polymerization and membrane binding of the CdvA:CdvB complex

(A) Schematic depicting the domain architecture of Cdv proteins. (B) Negative staining TEM image of elongated filaments formed by CdvA upon removal of the MBP tag in the absence of DNA. Scale bar: 80 nm. (C) Coomassie staining of flotation assay. CdvA is found to not bind lipid membranes on its own (i.e. lane 1 is empty). However, in presence of CdvB, both proteins bind liposomes. (D) Schematic representing the topology of Cdv proteins reconstituted inside dumbbell-shaped liposomes, recapitulating the shape of a dividing cell. (E) Spinning disk confocal images of the CdvA:CdvB complex reconstituted inside dumbbell-shaped liposomes. CdvB is fluorescently labelled, CdvA is unlabelled. CdvB is observed to localize at the neck. (F) Same as panel e, but with fluorescently labelled CdvA and unlabelled CdvB. CdvA is observed to localize at the neck. (G) Binding of His-tagged ZipA to dumbbell liposomes containing 18:1 DGS-NTA lipids. As expected for a protein binding the membrane via a His-tag, ZipA exhibits a homogeneous membrane distribution, without enrichment at the neck. Scale bars: 10 µm.

polymerization and membrane binding of the CDVB1: CdvB2ΔC complex.

(A) Negative staining TEM image of CdvB2ΔC filaments obtained upon removal of the MBP tag. (B) Negative staining TEM image of CdvB1:CdvB2ΔC co-polymer. Scale bars: 100 nm. (C) Ratio of the end-to-end distance to contour length. Lower ratio indicates more curved filaments. (D) Liposome flotation assays showing very limited membrane binding of CdvB2ΔC alone. (E) Microscopy image of fluorescently labelled CdvB2ΔC reconstituted inside dumbbell-shaped liposomes and localizing at necks. (F) Liposome flotation assays showing clear membrane binding of CdvB1 alone. (G) Microscopy image of fluorescently labelled CdvB1 reconstituted inside dumbbell-shaped liposomes and localizing at necks. (H) Liposome flotation assays showing CdvB2ΔC being recruited to membranes along with CdvB1. (I) Microscopy image of a fluorescently labelled CdvB1:CdvB2ΔC complex reconstituted inside dumbbell-shaped liposomes and localizing at necks.

membrane binding of the CdvA:CdvB:CdvB1:CdvB2ΔC quaternary complex

(A) Liposome flotation assays showing recruitment of CdvB1 and CdvB2ΔC to membranes, either alone or in combination, by the CdvA:CdvB complex. (B) Spinning disk confocal images of the ternary complexCdvA (unlabelled) + CdvB-Alexa568 + CdvB1-Alexa488 reconstituted inside the neck of dumbbell liposomes. (C) Spinning disk confocal images of the ternary complex CdvA (unlabelled) + CdvB-Alexa568 + CdvB2ΔC-Cy5 reconstituted inside the neck of dumbbell liposomes. (D) Spinning disk confocal images of the quaternary complex CdvA (unlabelled) + CdvB-Alexa568 + CdvB1-Alexa488 + CdvB2ΔC-Cy5 reconstituted inside the neck of dumbbell liposomes. Scale bars: 10 µm. (E) Schematic depicting the stepwise assembly and disassembly of the Cdv division ring. CdvA and CdvB cannot bind the membrane individually, but they are able to assemble at the neck by forming a complex. Once the CdvA:CdvB ring is assembled, CdvB1 and CdvB2 are both recruited at the neck. Subsequently, the proteasome removes CdvA, and CdvC removes CdvB1, leaving only CdvB2, which may be removed by CdvC, thus achieving membrane abscission.