title: Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membrane.
abstract: Although second harmonic generation (SHG) microscopy provides unique imaging advantages for voltage imaging and other biological applications, genetically-encoded SHG chromophores remain relatively unexplored. SHG only arises from non-centrosymmetric media, so an anisotropic arrangement of chromophores is essential to provide strong SHG signals. Here, inspired by the mechanism by which K-Ras4B associates with plasma membranes, we sought to achieve asymmetric arrangements of chromophores at the membrane-cytoplasm interface using the fluorescent protein mVenus. After adding a farnesylation motif to the C-terminus of mVenus, nine amino acids composing its β-barrel surface were replaced by lysine, forming an electrostatic patch. This protein (mVe9Knus-CVIM) was efficiently targeted to the plasma membrane in a geometrically defined manner and exhibited SHG in HEK293 cells. In agreement with its design, mVe9Knus-CVIM hyperpolarizability was oriented at a small angle (~7.3°) from the membrane normal. Genetically-encoded SHG chromophores could serve as a molecular platform for imaging membrane potential.
authors.affiliation: Laboratory of Integrative Physiology, Graduate School of Medicine, Osaka University Suita, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Jinno
authors.affiliation: Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN Wako, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Shoda
authors.affiliation: Department of Biological Sciences, Neurotechnology Center, Columbia University New York, NY, USA.
authors.forename: Emiliano
authors.initials: E
authors.lastname: Rial-Verde
authors.affiliation: Department of Biological Sciences, Neurotechnology Center, Columbia University New York, NY, USA.
authors.forename: Rafael
authors.initials: R
authors.lastname: Yuste
authors.affiliation: Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN Wako, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.affiliation: Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN Wako, Japan ; Formation of and Information Processing by Neural Networks, and Control, PRESTO, Japan Science and Technology Agency (JST) Kawaguchi, Japan ; Department of Material Science, Japan Advanced Institute of Science and Technology Nomi, Japan.
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Tsutsui
keywords: electrostatic surface potential; fluorescence protein; mutagenesis; second harmonic generation