A self-organized synthetic morphogenic liposome responds with shape changes to local light cues

Cells achieve their characteristic shapes, crucial for their functions, through dynamic cytoskeletal systems that deform the plasma membrane. Microtubules (MTs) and actin filaments drive these shape changes and motility, operating at different scales. Actin filaments govern rapid, local dynamics at the cell periphery, while MTs, longer-lived and spanning greater distances, are globally organized around microtubule-organizing centers (MTOCs). The MT network's dynamic organization allows for shape plasticity in undifferentiated cells responding to environmental cues, and shape stabilization after differentiation. Cytoskeletal reorganization is directed by extracellular morphogens, inducing localized signaling reactions that polarize cytoplasmic activity of MT-associated proteins. Prenylated Rho GTPases, such as Rac, act as recruitment factors, concentrating cytoplasmic kinases (e.g., PAK1) at morphogen-exposed membrane areas via dimensionality reduction. These kinases then phosphorylate the negative MT regulator stathmin, relieving its inhibitory effect on MT growth and promoting MT growth towards the signal. However, cells integrate past sensory experiences into their response, influencing shape commitment during differentiation. The mechanisms by which memory of previous morphogen patterns is maintained and its effect on signal-induced morphogenesis remain unclear. To study these principles, the researchers reconstituted a minimal out-of-equilibrium system based on the Rac1-Pak1-stathmin pathway in cell-sized liposomes with encapsulated dynamic MT-asters. This system responds to light cues, mimicking localized signal transduction from morphogens, generating intracellular MT-regulator signaling gradients. The aim was to investigate how this system influences astral-MT growth, membrane deformations, and the recursive interaction between the MT-cytoskeleton and signaling, ultimately leading to self-organized morphologies responsive to localized light cues, dependent on their initial shape.

The study builds upon existing knowledge of cytoskeletal dynamics and morphogen signaling. Previous research has established the roles of actin filaments and microtubules in cell motility and shape changes, highlighting their distinct temporal and spatial scales of action. The Rac1-Pak1-stathmin pathway has been well-characterized, demonstrating how localized signaling cascades can regulate microtubule dynamics and ultimately influence cell morphology. The principle of dimensionality reduction in cell signaling, where a three-dimensional signal is transduced into a two-dimensional response at the membrane, is also relevant. However, a gap existed in understanding how cells integrate past experiences into their morphogenic responses, and how these memories influence the dynamics of signal-induced shape changes. The authors review prior work illustrating the importance of cellular memory in processes such as chemotaxis and fibroblast migration, highlighting the need for a better understanding of these memory mechanisms in morphogenesis.

The researchers developed a Synthetic Morphogenic Membrane System (SynMMS) by encapsulating purified centrosomes, tubulin, GTP, and a light-inducible signaling system within giant unilamellar vesicles (GUVs). The signaling system uses the iLID/SspB optical dimerizer system. iLID, fused to a C2 phosphatidylserine-binding domain (C2-iLID), localizes to the membrane, while SspB is fused to the stathmin-phosphorylating kinase AuroraB (SspB-AuroraB). 488 nm light induces AuroraB translocation to the membrane, mimicking morphogen-induced kinase recruitment. The effects of stathmin phosphorylation on MT dynamics were quantified using single-filament total internal reflection fluorescence (TIRF) microscopy. GUVs were prepared using continuous Droplet Interface Crossing Encapsulation (cDICE), enabling precise control over encapsulated components and osmotic conditions (membrane rigidity). Confocal laser scanning microscopy (CLSM) was employed to visualize MT asters and GUV morphologies. The researchers employed agent-based Monte Carlo simulations and reaction-diffusion models to explore the underlying principles governing MT-membrane interactions and signaling gradients. Fluorescence lifetime imaging microscopy (FLIM) was used to monitor stathmin phosphorylation levels in GUVs using a FRET-based sensor (COPY). The morphometric analysis included measuring centrosome position, GUV eccentricity, MT length, and membrane curvature. Information entropy was used to quantify the regularity of SspB-AuroraB cluster patterns. The study included controls lacking stathmin (SynMMS-stat) to assess its role in coupling signaling to MT growth. Finally, numerical simulations were performed to explore the dynamics of the coupled MT-membrane and signaling system.

The study demonstrated that increasing stathmin concentration linearly decreased MT growth velocity and increased catastrophe frequency, effects reversible by increased phosphorylated stathmin (pStathmin). Encapsulation of stathmin resulted in smaller MT asters and spherical GUV morphology, while pStathmin led to larger asters and polar morphologies. Light-induced translocation of SspB-AuroraB generated a membrane-proximal gradient of pStathmin, creating a localized increase in tubulin concentration. This light-induced tubulin concentration promoted MT growth and altered GUV morphology. Three initial morphologies were observed in SynMMS: spherical, polar, and star-like, depending on MT density and basal signaling. The formation of membrane sheet protrusions (MSPs) depended on basal SspB-AuroraB signaling. Global irradiation of SynMMS led to centrosome decentering (rigid membrane) or drastic morphological changes (deformable membrane). Local irradiation triggered de novo formation of MSPs, which then accumulated SspB-AuroraB and guided MT growth. SspB-AuroraB preferentially recruited to preformed protrusions, demonstrating a positive feedback loop between membrane deformation and signaling. Mathematical modeling showed that the balance between self-induced capture (SIC) of MTs and cooperative clustering (CC) of SspB-AuroraB determined the initial morphology (polar vs. star-like) and responsiveness to stimuli. The study revealed that star-like SynMMS exhibited greater morphogenic plasticity and directional response to stimuli than polar SynMMS.

The findings demonstrate that the interplay between a light-responsive signaling system and a dynamic MT cytoskeleton within a synthetic protocell can generate self-organized morphologies responsive to both global and local stimuli. This system recapitulates key aspects of morphogen-guided cell morphogenesis, including the role of localized signaling gradients in regulating microtubule growth and the recursive coupling between cytoskeletal dynamics and signal transduction. The observed morphogenic plasticity, with star-like morphologies exhibiting greater responsiveness to local cues than polar morphologies, highlights the importance of initial system state in determining the response to external signals. The study provides a valuable model for investigating fundamental principles of morphogenesis, offering insights into how the interplay between self-organizing processes and external cues shapes cell form and function. The use of light as a controllable morphogen analogue also offers a powerful experimental tool for manipulating and studying these processes.

This study successfully reconstituted a synthetic morphogenic system that exhibits self-organized morphologies responsive to light cues. The findings underscore the critical role of a dynamic interplay between microtubule-cytoskeletal dynamics and localized signaling gradients in shaping cellular morphology. The balance between self-induced capture and cooperative clustering determines initial morphologies and responsiveness to external stimuli. The developed SynMMS system provides a powerful platform for future research exploring the complex interplay between signaling pathways and cytoskeletal organization in morphogenesis.

The study's limitations include the use of a simplified model system and the reliance on light as a proxy for morphogens. The variability in the encapsulation efficiency of certain components, such as C2-iLID, introduces some experimental variability in the initial conditions. The model does not fully incorporate the complex interactions of the actin cytoskeleton and their influence on overall morphogenesis. While the study provides valuable insights into fundamental principles, further research is needed to validate these findings in more complex cellular contexts.