Large-scale modulation of reconstituted Min protein patterns and gradients by defined mutations in MinE’s membrane targeting sequence

Abstract

The E. coli MinDE oscillator is a paradigm for protein self-organization and gradient formation. Previously, we reconstituted Min protein wave patterns on flat membranes as well as gradient-forming pole-to-pole oscillations in cell-shaped PDMS microcompartments. These oscillations appeared to require direct membrane interaction of the ATPase activating protein MinE. However, it remained unclear how exactly Min protein dynamics are regulated by MinE membrane binding. Here, we dissect the role of MinE’s membrane targeting sequence (MTS) by reconstituting various MinE mutants in 2D and 3D geometries. We demonstrate that the MTS defines the lower limit of the concentration-dependent wavelength of Min protein patterns while restraining MinE’s ability to stimulate MinD’s ATPase activity. Strikingly, a markedly reduced length scale—obtainable even by single mutations—is associated with a rich variety of multistable dynamic modes in cell-shaped compartments. This dramatic remodeling in response to biochemical changes reveals a remarkable trade-off between robustness and versatility of the Min oscillator.

Minimal in vitro systems shed light on cell polarity

“…2A). These studies experimentally illustrated the significance of parameter interplay, created comprehensive datasets for comparisons with simulations, and clarified, for example, the highly discussed role of membrane binding for MinE (Kretschmer et al, 2017).…”

“…Therefore, factors such as temperature (Touhami et al, 2006;Caspi and Dekker, 2016), membrane composition (Mileykovskaya and Dowhan, 2000;Koppelman et al, 2001;Mileykovskaya et al, 2003;Szeto et al, 2003;Hsieh et al, 2010;Weibel, 2011, 2012;Shih et al, 2011;Vecchiarelli et al, 2014;Zieske and Schwille, 2014), diffusion in the cytosol (Meacci et al, 2006;Schweizer et al, 2012;Martos et al, 2015;Caspi and Dekker, 2016) and on the membrane (Meacci et al, 2006;Martos et al, 2013), the concentration ratio of MinD to MinE (Raskin and de Boer, 1999b;Shih et al, 2002;Loose et al, 2008;Vecchiarelli et al, 2016;Kretschmer et al, 2017;Miyagi et al, 2018) and interaction of MinE with the membrane (Hsieh et al, 2010;Loose et al, 2011a;Park et al, 2011;Shih et al, 2011;Zieske and Schwille, 2014;Vecchiarelli et al, 2016;Kretschmer et al, 2017) can also modulate the Min behavior and cause a difference in the specific length scale in in vivo and reconstituted systems (see Table S2). Reconstitution experiments helped, for example, to characterize the role of the membranetargeting sequence of MinE; Kretschmer et al showed that membrane binding of MinE is not a requirement for Min oscillations, but that it modulates the length scale of the pattern (Kretschmer et al, 2017). Experiments with higher diffusion constants, representing the absence of molecular crowding in the cytosol and on the membrane, showed that these factors account for the increased length scale in vitro (Martos et al, 2013…”

“…Only in the past few years has the sensitivity of the system to parameter changes been considered as a factor itself, and systematic variations of geometry in interplay with other parameters were investigated (Caspi and Dekker, 2016;Kretschmer et al, 2017;Miyagi et al, 2018) ( Fig. 2A).…”

<em>In Vitro</em> Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

“…The proteins form traveling surface waves 6 and other kinds of patterns 17 19 under these conditions, albeit with a wavelength that is usually about a magnitude larger than in vivo . The use of an open chamber facilitates precise control over all aspects influencing pattern formation: protein concentrations 6 , protein properties 20 , membrane composition 10 , buffer composition, and ATP concentration 6 , as well as addition of other factors such as crowding agents 21 and other divisome proteins 22 . In comparison, the in vitro reconstitution of the MinCDE system in a flow-cell 18 19 23 can be used to probe the influence of flow 17 23 , protein limiting conditions 19 , membrane composition 19 and full 3D confinement 18 on protein patterns, but renders an exact control of protein/component concentration and sequential component addition much more complicated.…”

“…Using this open chamber, we also patterned the support of the planar lipid bilayers by which one can probe how geometrical boundaries influence pattern formation 21 , a phenomenon that has recently also been investigated in vivo using bacteria molded into microstructures 7 . We also employed this assay to investigate how defined mutations in MinE affect pattern formation of the system 20 . Furthermore, the same basic assay format has been employed to investigate how pattern formation can be controlled by light, introducing an azobenzene-crosslinked MinE peptide into the assay, and imaging with TIRF microscopy 24 .…”