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.