TY - BOOK

T1 - Lateral Organization of Plasma Membrane Proteins Studied Using Thermoplasmonics

AU - Arastoo, Mohammadreza

PY - 2023/5/16

Y1 - 2023/5/16

N2 - The eukaryotic cell membrane is a dynamic structure that holds around 30.000 proteins per μm2 of its surface area. Proteinsegregation in this crowded environment is crucial for theirproper functioning which otherwise influences the cell homeostasis.This is presumably doneby laterally organizing the plasma membraneinto nanoscopic local domains or so-called lipidrafts. Rafts are highly dynamic structures (milliseconds) with a very smallsize (10-200nm). These two traits make them difficult targets to directly visualize and investigate. Most of the methods reported so far for raftstudies 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 andensure 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-transferringtakes 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.

AB - The eukaryotic cell membrane is a dynamic structure that holds around 30.000 proteins per μm2 of its surface area. Proteinsegregation in this crowded environment is crucial for theirproper functioning which otherwise influences the cell homeostasis.This is presumably doneby laterally organizing the plasma membraneinto nanoscopic local domains or so-called lipidrafts. Rafts are highly dynamic structures (milliseconds) with a very smallsize (10-200nm). These two traits make them difficult targets to directly visualize and investigate. Most of the methods reported so far for raftstudies 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 andensure 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-transferringtakes 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.

M3 - Ph.D. thesis

BT - Lateral Organization of Plasma Membrane Proteins Studied Using Thermoplasmonics

PB - University of Copenhagen

CY - Copenhagen, Denmark

ER -