The eukaryotic cell membrane is a dynamic structure that holds
around 30.000 proteins per μm2 of its surface area. Protein
segregation in this crowded environment is crucial for their
proper functioning which otherwise influences the cell homeostasis.
This is presumably doneby laterally organizing the plasma membrane
into nanoscopic local domains or so-called lipidrafts.
Rafts are highly dynamic structures (milliseconds) with a very small
size (10-200nm). These two traits make them difficult targets to directly
visualize and investigate. Most of the methods reported so far for raft
studies involve procedures which can potentially influence protein
integrity and/or its localization on membranes.

Motivated by the challenges of raft visualization, we developed a new
technique to reconstitute rafts and membrane associated proteins into
a system of selective hybrid vesicles. Here, the protein of interest is
carried by vesicles derived from viable cells and delivered into
a phase-separated vesicle by thermoplasmonics based membrane fusion.
Phase-separated vesicles mirror the raft and therefore, provide a bed to
understand how the transferred proteins organize on the membrane. Also,
cell derived vesicles obviate the need for biochemical purifications and
ensure correct orientation of membrane associated proteins on the isolated
membrane.

Here, we combined cell culturing with optical manipulations and confocal
imaging to build the new platform. The whole process of protein-transferring
takes place within the cell culture by using a laser which operates within
the biological transparency window. By using the stablished method,
we showed that Neuraminidase (NA) from the influenza A virus and its two
truncated constructs exclusively partitions into non-raft resembled region
of the formed hybrid vesicle. Moreover, we inspected phase partitioning of
Hemagglutinin (HA), its transmembrane domain and a GPCR protein (KOR) by
the same method; they all showed the same tendency for the disordered
lipid phase of the hybrid vesicle. To extend the applicability of the
thermoplasmonic fusion method for protein research we investigated how
annexin A4 and A5 proteins are recruited to membrane ruptures performed
in either cell membranes or model membranes.

These experiments provided evidence for how cells respond to a photothermal damage. In both cases, they showed an upconcentration around the injured area and a pronounced rolling of the membrane surrounding the hole which may be caused by the protein’s ability to curve membranes. The use of plasmonic nanoparticles and optical trapping provides novel and efficient tool for investigation of protein dynamics in membranes.