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Reconstitution of self-organizing protein gradients as spatial cues in cell-free systems.

Zieske K, Schwille P - Elife (2014)

Bottom Line: Intracellular protein gradients are significant determinants of spatial organization.Reconstituting self-organized oscillations of MinCDE proteins in membrane-clad soft-polymer compartments, we demonstrate that distinct time-averaged protein concentration gradients are established.Moreover, we show that compartment geometry plays a major role in Min gradient establishment, and provide evidence for a geometry-mediated mechanism to partition Min proteins during bacterial development.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Martinsried, Munich, Germany.

ABSTRACT
Intracellular protein gradients are significant determinants of spatial organization. However, little is known about how protein patterns are established, and how their positional information directs downstream processes. We have accomplished the reconstitution of a protein concentration gradient that directs the assembly of the cell division machinery in E.coli from the bottom-up. Reconstituting self-organized oscillations of MinCDE proteins in membrane-clad soft-polymer compartments, we demonstrate that distinct time-averaged protein concentration gradients are established. Our minimal system allows to study complex organizational principles, such as spatial control of division site placement by intracellular protein gradients, under simplified conditions. In particular, we demonstrate that FtsZ, which marks the cell division site in many bacteria, can be targeted to the middle of a cell-like compartment. Moreover, we show that compartment geometry plays a major role in Min gradient establishment, and provide evidence for a geometry-mediated mechanism to partition Min proteins during bacterial development.

No MeSH data available.


Min oscillations in compartments with multiple septa.(A) Confocal image of MinD/E pattern in a compartment with two septa. The figure demonstrates that Min oscillations occur between each ‘constricting septum’. 5% MinE is labeled with Atto655. (B) The lipid membrane was labeled with DiI to visualize the surrounding of the compartment. The confocal images were taken with the focus level on the bottom, in the middle (5 µm above the bottom) and the upper level of membrane clad compartments (10 µm above the bottom of the compartment. (C) x/z-scans depict the profile of the compartment at the septum and at an unconstricted region. (A–C) Scale bar: 10 µm.DOI:http://dx.doi.org/10.7554/eLife.03949.021
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fig6s3: Min oscillations in compartments with multiple septa.(A) Confocal image of MinD/E pattern in a compartment with two septa. The figure demonstrates that Min oscillations occur between each ‘constricting septum’. 5% MinE is labeled with Atto655. (B) The lipid membrane was labeled with DiI to visualize the surrounding of the compartment. The confocal images were taken with the focus level on the bottom, in the middle (5 µm above the bottom) and the upper level of membrane clad compartments (10 µm above the bottom of the compartment. (C) x/z-scans depict the profile of the compartment at the septum and at an unconstricted region. (A–C) Scale bar: 10 µm.DOI:http://dx.doi.org/10.7554/eLife.03949.021

Mentions: (A) Pole to pole oscillations occur is narrow compartments. Scale bar: 20 µm. (B) In wider compartments the proteins oscillate parallel to the length axis of the compartment. Scale bar: 20 µm. In compartments with an even larger width more complex patterns occur as depicted in Figure 6—figure supplement 1. (C) Confocal images and kymographs of MinE pattern in compartments with different degrees of ‘constriction’ depict pole-to-pole oscillation in compartments with a constant width (upper panel). In compartments with a narrow width in the middle of the compartment double ascillations occur. In separated compartments independent pole-to-pole oscillations are observed. (D) Oscillation modes in compartments with different lengths and stepwise narrowing width at the middle of the compartment. Left: Compartments without constriction or only slight decrease of the width in the middle harbor pole-to-pole oscillations (asymmetric oscillations, yellow arrows). Compartments with a narrow width in the middle harbor double oscillations (symmetric oscillations, orange arrows). A series of time-lapse images as well as kymographs of the third and fourth compartment, in which the transition from pole-to-pole to double oscillation occurs are shown in Figure 6—figure supplement 2. Video 5 shows the dynamic pattern of the third and fourth compartment in this image. Middle: In longer compartments double-oscillations occur due to the increased length, even if the width along the length of the compartment is constant. Right: In very short cells the constriction of the compartments results in two connected compartments, with length axis perpendicular to the length axis in non-constricting compartments. Thus the Min proteins oscillate along the new length axis, which is perpendicular to the oscillation axis in non-constricting short compartments (blue arrows). 1 µM MinE (doped with 10% MinE.Atto655), 1 µM MinD, Scale bar: 20 µm Figure 6—figure supplement 3 demonstrates that oscillations happen between each ‘constricting septum’ if the compartment harbors two septa.


Reconstitution of self-organizing protein gradients as spatial cues in cell-free systems.

Zieske K, Schwille P - Elife (2014)

Min oscillations in compartments with multiple septa.(A) Confocal image of MinD/E pattern in a compartment with two septa. The figure demonstrates that Min oscillations occur between each ‘constricting septum’. 5% MinE is labeled with Atto655. (B) The lipid membrane was labeled with DiI to visualize the surrounding of the compartment. The confocal images were taken with the focus level on the bottom, in the middle (5 µm above the bottom) and the upper level of membrane clad compartments (10 µm above the bottom of the compartment. (C) x/z-scans depict the profile of the compartment at the septum and at an unconstricted region. (A–C) Scale bar: 10 µm.DOI:http://dx.doi.org/10.7554/eLife.03949.021
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215534&req=5

fig6s3: Min oscillations in compartments with multiple septa.(A) Confocal image of MinD/E pattern in a compartment with two septa. The figure demonstrates that Min oscillations occur between each ‘constricting septum’. 5% MinE is labeled with Atto655. (B) The lipid membrane was labeled with DiI to visualize the surrounding of the compartment. The confocal images were taken with the focus level on the bottom, in the middle (5 µm above the bottom) and the upper level of membrane clad compartments (10 µm above the bottom of the compartment. (C) x/z-scans depict the profile of the compartment at the septum and at an unconstricted region. (A–C) Scale bar: 10 µm.DOI:http://dx.doi.org/10.7554/eLife.03949.021
Mentions: (A) Pole to pole oscillations occur is narrow compartments. Scale bar: 20 µm. (B) In wider compartments the proteins oscillate parallel to the length axis of the compartment. Scale bar: 20 µm. In compartments with an even larger width more complex patterns occur as depicted in Figure 6—figure supplement 1. (C) Confocal images and kymographs of MinE pattern in compartments with different degrees of ‘constriction’ depict pole-to-pole oscillation in compartments with a constant width (upper panel). In compartments with a narrow width in the middle of the compartment double ascillations occur. In separated compartments independent pole-to-pole oscillations are observed. (D) Oscillation modes in compartments with different lengths and stepwise narrowing width at the middle of the compartment. Left: Compartments without constriction or only slight decrease of the width in the middle harbor pole-to-pole oscillations (asymmetric oscillations, yellow arrows). Compartments with a narrow width in the middle harbor double oscillations (symmetric oscillations, orange arrows). A series of time-lapse images as well as kymographs of the third and fourth compartment, in which the transition from pole-to-pole to double oscillation occurs are shown in Figure 6—figure supplement 2. Video 5 shows the dynamic pattern of the third and fourth compartment in this image. Middle: In longer compartments double-oscillations occur due to the increased length, even if the width along the length of the compartment is constant. Right: In very short cells the constriction of the compartments results in two connected compartments, with length axis perpendicular to the length axis in non-constricting compartments. Thus the Min proteins oscillate along the new length axis, which is perpendicular to the oscillation axis in non-constricting short compartments (blue arrows). 1 µM MinE (doped with 10% MinE.Atto655), 1 µM MinD, Scale bar: 20 µm Figure 6—figure supplement 3 demonstrates that oscillations happen between each ‘constricting septum’ if the compartment harbors two septa.

Bottom Line: Intracellular protein gradients are significant determinants of spatial organization.Reconstituting self-organized oscillations of MinCDE proteins in membrane-clad soft-polymer compartments, we demonstrate that distinct time-averaged protein concentration gradients are established.Moreover, we show that compartment geometry plays a major role in Min gradient establishment, and provide evidence for a geometry-mediated mechanism to partition Min proteins during bacterial development.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Martinsried, Munich, Germany.

ABSTRACT
Intracellular protein gradients are significant determinants of spatial organization. However, little is known about how protein patterns are established, and how their positional information directs downstream processes. We have accomplished the reconstitution of a protein concentration gradient that directs the assembly of the cell division machinery in E.coli from the bottom-up. Reconstituting self-organized oscillations of MinCDE proteins in membrane-clad soft-polymer compartments, we demonstrate that distinct time-averaged protein concentration gradients are established. Our minimal system allows to study complex organizational principles, such as spatial control of division site placement by intracellular protein gradients, under simplified conditions. In particular, we demonstrate that FtsZ, which marks the cell division site in many bacteria, can be targeted to the middle of a cell-like compartment. Moreover, we show that compartment geometry plays a major role in Min gradient establishment, and provide evidence for a geometry-mediated mechanism to partition Min proteins during bacterial development.

No MeSH data available.