1. Dynamic networks & clathrin-mediated endocytosis Gerrit Praefcke (now at Cologne) Marijn Ford (now at UC Davis) Eva Schmid (LMB Cambridge)

2. What is a Hub? Are they static? Why have them?

3. At the synapse speed and fidelity are important to ensure the quantal nature and reliability of synaptic vesicle exocytosis speedfidelity

4. Exo Endo Fidelity?What is

5. Clathrin-mediated endocytosis The overall process is a series of linear steps but at the same time it is a series of simultaneous micro-reactions (e.g. cargo recruitment, membrane invagination and coat assembly occurring in parallel)

6. Clathrin-mediated endocytosis The overall process is a series of linear steps but at the same time it is a series of simultaneous micro-reactions (e.g. cargo recruitment, membrane invagination and coat assembly occurring in parallel)

7. Clathrin-mediated endocytosis The overall process is a series of linear steps but at the same time it is a series of simultaneous micro-reactions (e.g. cargo recruitment, membrane invagination and coat assembly occurring in parallel) Involving clathrin, adaptors (AP2) and at least 20 different accessory proteins

8. The endocytic interactome Hubs Accessory Proteins (over 20 different proteins bind to the AP2  -appendage)

9. The AP2 hub binds to accessory proteins via it appendage domains

10. The  -appendage: two independent binding sites Top Site Side Site W840 F740 Peptide containing an FxDxF motif Binds with an affinity of 4.6  M Peptide containing a WVxF motif Binds with an affinity of 0.7  M

11. Motif domains Structured domains Protein:protein interaction domains with no obvious tertiary structure Contain multiple motifs, short amino acid sequences, Please don’t call them ‘unstructured domains’ as they may have some secondary structure!! Endocytic accessory proteins have a similar overall structure Epsin

12. Motif domains Structured domains AP2  -motifs

13. Eps15 affinity for the  -appendage Eps15 Motif Domain

14. Eps15 affinity for the  -appendage contains 15 repeats of the sequence DPF

15. Eps15 affinity for the  -appendage So not all 15 motifs are available for simultaneous interactions + 1 site of 20nM 2-3 sites of 16  M

16. Eps15 affinity for the  -appendage 1 site of 20nM 2-3 sites of 16  M From mutagenesis we know that the 20nM affinity is due to occupation of both top and side sites of one appendage Thus this is a novel way to gain high affinity yet a readily reversible interaction… ie. 2 linear peptides linked by a flexible linker 20nM 16  M

17. Eps15 affinity for the  -appendage 1 site of 20nM 2-3 sites of 16  M Eps15 with its simultaneous interactions with 4 appendage domains could help to cluster AP2s at sites of endocytosis

18. Motif domains are not unstructured and linear But neither are they stable globular domains. They are designed to package motifs in an efficient manner, such that when one motif is occupied then further motifs are exposed ‘structural cooperativity’ in motif binding Motif domain  -appen- dage Motif

19. This low structural stability means that these motif domains can search a wide range of space for potential ligands

20. Like a fishing line with lots of hooks…… But for entropic and statistical reasons the domain will prefer a more compact fold And thus the hooks will gather ligands back to the core folded domains

21. A novel way to gain relatively high affinity and yet reversibility Give rise to dynamic instability (a necessary characteristic of many cellular processes) Allow cross-linking/multimerisation of binding targets Efficient packaging of many different interactions surfaces Multiple interactions that filter noise A way to search space and draw ligands to a point Motif:domain interactions

22. The network behaviour makes sense….. Clathrin is an organising hub, not a protein recruitment hub. This ensures that empty clathrin cages do not form in the absence of membranes and cargo AP2 does not self assemble, and only weakly binds to cargo. This ensures that cargo recruitment, membrane bending and polymerisation are tightly coupled.

23. Properties of endocytic and other biological networks Noise reduction: Low affinity interactions ensure that processes are only activated on coincidence of several signals Information processing: The multimeric state of the AP2 hub allows it to bind multiple ligands according to their relative affinities and concentrations. Thus the hub integrates information. The competition between AP2 and clathrin also means that there is a sensing of the commitment along the endocytic pathway (the process gestalts). (feed forward and competitive loops)

24. Building the network around AP2….

25. There are 4 potential ligand interaction sites on each AP2 complex

26. Thus 4 potential ligand interaction sites on each AP2 complex. Does this make it a HUB?

27. Thus 4 potential ligand interaction sites on each AP2 complex. Does this make it a HUB? No

28. It is the concentration of AP2s on the membrane that gives it the ability to bind multiple partners according to affinities and concentrations AP2 hub zone

29. Changing hubs gives directionality AP2 in solution Recruitment of AP2 to membrane and concentration Clathrin polymerisation

30. The clathrin hub Amph WxxW  3 adaptor hinge LLDLD Miele et al 2004 Ter Haar et al 2002

31. Changing hubs gives directionality AP2 in solution Recruitment of AP2 to membrane and concentration Clathrin polymerisation Only on self-polymerisation does clathrin become a hub

32. Clathrin binding to the  -appendage displaces ligands, pushing accessory proteins to the edge of a clathrin-coated pit (appendage assembly zones) Clathrin  -appendage

33. Clathrin binding to the  -appendage displaces ligands, pushing accessory proteins to the edge of a clathrin-coated pit (appendage assembly zones) Clathrin terminal domain

34. Clathrin binding to the  -appendage displaces ligands, pushing accessory proteins to the edge of a clathrin-coated pit (appendage assembly zones)

36. How clathrin-coated pits mature… affinityaviditymatricity Sequential displacement of core and accessory proteins (affinity matures to avidity matures to matricity) The process is pulled forward from the end

37. How clathrin-coated pits mature… affinityaviditymatricity

38. How clathrin-coated pits mature… Sequential displacement of core and accessory proteins (affinity matures to avidity matures to matricity) The process is pulled forward from the end ATP GTP

39. AP2 A AP2 adaptors sense lipids, cargo, accessory proteins and other cargo adaptors A Network view of clathrin-coated vesicle formation

40. AP2 B Building the cage: AP2 network hub is stabilized through crosslinking by accessory proteins

41. AP2 C Clathrin is recruited and polymerisation stabilises the forming vesicle. AP2 loses its position as a hub. Clathrin is the new organising hub

42. AP2 D Dynamin and other late interacting partners (like uncoating factors) start to function The point of no return.

43. AP2 E Energy is used to re- prime the system for a new start.

44. Changing hubs gives directionality AP2 in solution Recruitment of AP2 to membrane and concentration Clathrin polymerisation Only on self-polymerisation does clathrin become a hub Note: in a clathrin-coated pit one has a snap- shot of the network at several different stages

45. AP2 hubs and clathrin hubs co-exist at the same time, but spatially separated

46. In a coated-pit there may even be the beginning stages of uncoating, as the lipid phosphatase begins to work under the clathrin lattice

47. This means that fluorescent imaging will frequently not have the resolution to deduce the time dependence of recruitment

48. But… we can deduce this information from the path-length in the network……

49. A short path-length gives an immediate response To put a time delay in the response an additional interaction step is added Cage formation Vesicle scission Uncoating and repriming of molecules TimeTime Early and late events can be predicted… 1 2 3 3’

50. This view maintains that: Overexpression of a pathway hub will have little phenotype Underexpression of a pathway hub will have a major phenotype Overexpression of an accessory node will have a major phenotype Underexpression of an accessory node will have little phenotype Hubs