Protein Patterns and Oscillations on Lipid Monolayers and in Microdroplets

Abstract

The Min proteins from E.coli position the bacterial cell-division machinery through pole-to-pole oscillations. In vitro,M in protein self-organization can be reconstituted in the presence of al ipid membrane as ac atalytic surface. However,M in dynamics have so far not been reconstituted in fully membrane-enclosed volumes.M icrodroplets interfaced by lipid monolayers were employed as as imple 3D mimic of cellular compartments to reconstitute Min protein oscillations. We demonstrate that lipid monolayers are sufficient to fulfil the catalytic role of the membrane and thus represent af acile platform to investigate Min protein regulated dynamics of the cell-division protein FtsZ-mts.I np articular,w es howt hat droplet containers reveal distinct Min oscillation modes,a nd reveal ad ependence of FtsZ-mts structures on compartment size. Finally,co-reconstitution of Min proteins and FtsZ-mts in droplets yields antagonistic localization, thus demonstrating that droplets indeed support the analysis of complex bacterial self-organization in confined volumes.The self-organization of proteins into large-scale structures and patterns is fundamental to all living systems,a nd is required to regulate complex cellular processes such as protein localization and cell division. As triking example of protein self-organization is the Min protein system (comprising the proteins MinC,M inD,a nd MinE) of the bacterium Escherichia coli, [1] which oscillates between the cell poles. These pole-to-pole oscillations generate atime-averaged nonhomogeneous concentration gradient with am inimum at the mid-cell plane,t he future cell division site. [2][3][4][5][6][7] Intriguingly, only am inimal set of components (the two membrane interacting proteins MinD and MinE, al ipid membrane, and ATP) has been shown to establish Min oscillations. [8][9][10][11][12] Theprotein MinC follows the oscillating MinDE patterns by binding to MinD,a nd it directly inhibits the assembly of the cell-division protein FtsZ at the poles. [3,7,[13][14][15] In this way,the main division initiator FtsZ, which assembles into aring-like structure (Z ring), is directed to the middle of the cell. [4] To study Min oscillations under well-defined conditions, cell-free systems are being developed that provide ahigh level of control over physicochemical parameters.P reviously,w e have reconstituted the pole-to-pole oscillations of the Min proteins,a sw ell as their ability to spatially direct FtsZ-mts (FtsZ fused to am embrane-targeting sequence) [16] to the middle of am icrofabricated compartment. [12,17] However, although the cell membrane of E. colii sac losed compartment, Min oscillations have thus far only been successfully reconstituted in volumes not fully enclosed by membranes, but open at least at one site. [12,18,19] Moreover,a lthough membrane-attached FtsZ rings have been observed in vesicles [16,20,21] and FtsZ bundles have been characterized in the lumen of droplets, [22] we are just beginning to understand how systems parameters,s uch as compartment ge...

Protein Pattern Formation

“…On flat lipid bilayers one observed spiral and travelling wave patterns, and a varying degree of spatial coherence sometimes verging on chemical turbulence [80]. Other experiments constrained the Min protein dynamics geometrically to small membrane patches [81], semi-open PDMS grooves with varying lipid composition [82], lipid-interfaced droplets [83], and bilayer coated three-dimensional chambers of various shapes and sizes [79]. Strikingly, the observed patterns show a very broad range of characteristics and varying degrees of sensitivity to the geometry of the enclosing membrane.…”

Tailoring the appearance: what will synthetic cells look like?

“…For example, crucial protein machineries (FtsZ and MinCDE) involved in bacterial cell division were reconstituted in droplets, where they exhibit the expected spatial anticorrelation between them (Fig. 3B) [41]•. Interestingly, division of droplets induced by chemical reaction at the water-oil interface was demonstrated, resulting in tunable, equal or unequal, daughter droplets [42].…”

Membrane functionalization in artificial cell engineering

“…The system consists of MinC, D and E which together positions the bacterial cell division machinery at the centre of the cell [99]. The Schwille group has successfully reconstituted MinD and E into surface-attached lipid bilayers (SLBs) [100], microemulsion droplets [101] and giant vesicles [102] in order to study system function ex vivo. This enabled observation of oscillatory protein complex assembly at membrane interfaces, generating multiple oscillatory behaviours in giant vesicles including 'breathing' , 'circling' and sphere to dumbbell morphological changes based on vesicle size and encapsulated protein concentration [102].…”