binding to negatively curved membranes

Cell biology with bacteria? 5 µm

Localization of cell division proteins

Rut Carballido-López

GFP-MinD

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

(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

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

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

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

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

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

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

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

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

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

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

Induction of curved membranes ? 200 nm

100 nm

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

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

E. coli division mutant MHD63

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

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

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

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 -

Monte Carlo simulation

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 k bT (equivalent to 1-4 kcal/mol) ~in range of typical weak protein-protein attractions

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

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

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

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

Modelling of doggy bones

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