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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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Decreasing medium osmolarity causes retraction as theoretically predicted.(A) Medial focal plane images of sporulating cells treated with lysozyme in osmotically protected medium but without sucrose (see Materials and Methods, Movie S4). Fully engulfed cells retract (cells 1 and 3) while partially engulfed cells (cells 2 and 4) fail to engulf and undergo retraction as well. (B) Percentage of forespore engulfment after lysozyme treatment in medium with 0.5 M sucrose and without sucrose. Average +/− STD of four different microscopy fields, each containing 136–160 cells in sucrose condition and 93–173 cells without sucrose. (C) Images of typical cell undergoing swelling and retraction. Time 0 minutes corresponds to fully rounded mother cell. (D) Mother-cell and forespore volume aligned in time based on 0 time point defined as in (C). After mother cells round, volume steadily increases. (Inset) Volume comparison for experiments with and without sucrose ().  is mother-cell volume  2 minutes after engulfment or retraction corresponding to 0.5 M surose or no sucrose, respectively.  is initial mother-cell volume  8 minutes before transition. (E) Mother-cell and forespore surface area. (Inset) Surface area comparison for cases with and without sucrose (). Average +/− SEM of   = 12 cells in panel (D, E). Scale bars 2 µm.
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pcbi-1003912-g007: Decreasing medium osmolarity causes retraction as theoretically predicted.(A) Medial focal plane images of sporulating cells treated with lysozyme in osmotically protected medium but without sucrose (see Materials and Methods, Movie S4). Fully engulfed cells retract (cells 1 and 3) while partially engulfed cells (cells 2 and 4) fail to engulf and undergo retraction as well. (B) Percentage of forespore engulfment after lysozyme treatment in medium with 0.5 M sucrose and without sucrose. Average +/− STD of four different microscopy fields, each containing 136–160 cells in sucrose condition and 93–173 cells without sucrose. (C) Images of typical cell undergoing swelling and retraction. Time 0 minutes corresponds to fully rounded mother cell. (D) Mother-cell and forespore volume aligned in time based on 0 time point defined as in (C). After mother cells round, volume steadily increases. (Inset) Volume comparison for experiments with and without sucrose (). is mother-cell volume 2 minutes after engulfment or retraction corresponding to 0.5 M surose or no sucrose, respectively. is initial mother-cell volume 8 minutes before transition. (E) Mother-cell and forespore surface area. (Inset) Surface area comparison for cases with and without sucrose (). Average +/− SEM of  = 12 cells in panel (D, E). Scale bars 2 µm.

Mentions: Our model predicts that high osmotic pressure differences across the mother-cell membrane lead to swelling, reduced membrane fluctuations and membrane retraction, therefore preventing the completion of engulfment (Fig. 5A and B). To assess the validity of these predictions, we followed the progression of engulfment in absence of the cell wall for different osmotic conditions by time-lapse fluorescence microscopy (see Materials and Methods). Briefly, we resuspended sporulating cells in SMM buffer, either with 0.5 M sucrose to support protoplast engulfment (Fig. 1A, Movie S1) [8] or without sucrose, leading to an increase in the osmotic pressure difference between the cytoplasm and the extracellular medium. We then stained cell membranes with FM 4–64, added lysozyme to remove the cell wall, and performed time-lapse microscopy as the cell wall was degraded (Fig. 7A). In the presence of 0.5 M sucrose, more than 50 of the cells completed engulfment as previously reported (Fig. 7B, Movie S1; [8]). However, in absence of sucrose the engulfing membrane retracted for almost all cells, and less than 2 of them completed engulfment (Fig. 7A and B, Movie S4). Furthermore, membrane retraction was accompanied by an increase in mother-cell volume and surface area of 30 and 5 , respectively (Fig. 7D and E). Hence, our data confirm the predictions of our biophysical model on the response to changes in medium osmolarity.


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)

Decreasing medium osmolarity causes retraction as theoretically predicted.(A) Medial focal plane images of sporulating cells treated with lysozyme in osmotically protected medium but without sucrose (see Materials and Methods, Movie S4). Fully engulfed cells retract (cells 1 and 3) while partially engulfed cells (cells 2 and 4) fail to engulf and undergo retraction as well. (B) Percentage of forespore engulfment after lysozyme treatment in medium with 0.5 M sucrose and without sucrose. Average +/− STD of four different microscopy fields, each containing 136–160 cells in sucrose condition and 93–173 cells without sucrose. (C) Images of typical cell undergoing swelling and retraction. Time 0 minutes corresponds to fully rounded mother cell. (D) Mother-cell and forespore volume aligned in time based on 0 time point defined as in (C). After mother cells round, volume steadily increases. (Inset) Volume comparison for experiments with and without sucrose ().  is mother-cell volume  2 minutes after engulfment or retraction corresponding to 0.5 M surose or no sucrose, respectively.  is initial mother-cell volume  8 minutes before transition. (E) Mother-cell and forespore surface area. (Inset) Surface area comparison for cases with and without sucrose (). Average +/− SEM of   = 12 cells in panel (D, E). Scale bars 2 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214620&req=5

pcbi-1003912-g007: Decreasing medium osmolarity causes retraction as theoretically predicted.(A) Medial focal plane images of sporulating cells treated with lysozyme in osmotically protected medium but without sucrose (see Materials and Methods, Movie S4). Fully engulfed cells retract (cells 1 and 3) while partially engulfed cells (cells 2 and 4) fail to engulf and undergo retraction as well. (B) Percentage of forespore engulfment after lysozyme treatment in medium with 0.5 M sucrose and without sucrose. Average +/− STD of four different microscopy fields, each containing 136–160 cells in sucrose condition and 93–173 cells without sucrose. (C) Images of typical cell undergoing swelling and retraction. Time 0 minutes corresponds to fully rounded mother cell. (D) Mother-cell and forespore volume aligned in time based on 0 time point defined as in (C). After mother cells round, volume steadily increases. (Inset) Volume comparison for experiments with and without sucrose (). is mother-cell volume 2 minutes after engulfment or retraction corresponding to 0.5 M surose or no sucrose, respectively. is initial mother-cell volume 8 minutes before transition. (E) Mother-cell and forespore surface area. (Inset) Surface area comparison for cases with and without sucrose (). Average +/− SEM of  = 12 cells in panel (D, E). Scale bars 2 µm.
Mentions: Our model predicts that high osmotic pressure differences across the mother-cell membrane lead to swelling, reduced membrane fluctuations and membrane retraction, therefore preventing the completion of engulfment (Fig. 5A and B). To assess the validity of these predictions, we followed the progression of engulfment in absence of the cell wall for different osmotic conditions by time-lapse fluorescence microscopy (see Materials and Methods). Briefly, we resuspended sporulating cells in SMM buffer, either with 0.5 M sucrose to support protoplast engulfment (Fig. 1A, Movie S1) [8] or without sucrose, leading to an increase in the osmotic pressure difference between the cytoplasm and the extracellular medium. We then stained cell membranes with FM 4–64, added lysozyme to remove the cell wall, and performed time-lapse microscopy as the cell wall was degraded (Fig. 7A). In the presence of 0.5 M sucrose, more than 50 of the cells completed engulfment as previously reported (Fig. 7B, Movie S1; [8]). However, in absence of sucrose the engulfing membrane retracted for almost all cells, and less than 2 of them completed engulfment (Fig. 7A and B, Movie S4). Furthermore, membrane retraction was accompanied by an increase in mother-cell volume and surface area of 30 and 5 , respectively (Fig. 7D and E). Hence, our data confirm the predictions of our biophysical model on the response to changes in medium osmolarity.

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