in-vitro-reconstitution-cytoskeletal-networks-inside-phase-separated

<em>In vitro</em> Reconstitution of Cytoskeletal Networks inside Phase Separated Giant Unilamellar Vesicles (GUVs)

Biomimetic lipid membranes in the form of giant unilamellar vesicles (GUVs) are commonly used to mimic cellular membrane behavior because of the ease of ...

Max Planck Institute of Biochemistry

A key challenge for bottom-up synthetic biology is engineering a minimal module for self-division of synthetic cells. Actin-based cytokinetic rings are considered a promising structure to produce the forces required for the controlled excision of cell-like compartments such as giant unilamellar vesicles (GUVs). Despite prior demonstrations of actin ring targeting to GUV membranes and myosin-induced constriction, large-scale vesicle deformation has been precluded due to the lacking spatial control of these contractile structures. Here we show the combined reconstitution of actomyosin rings and the bacterial MinDE protein system within GUVs. Incorporating this spatial positioning tool, able to induce active transport of membrane-attached diffusible molecules, yields self-organized equatorial assembly of actomyosin rings in vesicles. Remarkably, the synergistic effect of Min oscillations and the contractility of actomyosin bundles induces mid-vesicle deformations and vesicle blebbing. Our system showcases how functional machineries from various organisms may be combined in vitro, leading to the emergence of functionalities towards a synthetic division system.

Self-organized spatial targeting of contractile actomyosin rings for synthetic cell division.

Cell membranes in bacteria are laterally polarized to produce specific environments for membrane proteins, e.g., proteins involved in cell division which accumulate at mid-cell or the cell poles. An interesting result of such membrane-lipid interplay is the reorganization of lipid domains together with membrane-bound proteins at the onset of cell division, suggesting functional significance of membrane compartments in the cell cycle. Here, by adopting the key bacterial division proteins MinC, MinD, MinE, FtsA and FtsZ as an archetypal spatial patterning system, we present a simple vesicle-based in vitro model to explore the mutual dependence of protein pattern formation and membrane heterogeneity. Like many other peripheral membrane proteins, Min proteins exhibit preferential binding and macro-scale pattern formation at L domains, which leads to altered oscillation mode selection in phase-separated membrane compartments (GUVs). Moreover, incorporating bacterial division proteins within phase-separated GUVs leads to blebbing-like membrane deformations followed by the reorganization of L domains aligning at the neck region of the bleb, which agrees well with the domain rearrangement in bacterial membranes immediately preceding the radial constriction process. Overall, the presented in vitro model system showcases a basic framework to better comprehend the cellular division mechanism in consideration of complex cellular lipid environments.

Article	Year
Self-organized spatial targeting of contractile actomyosin rings for synthetic cell division. Nature communications\| PubMed ID: 39614082	2024
Mutual Dependence between Membrane Phase Separation and Bacterial Division Protein Dynamics in Synthetic Cell Models. Angewandte Chemie (International ed. in English)\| PubMed ID: 39623974	2025

Nishu Kanwa

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