembly Cargo Recognition Curvature sensing/ generating Actin regulatory Coat disassembly Dual role for Dynamin SNX9 Slepnev and DeCamilli (2000)Nat. Rev. Neuro. 1:165

30. Transient actin assembly associated with CCP dynamics Dafi Yarar

31. Actin assembles as coated vesicles form Dafi Yarar

32. latA Jasp Are actin assembly/disassembly dynamics required for endocytic CCV formation?

33. Disruption of Actin Dynamics Blocks Clathrin Dynamics

34. 80% reduction in CCP formation 80% reduction in internalization Disruption of Actin Dynamics Inhibits Internalization and Formation of CCPs Dafi Yarar

35. Actin Dynamics are Required at Multiple Stages of Clathrin-mediated Endocytosis assembly constriction fission and translocation actin Yarar et al. MBC (2005) 16:974

36. Coat disassembly A growing list of endocytic accessory proteins Clathrin assembly Cargo Recognition Curvature sensing/ generating Actin regulatory Coat disassembly Dual role for Dynamin SNX9 Slepnev and DeCamilli (2000)Nat. Rev. Neuro. 1:165

37. Auxilin 2/GAK AAK1-related kinase PTEN Clathrin binding J-domain The uncoating reaction: Hsc70 (the uncoating ATPase) Auxilin Dynamin binding

38. Auxilin places and activates hsc70 at the vertices Hsc70 interacts with and destabilizes tripod Interactions at the vertex

39. PTEN-like PIP binding Specificity? AAK1-related kinase Clathrin binding J-domain Auxilin 2/GAK Auxilin 1 How is the uncoating reaction regulated? (i.e. how does hsc70 distinguish a coated vesicle from a deeply-invaginated coated pit?) Auxilin regulates hsc70 uncoating activityy Dynamin binding Auxilin binds to dynamin•GTP: does this regulate auxilin activity? Synaptojanin (PI5 phosphatase) depletion inhibits uncoating Auxilin may recognize PIPs: does this regulate auxilin activity?

40. A growing list of endocytic accessory proteins Clathrin assembly Cargo Recognition Curvature sensing/ generating Actin regulatory Coat disassembly Dual role for Dynamin SNX9 Slepnev and DeCamilli (2000)Nat. Rev. Neuro. 1:165

41. Dynamin = Shibire Shibire flies (shits) provided the first evidence for dynamin’s involvement in endocytosis 19°C 29°C Shibire = dynamin

42. Collared pits accumulate at nerve terminal in shibirets flies Kazuo Ikeda

43. Model 1: Dynamin is a mechanochemical GTPase Assembly-stimulated GTPase activity drives a conformational change – akin to a powerstroke – that generates force necessary for membrane fission Hinshaw and Schmid, Nature, 1995

44. GED is required for assembly and functions as an assembly-stimulated GAP

45. For mechanochemical enzymes GAP-activity drives a Powerstroke For regulatory GTPases GAP activity Negatively regulates GTPase function The critical step in the NTPase cycle differs For Motors and GTPases

46. Dynamin GED mutants stimulate the formation of constricted coated pits GED negatively regulates dynamin function in vivo

47. Model 2: Dynamin is a regulatory GTPase • Self-assembly activates GED (a GAP), terminating dynamin’s interactions with effector molecules essential for CV formation Sever, Muhlberg and Schmid, Nature, 1999

48. Conflicting models? Dynamin is a mechanochemical enzyme Dynamin is a regulatory GTPase Hinshaw and Schmid, 1995 Sever, Mulhberg and Schmid, 1999

49. shits2 Suppressors of Shibire (Sushi)(Mani Ramaswami, U. Arizona) Suppressors of shibire (Sushi): • Fully rescue all shibire mutant phenotypes • Sushi mutations are intra-allelic: • One mapped to the GTPase domain • Two mapped to the GED domain sushi Narayanan et al 2005 JCB 169:117

50. shits2 (G146S) Switch 2 P-loop Switch 1 G4 C N Shits2(G146S) maps to Switch 2 • GAPs and/or effectors interact with the Switch 2 region of GTPases