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Bistable forespore engulfment in Bacillus subtilis by a zipper mechanism in absence of the cell wall.

Ojkic N, López-Garrido J, Pogliano K, Endres RG - PLoS Comput. Biol. (2014)

Bottom Line: By systematic exploration of model parameters, we predict regions of osmotic pressure and membrane-surface tension that produce successful engulfment.Indeed, decreasing the medium osmolarity in experiments prevents engulfment in line with our predictions.Forespore engulfment may thus not only be an ideal model system to study decision-making in single cells, but its biophysical principles are likely applicable to engulfment in other cell types, e.g. during phagocytosis in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College, London, United Kingdom; Centre for Integrative Systems Biology and Bioinformatics, Imperial College, London, United Kingdom.

ABSTRACT
To survive starvation, the bacterium Bacillus subtilis forms durable spores. The initial step of sporulation is asymmetric cell division, leading to a large mother-cell and a small forespore compartment. After division is completed and the dividing septum is thinned, the mother cell engulfs the forespore in a slow process based on cell-wall degradation and synthesis. However, recently a new cell-wall independent mechanism was shown to significantly contribute, which can even lead to fast engulfment in [Formula: see text] 60 [Formula: see text] of the cases when the cell wall is completely removed. In this backup mechanism, strong ligand-receptor binding between mother-cell protein SpoIIIAH and forespore-protein SpoIIQ leads to zipper-like engulfment, but quantitative understanding is missing. In our work, we combined fluorescence image analysis and stochastic Langevin simulations of the fluctuating membrane to investigate the origin of fast bistable engulfment in absence of the cell wall. Our cell morphologies compare favorably with experimental time-lapse microscopy, with engulfment sensitive to the number of SpoIIQ-SpoIIIAH bonds in a threshold-like manner. By systematic exploration of model parameters, we predict regions of osmotic pressure and membrane-surface tension that produce successful engulfment. Indeed, decreasing the medium osmolarity in experiments prevents engulfment in line with our predictions. Forespore engulfment may thus not only be an ideal model system to study decision-making in single cells, but its biophysical principles are likely applicable to engulfment in other cell types, e.g. during phagocytosis in eukaryotes.

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Bistable forespore engulfment of B. subtilis after cell-wall removal.(A and B) Images adopted from [8]. Medial focal plane images of sporulating bacteria treated with cell-wall removal lysozyme in osmotically protected medium with 0.5 M of sucrose [8]. Fluorescent membrane stain FM 4–64 was used to track the progressing mother-cell membrane engulfing the forespore. Arrows point to the moving edges of the mother membrane. Double-headed arrows show mother-forespore cell separation. (A) In wild-type (WT) cells upon cell-wall removal, mother cell either engulfs the forespore (top) or retracts (bottom), see Movie S1. Time 0 minutes (0') is assigned to the onset of volume loss (see Fig. 2A). (B) Absence of the zipper proteins SpoIIQ (top) or SpoIIIAH (bottom) prevents membrane from forward progression causing protoplast separation. Time 0 minutes is used as the time of physical separation of mother cell and forespore. (C) Cartoon of fast bistable forespore engulfment in WT cells. Mother-cell compartment and forespore are shown in blue and red, respectively. After cell-wall removal  60  of the sporulating cells engulf the forespore, while  40  fail to engulf [8]. (D) Cartoon showing the protoplast separation as observed in mutants of panel B. Scale bars: 2 m.
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pcbi-1003912-g001: Bistable forespore engulfment of B. subtilis after cell-wall removal.(A and B) Images adopted from [8]. Medial focal plane images of sporulating bacteria treated with cell-wall removal lysozyme in osmotically protected medium with 0.5 M of sucrose [8]. Fluorescent membrane stain FM 4–64 was used to track the progressing mother-cell membrane engulfing the forespore. Arrows point to the moving edges of the mother membrane. Double-headed arrows show mother-forespore cell separation. (A) In wild-type (WT) cells upon cell-wall removal, mother cell either engulfs the forespore (top) or retracts (bottom), see Movie S1. Time 0 minutes (0') is assigned to the onset of volume loss (see Fig. 2A). (B) Absence of the zipper proteins SpoIIQ (top) or SpoIIIAH (bottom) prevents membrane from forward progression causing protoplast separation. Time 0 minutes is used as the time of physical separation of mother cell and forespore. (C) Cartoon of fast bistable forespore engulfment in WT cells. Mother-cell compartment and forespore are shown in blue and red, respectively. After cell-wall removal 60 of the sporulating cells engulf the forespore, while 40 fail to engulf [8]. (D) Cartoon showing the protoplast separation as observed in mutants of panel B. Scale bars: 2 m.

Mentions: To survive starvation the Gram-positive bacterium Bacillus subtilis develops durable spores among other survival strategies [1]. During sporulation, bacteria go through a costly developmental process under limited energy resources. The initial morphological step of sporulation is asymmetric cell division, resulting in a large mother-cell and a small forespore compartment [2]. Subsequently, the dividing septum is largely degraded and the mother-cell membrane moves around the forespore. This membrane movement is similar to phagocytosis whereby immune cells clear our bodies from pathogens and other particles [3], [4]. Finally, the engulfed forespore matures into a spore and the mother cell lyzes for its release. The origin of the engulfment force has been a topic of current research [5]–[11]. Cell-wall degradation and new cell-wall deposition were shown to play a significant role in advancing the mother-cell membrane leading edge. Strikingly, when the cell wall is enzymatically removed engulfment still occurs, surprisingly taking only 1–2 min compared to 45 min with the cell wall (see Fig. 1, Movie S1) [8]. Furthermore, engulfment is successful in 60 of cells while the remaining 40 retract. This observation raises questions on the origin of bistability and decision-making in relatively simple systems under severe energy limitations.


Bistable forespore engulfment in Bacillus subtilis by a zipper mechanism in absence of the cell wall.

Ojkic N, López-Garrido J, Pogliano K, Endres RG - PLoS Comput. Biol. (2014)

Bistable forespore engulfment of B. subtilis after cell-wall removal.(A and B) Images adopted from [8]. Medial focal plane images of sporulating bacteria treated with cell-wall removal lysozyme in osmotically protected medium with 0.5 M of sucrose [8]. Fluorescent membrane stain FM 4–64 was used to track the progressing mother-cell membrane engulfing the forespore. Arrows point to the moving edges of the mother membrane. Double-headed arrows show mother-forespore cell separation. (A) In wild-type (WT) cells upon cell-wall removal, mother cell either engulfs the forespore (top) or retracts (bottom), see Movie S1. Time 0 minutes (0') is assigned to the onset of volume loss (see Fig. 2A). (B) Absence of the zipper proteins SpoIIQ (top) or SpoIIIAH (bottom) prevents membrane from forward progression causing protoplast separation. Time 0 minutes is used as the time of physical separation of mother cell and forespore. (C) Cartoon of fast bistable forespore engulfment in WT cells. Mother-cell compartment and forespore are shown in blue and red, respectively. After cell-wall removal  60  of the sporulating cells engulf the forespore, while  40  fail to engulf [8]. (D) Cartoon showing the protoplast separation as observed in mutants of panel B. Scale bars: 2 m.
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pcbi-1003912-g001: Bistable forespore engulfment of B. subtilis after cell-wall removal.(A and B) Images adopted from [8]. Medial focal plane images of sporulating bacteria treated with cell-wall removal lysozyme in osmotically protected medium with 0.5 M of sucrose [8]. Fluorescent membrane stain FM 4–64 was used to track the progressing mother-cell membrane engulfing the forespore. Arrows point to the moving edges of the mother membrane. Double-headed arrows show mother-forespore cell separation. (A) In wild-type (WT) cells upon cell-wall removal, mother cell either engulfs the forespore (top) or retracts (bottom), see Movie S1. Time 0 minutes (0') is assigned to the onset of volume loss (see Fig. 2A). (B) Absence of the zipper proteins SpoIIQ (top) or SpoIIIAH (bottom) prevents membrane from forward progression causing protoplast separation. Time 0 minutes is used as the time of physical separation of mother cell and forespore. (C) Cartoon of fast bistable forespore engulfment in WT cells. Mother-cell compartment and forespore are shown in blue and red, respectively. After cell-wall removal 60 of the sporulating cells engulf the forespore, while 40 fail to engulf [8]. (D) Cartoon showing the protoplast separation as observed in mutants of panel B. Scale bars: 2 m.
Mentions: To survive starvation the Gram-positive bacterium Bacillus subtilis develops durable spores among other survival strategies [1]. During sporulation, bacteria go through a costly developmental process under limited energy resources. The initial morphological step of sporulation is asymmetric cell division, resulting in a large mother-cell and a small forespore compartment [2]. Subsequently, the dividing septum is largely degraded and the mother-cell membrane moves around the forespore. This membrane movement is similar to phagocytosis whereby immune cells clear our bodies from pathogens and other particles [3], [4]. Finally, the engulfed forespore matures into a spore and the mother cell lyzes for its release. The origin of the engulfment force has been a topic of current research [5]–[11]. Cell-wall degradation and new cell-wall deposition were shown to play a significant role in advancing the mother-cell membrane leading edge. Strikingly, when the cell wall is enzymatically removed engulfment still occurs, surprisingly taking only 1–2 min compared to 45 min with the cell wall (see Fig. 1, Movie S1) [8]. Furthermore, engulfment is successful in 60 of cells while the remaining 40 retract. This observation raises questions on the origin of bistability and decision-making in relatively simple systems under severe energy limitations.

Bottom Line: By systematic exploration of model parameters, we predict regions of osmotic pressure and membrane-surface tension that produce successful engulfment.Indeed, decreasing the medium osmolarity in experiments prevents engulfment in line with our predictions.Forespore engulfment may thus not only be an ideal model system to study decision-making in single cells, but its biophysical principles are likely applicable to engulfment in other cell types, e.g. during phagocytosis in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College, London, United Kingdom; Centre for Integrative Systems Biology and Bioinformatics, Imperial College, London, United Kingdom.

ABSTRACT
To survive starvation, the bacterium Bacillus subtilis forms durable spores. The initial step of sporulation is asymmetric cell division, leading to a large mother-cell and a small forespore compartment. After division is completed and the dividing septum is thinned, the mother cell engulfs the forespore in a slow process based on cell-wall degradation and synthesis. However, recently a new cell-wall independent mechanism was shown to significantly contribute, which can even lead to fast engulfment in [Formula: see text] 60 [Formula: see text] of the cases when the cell wall is completely removed. In this backup mechanism, strong ligand-receptor binding between mother-cell protein SpoIIIAH and forespore-protein SpoIIQ leads to zipper-like engulfment, but quantitative understanding is missing. In our work, we combined fluorescence image analysis and stochastic Langevin simulations of the fluctuating membrane to investigate the origin of fast bistable engulfment in absence of the cell wall. Our cell morphologies compare favorably with experimental time-lapse microscopy, with engulfment sensitive to the number of SpoIIQ-SpoIIIAH bonds in a threshold-like manner. By systematic exploration of model parameters, we predict regions of osmotic pressure and membrane-surface tension that produce successful engulfment. Indeed, decreasing the medium osmolarity in experiments prevents engulfment in line with our predictions. Forespore engulfment may thus not only be an ideal model system to study decision-making in single cells, but its biophysical principles are likely applicable to engulfment in other cell types, e.g. during phagocytosis in eukaryotes.

Show MeSH
Related in: MedlinePlus