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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.


Protein gradients in vitro are established by dynamic redistribution of proteins.(A–D) Concentration profiles of MinD along the length axis of a cell-shaped container were measured in intervals of 5 s. The whole oscillation cycle takes about 1 min. (A) Initially MinD forms a cap at the left pole of the compartment (light blue). Then the concentration of the MinD at the left pol decreases whereas it increases at the opposite pole. (B) The trail of the MinD zone at the right pol moves towards the right pole. (C) The concentration of MinD at the right pol decreases whereas it increases at the left pole. (D) The trail of the MinD cap at the left pole moves towards the left pole. (E) MinD (black) and MinE (red) concentration profiles during an oscillation from the left to the right pole. Dotted line: reference line to better visualize the movement of the MinE peak and MinD tail to the right pole.DOI:http://dx.doi.org/10.7554/eLife.03949.006
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fig1s3: Protein gradients in vitro are established by dynamic redistribution of proteins.(A–D) Concentration profiles of MinD along the length axis of a cell-shaped container were measured in intervals of 5 s. The whole oscillation cycle takes about 1 min. (A) Initially MinD forms a cap at the left pole of the compartment (light blue). Then the concentration of the MinD at the left pol decreases whereas it increases at the opposite pole. (B) The trail of the MinD zone at the right pol moves towards the right pole. (C) The concentration of MinD at the right pol decreases whereas it increases at the left pole. (D) The trail of the MinD cap at the left pole moves towards the left pole. (E) MinD (black) and MinE (red) concentration profiles during an oscillation from the left to the right pole. Dotted line: reference line to better visualize the movement of the MinE peak and MinD tail to the right pole.DOI:http://dx.doi.org/10.7554/eLife.03949.006

Mentions: (A) Experimental setup. Purified proteins of the MinC/D/E system were reconstituted in membrane-clad soft-polymer compartments and imaged by confocal microscopy. Profiles and electron micrographs of the compartments are shown in Figure 1—figure supplement 1 and a more detailed description of the assay to reconstitute Min protein oscillations is presented in Figure 1—figure supplement 2. Figure 1—figure supplement 3 demonstrates how the intensity profiles of MinD and MinE along the length axis of the compartments are modulated with time. To mimic a bacterial membrane we used E. coli polar lipids to generate supported lipid membranes. A more detailed description of how lipid composition influences pattern formation of Min proteins is presented in Figure 1—figure supplement 4. (B) In cell-shaped compartments, eGFP-MinC (yellow) follows the oscillations of MinD and MinE (red). Comparable results were obtained in more than a hundred soft-polymer compartments. Confocal time-lapse images with the image plane at the bottom of the compartment. Protein concentrations: 1 µM MinD, 0.9 µM MinE, 0.1 µM MinE.Atto655 (red), 0.05 µM eGFP-MinC (yellow). Time between individual frames: 30 s. Scale bar: 5 µm. (C) The time-averaged concentration profile of MinC along the long axis of a compartment has a distinct concentration minimum in the middle of the compartment. The time averaged distribution of protein concentrations was calculated by acquiring time-lapse-images with the focal plane at the middle of the compartment and averaging the intensity of the acquired frames. (D) Time-averaged fluorescent signal of eGFP-MinD with image plane in the middle of a compartment. An intensity offset is subtracted to better visualize the gradient along the boundary of the compartment. 0.9 µM MinD, 0.1 µM eGFP-MinD, 1 µM MinE. Scale bar: 5 µm. Stable pole-to-pole oscillations which result in the time-averaged gradient of MinD are severely affected if the membrane targeting sequence of MinE is deleted (Figure 1—figure supplement 5).


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

Zieske K, Schwille P - Elife (2014)

Protein gradients in vitro are established by dynamic redistribution of proteins.(A–D) Concentration profiles of MinD along the length axis of a cell-shaped container were measured in intervals of 5 s. The whole oscillation cycle takes about 1 min. (A) Initially MinD forms a cap at the left pole of the compartment (light blue). Then the concentration of the MinD at the left pol decreases whereas it increases at the opposite pole. (B) The trail of the MinD zone at the right pol moves towards the right pole. (C) The concentration of MinD at the right pol decreases whereas it increases at the left pole. (D) The trail of the MinD cap at the left pole moves towards the left pole. (E) MinD (black) and MinE (red) concentration profiles during an oscillation from the left to the right pole. Dotted line: reference line to better visualize the movement of the MinE peak and MinD tail to the right pole.DOI:http://dx.doi.org/10.7554/eLife.03949.006
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1s3: Protein gradients in vitro are established by dynamic redistribution of proteins.(A–D) Concentration profiles of MinD along the length axis of a cell-shaped container were measured in intervals of 5 s. The whole oscillation cycle takes about 1 min. (A) Initially MinD forms a cap at the left pole of the compartment (light blue). Then the concentration of the MinD at the left pol decreases whereas it increases at the opposite pole. (B) The trail of the MinD zone at the right pol moves towards the right pole. (C) The concentration of MinD at the right pol decreases whereas it increases at the left pole. (D) The trail of the MinD cap at the left pole moves towards the left pole. (E) MinD (black) and MinE (red) concentration profiles during an oscillation from the left to the right pole. Dotted line: reference line to better visualize the movement of the MinE peak and MinD tail to the right pole.DOI:http://dx.doi.org/10.7554/eLife.03949.006
Mentions: (A) Experimental setup. Purified proteins of the MinC/D/E system were reconstituted in membrane-clad soft-polymer compartments and imaged by confocal microscopy. Profiles and electron micrographs of the compartments are shown in Figure 1—figure supplement 1 and a more detailed description of the assay to reconstitute Min protein oscillations is presented in Figure 1—figure supplement 2. Figure 1—figure supplement 3 demonstrates how the intensity profiles of MinD and MinE along the length axis of the compartments are modulated with time. To mimic a bacterial membrane we used E. coli polar lipids to generate supported lipid membranes. A more detailed description of how lipid composition influences pattern formation of Min proteins is presented in Figure 1—figure supplement 4. (B) In cell-shaped compartments, eGFP-MinC (yellow) follows the oscillations of MinD and MinE (red). Comparable results were obtained in more than a hundred soft-polymer compartments. Confocal time-lapse images with the image plane at the bottom of the compartment. Protein concentrations: 1 µM MinD, 0.9 µM MinE, 0.1 µM MinE.Atto655 (red), 0.05 µM eGFP-MinC (yellow). Time between individual frames: 30 s. Scale bar: 5 µm. (C) The time-averaged concentration profile of MinC along the long axis of a compartment has a distinct concentration minimum in the middle of the compartment. The time averaged distribution of protein concentrations was calculated by acquiring time-lapse-images with the focal plane at the middle of the compartment and averaging the intensity of the acquired frames. (D) Time-averaged fluorescent signal of eGFP-MinD with image plane in the middle of a compartment. An intensity offset is subtracted to better visualize the gradient along the boundary of the compartment. 0.9 µM MinD, 0.1 µM eGFP-MinD, 1 µM MinE. Scale bar: 5 µm. Stable pole-to-pole oscillations which result in the time-averaged gradient of MinD are severely affected if the membrane targeting sequence of MinE is deleted (Figure 1—figure supplement 5).

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.