polymerization and membrane binding of CdvA and CdvB

(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. (C) Flotation assay showing recombinant Cdv proteins binding to multilamellar vesicles. CdvA alone is distributed in all fractions, while CdvB alone is found predominantly in the liposome-bound fraction. When both CdvA and CdvB are present, both proteins are found predominantly in the liposome-bound fraction. A representative gel from 3 independent experiments is shown. (D) Schematic representing the topology of Cdv proteins reconstituted inside dumbbell-shaped liposomes, recapitulating the shape of a dividing cell. (E) Confocal image of CdvA reconstituted inside dumbbell-shaped liposomes. CdvA localizes at the neck. A representative image from 2 independent experimental preparations is shown. (F) Representative confocal image of CdvB reconstituted inside dumbbell-shaped liposomes. CdvB localizes at the neck. A representative image from 2 independent experimental preparations is shown.(G) Representative confocal image of the CdvA:CdvB complex reconstituted inside dumbbell-shaped liposomes. Both proteins localize at the neck. A representative image from 2 independent experimental preparations is shown. (H) 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.

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. (C) Liposome flotation assays showing very limited membrane binding of CdvB2ΔC alone. A representative gel from 2 independent experimental preparations is shown. (D) Microscopy image of fluorescently labelled CdvB2ΔC reconstituted inside dumbbell-shaped liposomes and localizing at necks. (E) Liposome flotation assays showing clear membrane binding of CdvB1 alone. A representative gel from 2 independent experimental preparations is shown. (F) Microscopy image of fluorescently labelled CdvB1 reconstituted inside dumbbell-shaped liposomes and localizing at necks. (G) Liposome flotation assays showing CdvB2ΔC being recruited to membranes along with CdvB1. A representative gel from 2 independent experimental preparations is shown. (H) 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 complex CdvA (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 can bind the membrane individually, but binding of CdvA is enhanced by CdvB, and the two proteins 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.