title: A tunable and reversible platform for the intracellular formation of genetically engineered protein microdomains.
abstract: From mitochondria to the nuclear envelope, the controlled assembly of micro- and nanostructures is essential for life; however, the level at which we can deliberately engineer the assembly of microstructures within intracellular environments remains primitive. To overcome this obstacle, we present a platform to reversibly assemble genetically engineered protein microdomains (GEPMs) on the time scale of minutes within living cells. Biologically inspired from the human protein tropoelastin, these protein polymers form a secondary aqueous phase above a tunable transition temperature. This assembly process is easily manipulated to occur at or near physiological temperature by adjusting molecular weight and hydrophobicity. We fused protein polymers to green fluorescent protein (GFP) to visualize their behavior within the cytoplasm. While soluble, these polymers have a similar intracellular diffusion constant as cytosolic proteins at 7.4 μm(2)/s; however, above their phase transition temperature, the proteins form distinct microdomains (0.1-2 μm) with a reduced diffusion coefficient of 1.1 μm(2)/s. Microdomain assembly and disassembly are both rapid processes with half-lives of 3.8 and 1.0 min, respectively. Via selection of the protein polymer, the assembly temperature is tunable between 20 and 40 °C. This approach may be useful to control intracellular formation of genetically engineered proteins and protein complexes into concentrated microdomains.
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033-9121, USA.
authors.forename: Martha K
authors.initials: MK
authors.lastname: Pastuszka
authors.forename: Siti M
authors.initials: SM
authors.lastname: Janib
authors.forename: Isaac
authors.initials: I
authors.lastname: Weitzhandler
authors.forename: Curtis T
authors.initials: CT
authors.lastname: Okamoto
authors.forename: Sarah
authors.initials: S
authors.lastname: Hamm-Alvarez
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Mackay
mesh_terms: D002460:Cell Line; D004549:Elastin / Q000737:chemistry*; D049452:Green Fluorescent Proteins; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D049329:Nanostructures; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014333:Tropoelastin / Q000737:chemistry
chemical_list: D010455:Peptides; D011993:Recombinant Fusion Proteins; D014333:Tropoelastin; D049452:Green Fluorescent Proteins; D004549:Elastin