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

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.DOI: http://dx.doi.org/10.7554/eLife.03949.001

Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD

“…Furthermore, the observed patterns lack standing wave dynamics with nodes where the timeaveraged local MinD concentration is minimum, as observed at midcell in vivo. Recently, standing wave dynamics were reconstituted by isolating the reaction in small volumes (30,31). However, the mechanistic basis for standing wave oscillation has not been experimentally addressed.…”

“…S3B). The MinE variant containing the MinD interaction interface but lacking both the dimerization and MTS domains, MinE [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] , stimulated the fastest MinD release rate and neither stabilized MinD nor lingered after MinD release (Fig. 6B and Fig.…”

“…Recently, the Schwille group has shown that Min spirals on the bottom of an SLB-coated well are converted to an in vivo-like oscillating pattern (30,31). This was achieved by aspirating the sample solution so that the remaining volume was confined to 10-μm-deep × 10-μm-wide troughs with varying lengths (30,31) .…”

Oscillation helps to get division right

“…Nevertheless, recent work using cell-shaped membrane-bound compartments of picoliter reaction volumes and large lipid surface areas were able to reconstitute the standing wave dynamics (18), thereby underlining the importance of capturing as many in vivo characteristics as possible when developing in vitro reaction assays.…”

Classic Spotlight: Huge Genetic Inferences from Miniature Cells

“…Many other species lack Min homologs but use alternative septum positioning mechanisms that are only now coming to light. All the mechanistic understanding of these self-organizing gradients in bacteria, including mathematical modeling and exciting recent experiments that reconstitute Min-mediated FtsZ positioning in vitro (8), stems from the genetic inferences in the two Rothfield lab papers from JB.…”