Bacterial division proteins FtsZ and ZipA induce vesicle shrinkage and cell membrane invagination

Abstract

Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles.

Keywords: Bacterial Membrane; Cell Division; Escherichia coli; FtsZ; Giant Vesicles; Membrane Function; Protein-Protein Interactions; Synthetic Biology; ZipA.

FIGURE 1.

Encapsulation and polymerization of FtsZ inside permeable vesicles (A) and vesicle shrinkage and collapse induced by interaction of membrane bound ZipA with FtsZ polymers (B). A, panel a shows an equatorial confocal image of permeable vesicles containing 12 μm FtsZ-Alexa Fluor 488 in the presence of GDP; the membrane layer is stained with the lipid dye DiIC₁₈. Panel b shows a similar image of FtsZ polymers after addition of α-hemolysin, GTP, and magnesium, whereas panel c is a full reconstruction of all confocal sections. The differential interference contrast image of the vesicle containing FtsZ polymers is shown in panel d. B, equatorial cross-sectional merged images of permeable vesicles containing 5 μm sZipA-Alexa Fluor 647 attached to the membrane through DOGS-NTA lipids and 12 μm Alexa Fluor 488-FtsZ in the absence (top row) or presence of the CTZ-MUT peptide inhibitor of FtsZ-ZipA interaction (middle row). Vesicles similar to those shown in top row containing 2 μm sZipA-Alexa Fluor 647 are shown in the bottom row. Frames were taken at the times (seconds) indicated after external addition of GTP and Mg²⁺. Further details are under “Results.” Movies reconstructed from the whole collection of images can be found in supplemental Movies S1–S4).

FIGURE 2.

Membrane invagination of vesicle during shrinkage induced by interaction of membrane bound ZipA with FtsZ polymers in the presence of GMP-CPP. Equatorial cross-sectional images show a permeable vesicle containing 10 μm sZipA-Alexa Fluor 647 (middle row) attached to the membrane through DOGS-NTA lipids and 12 μm FtsZ-Alexa Fluor 488 (upper row) in the presence of 1 mm GMP-CPP. Columns show the frames that were taken at the times (seconds) indicated after GMP-CPP and Mg²⁺ addition. Merged images are shown in bottom row. Supplemental Movies S5 and 6 show vesicle collapse mediated by the interaction of GMPCPP-FtsZ polymers and ZipA bound to the inner face of the membrane. The two movies correspond to the time course of the collapse progression as detected by observing FtsZ (supplemental Movie S5) or ZipA (supplemental Movie S6).

FIGURE 3.

Effects of elevated ZipA levels on ultracellular structure. The four left side columns show immunofluorescence images obtained from cells in which expression of an extrachromosomal copy of zipA had been induced during 0 (top row), 60, 120, 150 min (bottom row) before fixation. The images have been developed using antibodies against ZipA and Alexa Fluor 594 anti-rabbit conjugated to detect the total amount of ZipA (first column), against His tail and Alexa Fluor 488-anti-mouse conjugated to specifically detect the overproduced protein (second column). The third and fourth columns are overlays showing the overproduced protein over the total protein (third column) and the DAPI-stained nucleoid image over the total amount of ZipA (fourth column). The panels on the right show transmission electron micrographs of thin sections of cells overproducing ZipA. The top row is a longitudinal section of a cell before zipA induction. The second row is a longitudinal section of a segment of a filamentous cell after 150-min induction of zipA. The two framed sections reproduced at higher magnification are shown in the third row. The bottom row shows immunogold staining of ZipA in a filamentous cell 150 min after induction of zipA. See “Results” for growth and induction conditions.

FIGURE 4.

Effects of elevated ZipA levels on localization of other cell division proteins. Immunofluorescence images were obtained from cells in which expression of an extrachromosomal copy of zipA had been induced during 0 (top row) or 150 min (bottom row) before fixation. The images have been developed using antibodies against FtsZ, FtsA, or FtsN and Alexa Fluor 594 anti-rabbit conjugated to detect FtsZ (first column), against His tail and Alexa Fluor 488-anti-mouse conjugated to specifically detect the overproduced His-ZipA protein (second column). The third and fourth columns are overlays showing the overproduced His-ZipA protein (third column) and the DAPI-stained nucleoid image (fourth column) over FtsZ, FtsA, or FtsN.

FIGURE 5.

Modification of membrane permeability during ZipA overproduction. Shown is the percentage of propidium iodide-stained cells present at different times in a population in which ZipA is overproduced. Error bars indicate the S.D.