Regulating Biocondensates within Synthetic Cells via Segregative Phase Separation

Living cells orchestrate a myriad of biological reactions
within
a highly complex and crowded environment. A major factor responsible
for such seamless assembly is the preferential interactions between
the constituent macromolecules, that can drive demixing to produce
coexisting phases and thus provide dynamic intracellular compartmentalization.
However, the way multiple-phase separation phenomena, occurring simultaneously
within the cytoplasmic space, influence each other is still largely
unknown. Here, we show that the interplay between segregative and
associative phase separation within cell-mimicking confinements can
lead to rich dynamics between multiple phases and the lipid boundary.
Using on-chip microfluidic systems, we encapsulate the associative
and segregative components and externally trigger their phase separation
within cell-sized vesicles. We find that segregative phases create
microdomains and tend to dictate the fate of associative components
by acting as molecular recruiters, membrane-targeting agents, and
initiators of condensation. The obtained multiphase architecture provides
an isolated microenvironment for condensates, restricting their molecular
communication as well as diffusive motion, and can further lead to
global shape transformation of the confinement itself in the form
of wetted, hierarchical domains at the lipid membrane. In conclusion,
we propose segregative phase separation as a universal condensation
regulation strategy by managing their molecular distribution, process
initiation, and spatial localization, including membrane interaction.
The presented interplay between the two phase separation systems suggests
a distinct design principle in constructing complex synthetic cells
and controlling the behavior of artificial membraneless organelles
within.

活细胞在高度复杂且拥挤的微环境中，统筹调控着海量的生物化学反应。促成这种无缝衔接组装的核心因素，是组成大分子间的偏好性相互作用：这类相互作用可驱动脱混合（demixing）过程，形成多种共存相，进而实现动态的细胞内区室化。然而，细胞质内同时发生的多相分离现象之间的相互调控机制，目前仍在很大程度上尚不明确。本研究证实，在细胞模拟限域环境中，分离型相分离（segregative phase separation）与缔合型相分离（associative phase separation）之间的相互作用，可在多相体系与脂质边界之间催生丰富的动态行为。我们借助芯片微流控系统（on-chip microfluidic systems），将缔合型与分离型组分封装于细胞尺寸囊泡（vesicles）中，并通过外部触发手段诱导其发生相分离。研究发现，分离型相可形成微结构域，并通过充当分子招募因子、膜靶向因子及凝聚起始因子，主导缔合型组分的行为走向。所构建的多相结构可为凝聚体（condensates）提供孤立的微环境，限制其分子交流与扩散行为；同时还可通过脂质膜上形成的浸润型层级结构域，引发限域环境本身的整体形态转变。综上，本研究提出：分离型相分离可通过调控分子分布、过程起始与空间定位（包括膜相互作用），成为一种普适性的凝聚调控策略。这两种相分离体系间的相互作用，为构建复杂合成细胞以及调控其内人工无膜细胞器的行为，提供了全新的设计思路。