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

Bach JN, Albrecht N, Bramkamp M - Front Microbiol (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology I, Ludwig-Maximilians-University Munich, Planegg-Martinsried, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Protein mobility in vivo can be quantified with fluorescence recovery after photo-bleaching (FRAP). We bleached part of the DivIVA-GFP signal at one cell pole or at the septum and measured reoccurrence of GFP signals. Within few minutes the majority of the signal recovered in the bleached area (Figures 2A–D). This recovery is in contrast to results published earlier (Eswaramoorthy et al., 2011) where FRAP experiments were reported to have only little recovery. However, images in that publication reveal already slight recovery after 1 min (Eswaramoorthy et al., 2011). In order to rule out the possibility that recovery of fluorescence might exclusively be due to new synthesis of DivIVA-GFP, we blocked protein biosynthesis (see material and methods) before performing FRAP experiments. Strikingly, recovery of DivIVA-GFP in cells with inhibited protein biosynthesis was almost identical to the FRAP experiments without inhibitor (Figure S1). These results support that DivIVA dynamically reassembles at new division sites with material recruited from cell poles.


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

Bach JN, Albrecht N, Bramkamp M - Front Microbiol (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927310&req=5

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.
Mentions: Protein mobility in vivo can be quantified with fluorescence recovery after photo-bleaching (FRAP). We bleached part of the DivIVA-GFP signal at one cell pole or at the septum and measured reoccurrence of GFP signals. Within few minutes the majority of the signal recovered in the bleached area (Figures 2A–D). This recovery is in contrast to results published earlier (Eswaramoorthy et al., 2011) where FRAP experiments were reported to have only little recovery. However, images in that publication reveal already slight recovery after 1 min (Eswaramoorthy et al., 2011). In order to rule out the possibility that recovery of fluorescence might exclusively be due to new synthesis of DivIVA-GFP, we blocked protein biosynthesis (see material and methods) before performing FRAP experiments. Strikingly, recovery of DivIVA-GFP in cells with inhibited protein biosynthesis was almost identical to the FRAP experiments without inhibitor (Figure S1). These results support that DivIVA dynamically reassembles at new division sites with material recruited from cell poles.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology I, Ludwig-Maximilians-University Munich, Planegg-Martinsried, Germany.

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.

No MeSH data available.


Related in: MedlinePlus