Imaging DivIVA dynamics using photo-convertible and activatable fluorophores in Bacillus subtilis

Abstract

Most rod-shape model organisms such as Escherichia coli or Bacillus subtilis utilize two inhibitory systems for correct positioning of the cell division apparatus. While the nucleoid occlusion system acts in vicinity of the nucleoid, the Min system was thought to protect the cell poles from futile division leading to DNA-free miniature cells. The Min system is composed of an inhibitory protein, MinC, which acts at the level of the FtsZ ring formation. MinC is recruited to the membrane by MinD, a member of the MinD/ParA family of Walker-ATPases. Topological positioning of the MinCD complex depends on MinE in E. coli and MinJ/DivIVA in B. subtilis. While MinE drives an oscillation of MinCD in the E. coli cell with a time-dependent minimal concentration at midcell, the B. subtilis system was thought to be stably tethered to the cell poles by MinJ/DivIVA. Recent developments revealed that the Min system in B. subtilis mainly acts at the site of division, where it seems to prevent reinitiation of the division machinery. Thus, MinCD describe a dynamic behavior in B. subtilis. This is somewhat inconsistent with a stable localization of DivIVA at the cell poles. High resolution imaging of ongoing divisions show that DivIVA also enriches at the site of division. Here we analyze whether polar localized DivIVA is partially mobile and can contribute to septal DivIVA and vice versa. For this purpose we use fusions with green to red photoconvertible fluorophores, Dendra2 and photoactivatable PA-GFP. These techniques have proven very powerful to discriminate protein relocalization in vivo. Our results show that B. subtilis DivIVA is indeed dynamic and moves from the poles to the new septum.

Keywords: DivIVA; PA-GFP; dendra2; division-site selection; photoactivation; photoconversion.

Figure 1

Time lapse analysis reveals DivIVA-GFP dynamics in B. subtilis. Cells expressing DivIVA-GFP under its native promoter were grown on agarose slides supplemented with LB at 37°C and analyzed microscopically. Pictures were taken every 2 min. After division DivIVA-GFP is mostly located at midcell or at one pole (white arrow). After 14 min DivIVA-GFP is recruited to new forming septa (red and blue arrow) and fluorescence intensity at the old septa decreases stepwise. For plotting the fluorescence measured at the old and new formed septa ROIs of identical size were drawn and the mean fluorescence of every spot was calculated individually.

Figure 2

Fluorescence recovery after photobleaching of DivIVA-GFP. Cells expressing DivIVA-GFP under control of the native promoter were grown on agarose slides supplemented with LB. (A) DIC images of B. subtilis cells expressing DivIVA-GFP directly after a bleaching event and after 18 min are shown (B) Heat maps of the GFP signal and time lapse images indicate fluorescence distribution (C). DivIVA-GFP was bleached (red circles) as described in material and methods. Pictures were taken every 3 min after the bleaching event. Images show GFP fluorescence. (D) Quantification of the recovery rate of bleached spots; n = 6.

Figure 3

DivIVA-PA-GFP is dynamically recruited from the cell pole to the septa. DivIVA-PA-GFP fluorescence was imaged before photo-activation using DIC and FITC specific filters. Photoactivation was performed using a laser at 405 nm (red circle). After photoactivation DivIVA-PA-GFP (white arrow) is localized at the cell pole. Although, the signal gets more diffuse over time, accumulation at a new septum after 20 min becomes evident (red arrow). A cartoon of DivIVA-PA-GFP dynamics is drawn below. The relative fluorescence of the pole (white arrow) and the new formed septa (red arrow) was measured. The relative fluorescence of the according spots were calculated (CTF was calculated and the highest CTF of all spots were set as 1) and plotted. For every time point spots were chosen individually.

Figure 4

DivIVA-Dendra2 is dynamically recruited from the cell pole to the septa. DivIVA-Dendra2 fluorescence (green and red) was imaged before photoconversion using DIC, FITC, or TRITC specific filters. After photoconversion using a 405 nm laser (cyan circles) only red fluorescence (TRITC) and DIC was monitored to prevent additional photoconversion. After 5 min DivIVA-Dendra2 is recruited from the place of photoconversion (black and blue circle) to new septa forming (red arrow). A cartoon of the photoconversion is shown. The relative fluorescence of the left pole (black circle), the right pole (blue circle), and the new formed septa (red arrow) was measured. The relative fluorescence of the corresponding spots were calculated (CTF was calculated and the highest CTF of each spot was set as 1) and plotted. For every time point spots were chosen individually. Heat maps and corresponding histograms are shown below.

Figure 5

Model of DivIVA dynamics in B. subtilis. During cytokinesis DivIVA rings at constricting septa collapse into foci/patches (Eswaramoorthy et al., 2011). Exponentially growing B. subtilis cells redeploy at least a fraction of DivIVA molecules from old division sites (cell poles) to nascent septa (broken white lines). Only in non-dividing cells DivIVA is clearly seen as accumulation at both cell poles.