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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

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.
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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.

Mentions: Milder conditions are apparently needed to avoid phototoxic effects. Therefore, we turned our attention to a photoconvertible fluorophore, Dendra2 (Gurskaya et al., 2006; Chudakov et al., 2007). Dendra2 is a monomeric protein with a green-to-red photoconversion upon blue light exposure (Gurskaya et al., 2006). Dendra2 was reported to fold efficiently in bacteria and its photostability makes it ideal for long-term protein tracking (Gurskaya et al., 2006). We have constructed a DivIVA-Dendra2 and analyzed localization of the translational fusion protein in growing cells (Figure 4). Green fluorescence was readily observed at cell poles and septa, undistinguishable from the DivIVA-GFP fusion. DivIVA-Dendra was converted by a very fast (0.05 s) 405 nm laser flash. Immediately after the conversion time lapse analysis revealed the generation of a red fluorophore at the site of conversion. Since imaging of green fluorescence at 488 nm exposure slowly, but significantly convert more DivIVA-Dendra2 from green to red, we only followed the red signal. Clearly, the red, converted DivIVA redistributed and accumulated over time at a new septum that was formed (Figure 4 and Figure S2). Control experiments using the same imaging conditions without laser event revealed no photoconversion (data not shown). Thus, DivIVA-Dendra2 which was converted at an ongoing site of division or a cell pole redistributes to new division sites.


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

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

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

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

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.
Mentions: Milder conditions are apparently needed to avoid phototoxic effects. Therefore, we turned our attention to a photoconvertible fluorophore, Dendra2 (Gurskaya et al., 2006; Chudakov et al., 2007). Dendra2 is a monomeric protein with a green-to-red photoconversion upon blue light exposure (Gurskaya et al., 2006). Dendra2 was reported to fold efficiently in bacteria and its photostability makes it ideal for long-term protein tracking (Gurskaya et al., 2006). We have constructed a DivIVA-Dendra2 and analyzed localization of the translational fusion protein in growing cells (Figure 4). Green fluorescence was readily observed at cell poles and septa, undistinguishable from the DivIVA-GFP fusion. DivIVA-Dendra was converted by a very fast (0.05 s) 405 nm laser flash. Immediately after the conversion time lapse analysis revealed the generation of a red fluorophore at the site of conversion. Since imaging of green fluorescence at 488 nm exposure slowly, but significantly convert more DivIVA-Dendra2 from green to red, we only followed the red signal. Clearly, the red, converted DivIVA redistributed and accumulated over time at a new septum that was formed (Figure 4 and Figure S2). Control experiments using the same imaging conditions without laser event revealed no photoconversion (data not shown). Thus, DivIVA-Dendra2 which was converted at an ongoing site of division or a cell pole redistributes to new division sites.

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