Researchers Recreate Chemically Regulated Actin Polymerization to Study Membrane Dynamics

The forces induced by actin polymerization drive the movement of the plasma membrane and are essential for cellular functions such as cell division, chemotaxis, cell-cell fusion, and phagocytosis. Symmetry breaking induced by the rearrangement of proteins in the actin cytoskeleton regulates these cellular processes.

In a recent study published in Science Advances, researchers used a synthetic platform that uses dimerization modules consisting of chemical proteins to precisely control the polymerization of actin and demonstrate symmetry breaking.

​​​​​​​Study: Synthetic control of actin polymerization and symmetry breaking in active protocells. Image Credit: matthewjohn5539/Shutterstock.com​​​​​​​Study: Synthetic control of actin polymerization and symmetry breaking in active protocells. Image Credit: matthewjohn5539/Shutterstock.com

Background

Essential cellular processes such as the fusion of cells, phagocytosis, chemotaxis, and cell division require the movement of the plasma membrane.

Symmetry breaking, where spatial patterns are formed due to the proteins in the actin cytoskeleton being arranged asymmetrically, is fundamental to all these processes and eventually also regulates cellular differentiation and growth.

Biomolecular asymmetries in eukaryotic cells are regulated through multiple mechanisms, such as allosteric regulation and protein switches. Membrane-bound simplified systems without cellular complexities have been used in vitro to understand the mechanisms that regulate actin dynamics and study the symmetry breaking involving actin.

Previous studies have found that vesicle membranes undergo deformation when subjected to actin polymerization and regulatory proteins, but these studies have not been able to perturb and observe the membrane remodeling spatially and temporally.

About the Study

In the present study, the team used a synthetic platform consisting of giant unilamellar vesicles to control and study actin polymerization precisely using chemical induction. In this platform, external chemical signals were used to control the actin polymerization precisely and remodel the membrane.

An actuation and sensing model designed based on protein dimerization was encapsulated inside the giant unilamellar vesicles to build this synthetic platform.

Chemically inducible dimerization, previously used for phase separation during emulsion, tethering artificial membranes, and manipulating biochemical reactions on membrane surfaces, was used to design this model.

Proteins that bind to the immunosuppressive drug FK506, known as FKBPs, as well as the FKBP-rifampicin binding domain, which heterodimerizes when a small-rifampicin derived molecule is present, were used in the chemically inducible dimerization process.

FKBP and the FKBP-rifampicin binding domain were linked to fluorescent proteins and encapsulated inside giant unilamellar vesicles formed using inverted emulsion.

Conjugates of the cyan fluorescent protein and FKBP-rifampicin binding domain (CFP-FRB) were then localized near the FKBP anchored in the membrane, using rifampicin-based induction.

The researchers observed the temporal dynamics of actin using a system consisting of CFP-FRB, FKBP, and myristoylated alanine-rich kinase substrate (MARCKS).

The chemically inducible dimerization-based constructs were then linked to actin polymerization by fusing the FKBP-rifampicin binding domain to a re-engineered domain of ActA, an actin assembly-inducing protein. This system was used to study the asymmetric and stochastic deformities that occur on the giant unilamellar vesicle boundaries due to actin polymerization.

The role of actin polymerization in membrane deformation was confirmed using the toxin latrunculin A, which prevents actin assembly by binding and depolymerizing G- and F-actin, respectively.

Kymographs, which graphically represent spatial changes over time, were built for studying actin fluorescence and membrane curvature over time.

Correlations between ActA and actin were analyzed to determine whether the distribution of ActA upstream influenced actin nucleation heterogeneity. Similar analyses were conducted to determine if membrane curvature was associated with actin and ActA protein.

Major Findings

The study showed that the synthetic platform consisting of a giant unilamellar vesicle and an actuation and sensing model that can be modulated using chemical cues could spatiotemporally control the lipid and protein modules to achieve symmetry breaking.

The findings established that a design without phase-separated lipids, myosin, and capping protein, which was previously considered essential, could produce symmetry breaking in physiologically relevant contexts, such as chemotaxis.

Using the synthetic platform, the researchers demonstrated how external chemical cues applied in an undirected manner resulted in the polymerization of actin and asymmetric deformations in the membrane.

Furthermore, these deformations in the membrane and actin polymerization were not correlated with biochemical cues found upstream.

Contrary to previous studies that suggested that myosin or capping protein were required to break the actin symmetry, the present study showed that amplifying the signal through upstream chemical components, such as actin-related protein 2/3, was sufficient to break the actin symmetry.

Conclusions

To conclude, the study demonstrated that the synthetic platform comprising an actuation and sensing model and a giant unilamellar vesicle could be used to study chemical cue-induced symmetry breaking.

Furthermore, the platform allows the localization of the molecular components during symmetry breaking to be visualized spatiotemporally, improving our understanding of cellular processes such as chemotaxis and cell division.

The findings reported that symmetry breaking and membrane deformation occur when chemical cues are applied externally, causing actin polymerization without myosin or capping protein.

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