ABSTRACT
Generation of artificial cells provides the bridge needed to cover the gap between studying the complexity of biological processes in whole cells and studying these same processes in an in vitro reconstituted system. Artificial cells are defined as the encapsulation of biologically active material in a biological or synthetic membrane. Here, we describe a robust and general method to produce artificial cells for the purpose of mimicking one or more behaviors of a cell. A microfluidic double emulsion system is used to encapsulate a mammalian cell-free expression system that is able to express membrane proteins into the bilayer or soluble proteins inside the vesicles. The development of a robust platform that allows the assembly of artificial cells is valuable in understanding subcellular functions and emergent behaviors in a more cell-like environment as well as for creating novel signaling pathways to achieve specific cellular behaviors.
Subject(s)
Artificial Cells/cytology , Cell-Free System/metabolism , Lipid Bilayers/metabolism , Membrane Proteins/metabolism , Microfluidics/methods , Cell Line, Tumor , DNA/genetics , Emulsions/metabolism , Green Fluorescent Proteins/genetics , HeLa Cells , Humans , Plasmids/genetics , RNA, Messenger/geneticsABSTRACT
Dimerization of G protein-coupled receptors (GPCRs) represents a potential mechanism by which GPCR functions are regulated. Several resonance energy transfer (RET)-based methods have revealed GPCR homo- and heterodimerization. However, interpretation of an increase in FRET efficiency could be attributed to either dimerization/oligomerization events or conformational changes within an already dimerized/oligomerized receptor complex. Furthermore, RET-based methods can only measure pairwise dimerization, and cannot easily achieve multiplex detection. In this study, we applied proximity-based biotinylation for detecting receptor dimerization by utilizing a specific enzyme-substrate pair that are fused to GPCRs. The biotin ligase BirA is fused to CXCR4 and site-specifically biotinylates an acceptor peptide (AP) in the presence of biotin. As a test case for our newly developed assay, we have characterized the homo-dimerization of chemokine receptor CXCR4 and heterodimerization of CXCR4 with CCR2 or CCR5. The degree of biotinylation varies with the amount of GPCR-AP as well as biotinylation time. Using enzyme/substrate receptor pairs and measuring receptor biotinylation, we demonstrate that CXCR4 can homo-dimerize and hetero-dimerize with CCR2 and CCR5. The effect of CXCL12, agonist for CXCR4, was found to decrease surface biotinylation of CXCR4-AP. This effect is due to a combination of CXCR4 endocytosis and stabilization of CXCR4 homodimers. Finally, when CXCR4-AP, CCR2-AP, and CCR5-AP were expressed together, we observed CXCR4-CXCR4 homodimers and CXCR4-CCR2 and CXCR4-CCR5 heterodimers. The newly developed assay opens new opportunity for multiplex detection for GPCR homo- and heterodimerization within the same cellular context.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Protein Binding , Protein Multimerization , Receptors, G-Protein-Coupled/chemistry , Animals , Biotinylation , Carbon-Nitrogen Ligases/chemistry , Chemokine CXCL12/chemistry , Endocytosis , Escherichia coli Proteins/chemistry , Protein Conformation , Receptors, CCR2/chemistry , Receptors, CCR5/chemistry , Receptors, CXCR4/chemistry , Repressor Proteins/chemistry , Signal TransductionABSTRACT
Cells can sense a myriad of mechanical stimuli. Mechanosensitive channel of large conductance (MscL) found in bacteria is a well-characterized mechanosensitive channel that rapidly responds to an increase in turgor pressure. Functional expression of MscL in mammalian cells has recently been demonstrated, revealing that molecular delivery or transport can be achieved by charge-induced activation of MscL. Despite a well-accepted mechanism for MscL activation by membrane tension in bacteria, it is not clear whether and how MscL can be opened by other modes of force transduction in mammalian cells. In this work, we used a variety of techniques to characterize the gating of MscL expressed in mammalian cells, using both wild type and a G22S mutant which activates at a lower threshold. In particular, employing a new technique, acoustic tweezing cytometry (ATC), we show that ultrasound actuation of integrin-bound microbubbles can lead to MscL opening and that ATC induced MscL activation was dependent on the functional linkage of the microbubbles with an intact actin cytoskeleton. Our results indicate that localized mechanical stress can mediate opening of MscL that requires force transduction through the actin cytoskeleton, revealing a new mode of MscL activation that may prove to be a useful tool for mechanobiology and drug delivery research.
