1. binding to negatively curved membranes

3. Cell biology with bacteria? 5 µm

4. Localization of cell division proteins

6. Rut Carballido-López

7. GFP-MinD

8. How do proteins localize to cell poles ? (DivIVA as model system) DivIVA-GFP

9. (lack of) Information from secondary structure prediction 164 amino acids, mostly helical coiled coil prediction by LUPAS secondary structure prediction by PSIPRED multimerization via coiled coil regions

10. Possible mechanisms: 1) binding to another (cell division) protein 2) binding to a specific lipid species 3) affinity for curved membranes

11. DG = DivIVA-GFP V = membrane vesicles Lip = liposomes D = DivIVA G = GFP Binding to another (membrane) protein? 70 % 20 % 30 % membrane vesicles

12. Biacore (surface plasmon resonance) with L1-chip T = min amphipathic helix of N-terminus (60 aa)

13. Possible mechanisms: 1) binding to another (cell division) protein 2) binding to a specific lipid species 3) affinity for curved membranes Edwards, 2000, EMBO

14. Cardiolipin Domains in Bacillus subtilis Kawai, 2003, J. Bac.

15. DivIVA localization in B. subtilis strains lacking certain lipids wt- PG- CL-PE

16. Possible mechanisms: 1) binding to another (cell division) protein 2) binding to a specific lipid species 3) affinity for curved membranes

17. Affinity for curvature = induces curvature ‘BAR domains as sensors or membrane curvature’ Peter et al., 2004, Science

18. Affinity for curvature = induces curvature ‘BAR domains as sensors or membrane curvature’ Peter et al., 2004, Science

19. Induction of curved membranes ? liposomes liposomes + DivIVA D D D DD DD D D liposomes DivIVA 200 nm

20. Induction of curved membranes ? 200 nm

21. 100 nm

22. Possible mechanisms: 1) binding to another (cell division) protein 2) binding to a specific lipid species 3) affinity for curved membranes ?

23. Does curvature really not play a role? B. subtilisE. coli

24. E. coli division mutant MHD63

25. Possible mechanisms: 1) binding to another (cell division) protein 2) binding to a specific lipid species 3) affinity for curved membranes….., but not as we know it

26. Higher order DivIVA structures Stahlberg, 2004, Mol. Mic. ( Cryo-negative stain EM ) ‘Doggy bones’ Ø ~ 25 nm

27. Ø ~ 100 nm ~ 25 nm ? ? Conceptual simplification:

28. 1) self interaction (clustering) of subunits 2) subunits should be large (relative to curvature) 3) membrane interaction (weak) ‘Molecular Bridging’ - no other proteins / lipids / or curved proteins necessary -

29. Monte Carlo simulation

30. Rules: - cylinder 1 x 4 µm - DivIVA oligomers (green) = spheres of 25 nm diameter - curvature of membranes at transition from lateral wall to sides = diameter of 100 nm - spheres can make max 8 contacts (doggy bone contains at least 8 DivIVA molecules) - 2 membrane contacts maximal (based on our EM data) - Epp and Epm in the range 1.5-6 k bT (equivalent to 1-4 kcal/mol) ~in range of typical weak protein-protein attractions

31. - spheres can make 8 contacts - 2 membrane contacts maximal - spheres can make 4 contacts - no limitations in membrane contacts

32. d = 50 nm d =100 nm - No restrictions in nr. of interactions Epp = 2 k bT Epm = 6 k bT - 4 pp bonds - membrane contact = 1 pp contact Epp = 2.5 k bT Epm = 5.5 k bT

33. d = 50 nm d =100 nm - max 4 pp bonds - membrane contact = 2 pp contact Epp = 3 k bT Emp = 5.5 k bT - max 6 pp bonds - membrane contact = 3 pp contacts Epp = 3.5 k bT Epm = 5.5 k bT

34. d = 50 nm d =100 nm -Max 8 pp bonds -membrane contact = 4 pp contacts Epp = 3.5 k bT Epm = 5.5 k bT

35. Modelling of doggy bones

36. CBCB - Newcastle University Rok Lenarcic Ling Wu Jeff Errington Sven Halbedel University of Oxford Wouter de Jong Loek Visser Michael Shaw University of Edinburgh Davide Marenduzzo