ABSTRACT
Functional membrane proteins in the plasma membrane are suggested to have specific membrane environments that play important roles to maintain and regulate their function. However, the local membrane environments of membrane proteins remain largely unexplored due to the lack of available techniques. We have developed a method to probe the local membrane environment surrounding membrane proteins in the plasma membrane by covalently tethering a solvatochromic, environment-sensitive dye, Nile Red, to a GPI-anchored protein and the insulin receptor through a flexible linker. The fluidity of the membrane environment of the GPI-anchored protein depended upon the saturation of the acyl chains of the lipid anchor. The local environment of the insulin receptor was distinct from the average plasma membrane fluidity and was quite dynamic and heterogeneous. Upon addition of insulin, the local membrane environment surrounding the receptor specifically increased in fluidity in an insulin receptor-kinase dependent manner and on the distance between the dye and the receptor.
Subject(s)
Cell Membrane , Membrane Proteins , Receptor, Insulin , Cell Membrane/metabolism , Glycosylphosphatidylinositols/metabolism , GPI-Linked Proteins/metabolism , Membrane Proteins/metabolism , Receptor, Insulin/metabolism , Molecular Probe TechniquesABSTRACT
Optical monitoring of neuronal voltage using fluorescent indicators is a powerful approach for the interrogation of the cellular and molecular logic of the nervous system. Herein, a semisynthetic tethered voltage indicator (STeVI1) based upon nile red is described that displays voltage sensitivity when genetically targeted to neuronal membranes. This environmentally sensitive probe allows for wash-free imaging and faithfully detects supra- and sub-threshold activity in neurons.
Subject(s)
Fluorescent Dyes/chemistry , Neurons/metabolism , Optical Imaging , Oxazines/chemistry , HEK293 Cells , Humans , Molecular Structure , Neurons/cytologyABSTRACT
A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.
Subject(s)
Cell Membrane/metabolism , Fluorescent Dyes/chemistry , Membrane Proteins/chemistry , Molecular Imaging/methods , O(6)-Methylguanine-DNA Methyltransferase/chemistry , Oxazines/chemistry , Animals , Antigens, CD/chemistry , Antigens, CD/genetics , Antigens, CD/metabolism , CHO Cells , Carbonic Anhydrases/chemistry , Carbonic Anhydrases/genetics , Carbonic Anhydrases/metabolism , Cricetinae , Cricetulus , Electron Transport , Fluorescent Dyes/chemical synthesis , HEK293 Cells , Humans , Lipid Bilayers/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Confocal , O(6)-Methylguanine-DNA Methyltransferase/genetics , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Receptor, Insulin/chemistry , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
Dynamic amphiphiles provide access to transmembrane ion transport, differential sensing and cellular uptake. In this report, we introduce dynamic amphiphiles with fluorescent tails. Core-substituted naphthalenediimides (cNDIs) and perylenediimides (cPDIs) are tested. Whereas the latter suffer from poor partitioning, dynamic cNDI amphiphiles are found to be purifiable by RP-HPLC, to partition selectively into liquid-disordered (Ld) microdomains of mixed lipid bilayers and to activate DNA as transporters. Importantly, fluorescence properties, partitioning and activity can be modulated by changes in the structure of mixed amphiphiles. These results confirm the potential of dynamic fluorescent amphiphiles to selectively label extra- and intracellular membrane domains and visualize biological function.
Subject(s)
Hydrophobic and Hydrophilic Interactions , Imides/chemistry , Imides/metabolism , Membrane Microdomains/metabolism , Naphthalenes/chemistry , Naphthalenes/metabolism , Perylene/analogs & derivatives , Unilamellar Liposomes/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Molecular Imaging , Perylene/chemistry , Perylene/metabolism , Unilamellar Liposomes/chemistryABSTRACT
The study of membranes is at a turning point. New theories about membrane structure and function have recently been proposed, however, new technologies, combining chemical, physical, and biochemical approaches are necessary to test these hypotheses. In particular, the NCCR in chemical biology aims to visualize and characterize membrane microdomains and determine their function during hormone signaling.
