Dynamic SpoIIIE assembly mediates septal membrane fission during Bacillus subtilis sporulation

Abstract

SpoIIIE is an FtsK-related protein that transports the forespore chromosome across the Bacillus subtilis sporulation septum. We use membrane photobleaching and protoplast assays to demonstrate that SpoIIIE is required for septal membrane fission in the presence of trapped DNA, and that DNA is transported across separate daughter cell membranes, suggesting that SpoIIIE forms a channel that partitions the daughter cell membranes. Our results reveal a close correlation between septal membrane fission and the assembly of a stable SpoIIIE translocation complex at the septal midpoint. Time-lapse epifluorescence, total internal reflection fluorescence (TIRF) microscopy, and live-cell photoactivation localization microscopy (PALM) demonstrate that the SpoIIIE transmembrane domain mediates dynamic localization to active division sites, whereas the assembly of a stable focus also requires the cytoplasmic domain. The transmembrane domain fails to completely separate the membrane, and it assembles unstable foci. TIRF microscopy and biophysical modeling of fluorescence recovery after photobleaching (FRAP) data suggest that this unstable protein transitions between disassembled and assembled oligomeric states. We propose a new model for the role of SpoIIIE assembly in septal membrane fission that has strong implications for how the chromosome terminus crosses the septum.

Figure 1.

SpoIIIE is required for septal membrane fission. (A) Cartoon showing membrane and DNA dynamics during sporulation and the structure of ΔspoIIIE sporangia. SpoIIIE is shown in green (Wu and Errington 1997; Sharp and Pogliano 1999). (B,C) Protoplast assays for septal membrane fission in wild type (B) and ΔspoIIIE (C), showing cell membranes (red) and DNA (green). (Chevrons) Sporangia that generated separate protoplasts; (arrowheads) septa that retracted; (arrows) protoplasts that separated after septal retraction (see also Supplemental Movies S1, S2). (D,E) FRAP assays for septal membrane fission on a mixed culture of CFP-tagged wild-type (blue) and YFP-tagged ΔspoIIIE (green) cells. (D) Prebleach image also showing FM 4-64-stained membranes in red. (E) Membranes (white) imaged at various times show no recovery for wild type (top) and rapid recovery for ΔspoIIIE (bottom) forespore membrane fluorescence. Photobleaching of the bleached region (red circle) occurred at 0 sec. (F) Diagrams illustrate membrane structure of wild type and ΔspoIIIE allowing diffusion from the mother cell to the forespore (arrows), and SpoIIIE^ATP− (green) blocking diffusion. (G) Plot showing the cFR (see the Materials and Methods) of forespore membrane fluorescence for wild-type (blue) and ΔspoIIIE (green) sporangia. Each green and blue line represents fluorescence recovery of an individual forespore. The thick black lines show the average recovery for each genotype. (H) Plot showing the average cFR and standard deviation of wild-type, ΔspoIIIE, and whole-cell bleach events (WC bleach) for 84 sec of recovery time. (I) Average cFR for spoIIIE^ATP− (red) with standard deviation error bars compared with wild-type average (blue).

Figure 2.

Septal fission defects occur only in ΔspoIIIE sporangia with trapped DNA. (A) Plot of cFR for ΔracA sporangia with (green) or without (black) DNA in the forespore show similarly limited fluorescence recovery. (Gray curve) Whole-cell bleach control. (B) Plot of cFR for ΔspoIIIE ΔracA sporangia with DNA in the forespore (green) show recovery, and those without DNA (black) show limited recovery similar to wild type. (C) Cartoon showing possible membrane and DNA configurations for ΔspoIIIE ΔracA and the impact on FM 4-64 diffusion (arrows).

Figure 3.

Role of transmembrane and cytoplasmic domains in septal fission and SpoIIIE assembly. (A–D) Average cFR for wild type (blue) and ΔspoIIIE (green) are shown as references. Plot of cFR for individual cells (black lines) expressing SpoIIIE^1–192 (A), SpoIIIE^Fis− (B), and SpoIIIE^Fis−ATP− (C). (D) Average cFR and standard deviations for spoIIIE^{1–192 Fis−} (orange). (E–J) Fluorescent images of membrane (red) and SpoIIIE-GFP (green) for wild type (E), SpoIIIE^ATP− (F), SpoIIIE^1–192 (G), SpoIIIE^Fis− (H), SpoIIIE^Fis−ATP− (I), and SpoIIIE^{1–192 Fis−} (J). The third panel is a three-dimensional plot of the normalized GFP intensity in each field. (K) Histogram of average GFP intensity for wild-type and mutant proteins, with 99% CI error bars. (L,M) Rank correlation plot of GFP focus intensity versus FRAP recovery in individual cells expressing SpoIIIE^Fis−-GFP (L) and SpoIIIE^Fis−ATP− GFP (M), with the brightest focus and the lowest cFR ranked separately as 1 (Supplemental Table S1). The black line of best fit and Spearman's rank correlation (Rs values of 0.576 and 0.697, with P < 0.05 for SpoIIIE^Fis and SpoIIIE^Fis−ATP) show a correlation between dim foci and rapid recovery.

Figure 4.

SpoIIIE dynamics. (A) Time-lapse epifluorescence microscopy of SpoIIIE-GFP. Images of FM 4-64 staining (top) and GFP fluorescence (bottom) were collected every 90 sec with time (minutes) shown below the images. The asterick denotes the apparent completion of septation, based on membrane images (see also Supplemental Movie S5). (B,C) Diagrams show construction of septal kymographs in cells with unstable (B) or stable (C) foci. Slices the width of septum were taken for each time point and then stacked, with the first time point at the top and the last at the bottom. (D–G) TIRF microscopy of SpoIIIE-GFP (D,E) and SpoIIIE^1–192-GFP (F,G) sporangia with clearly incomplete septa (D,F) or flat septa (E,G), based on FM 4-64 staining. Images were collected approximately every 0.5 sec for >37 sec. Septal kymographs for nine individual cells, including three incomplete and six flat septa, were generated by stacking 1 × 18 pixel slices of the septum for each time. Arrowheads indicate the times on the kymographs that correspond to the full cell images shown to the left of the kymographs. The first image shows the membrane, and the three following images show TIRF GFP fluorescence at the indicated times (see also Supplemental Movies S6, S7).

Figure 5.

SpoIIIE localization by live-cell PALM. (A–I) Localization of tdEOS fusions to SpoIIIE (A–C), SpoIIIE^ATP− (D–F), and SpoIIIE^1–192 (G–I) at incomplete (I) and flat (II and III) sporulation septa visualized by FM 5-95 staining. PALM images of SpoIIIE-tdEOS (B,E,H) and diffraction-limited image of membranes (gray) were overlaid (A,D,G) to show the localization of SpoIIIE molecules at the septum (arrowhead). Enlarged PALM images (C,F,I) show individual SpoIIIE-tdEOS molecules not apparent in the diffraction-limited images (B,E,H, inset). Bars: A, 1 μm; C, 0.2 μm. (J,K) The percent of cells with noted range of tdEOS molecules for constricting (class I) and flat septa (class II and III). (L) Number and percent of sporangia with tdEOS fusions proteins at constricting and flat septa. For class 1, a SpoIIIE cluster at one or both sides of an incomplete septum was scored as localized. For cells with flat septa, the presence of a SpoIIIE cluster that spread across the septum or more than one cluster were scored as class II, and those with one compact cluster were scored as class III. Only cells with >11 tdEOS molecules were included in classes I–III. tdEOS molecules with localization precision <50 nm were selected for analysis.

Figure 6.

Modeling FM 4-64 diffusion in various cell geometries. (A) Modeling of FRAP on the poles of cells 2.8 μm long by 0.854 μm wide, represented by a cylindrical surface with hemispherical poles. Simulated recovery curves (green) follow the functional form . (A) The simulated cFR (cFR^S, green) of lipid molecules with a diffusion constant D = 0.4 μm²/sec in a vegative cell geometry closely matches experimental data (blue circles) and has a recovery time constant of τ_1/2 = 1 sec (exponential fit shown as dashed line). (B–D) FRAP simulations of an invaginated cell with a pore radius of 10 nm (see Supplemental Fig. S6 for other pore radii). (B) cFR^S of lipids with D = 0.4 μm²/sec predicts a recovery time constant of τ_1/2 = 5 sec that also closely matches ΔspoIIIE experimental data (blue circles). (C) cFR^S assuming a leaky complex with a range of diffusion constants, D, at the tip of the septal invagination. (D) cFR^S with different probabilities (p) of SpoIIIE assembly and consequent inhibition of diffusion.

Figure 7.

Model for SpoIIIE function in septal membrane fission. Diagrams of septation show the whole cell (A), with the region of interest indicated by red circles, and the septum as a side view (B) and as a top view (C), showing the lipid bilayer (green circles), DNA (gold), and SpoIIIE (blue). SpoIIIE transmembrane domain is shown as a wedge in the lipid bilayer, with the linker as a black line and the motor domain as a sphere. (Step 1) Initially, SpoIIIE dynamically localizes to the leading edge of constricting septa, but does not block lipid diffusion. As the membrane contacts DNA, the SpoIIIE transmembrane domain assembles around the trapped DNA (step 2) forming an unstable channel that can separate membranes (step 3). (Step 4) The cytoplasmic motor domain then assembles on the DNA (likely only in the mother cell) (Becker and Pogliano 2007), stabilizing the channel and forming a channel that blocks diffusion between the daughter cell membranes during DNA translocation. We show this structure to be a paired channel, although other arrangements are possible. (Step 5) After DNA translocation, the motor domain releases DNA, and the complex disassociates to allow the terminus across the septum. (Step 6) Finally, irreversible membrane fission occurs.