Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membrane
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 chromopho...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2014-11-01
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| Series: | Frontiers in Molecular Neuroscience |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fnmol.2014.00093/full |
| _version_ | 1811300954150010880 |
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| author | Yuka eJinno Keiko eShoda Emiliano eRial-Verde Rafael eYuste Miyawaki eAtsushi Hidekazu eTsutsui Hidekazu eTsutsui Hidekazu eTsutsui |
| author_facet | Yuka eJinno Keiko eShoda Emiliano eRial-Verde Rafael eYuste Miyawaki eAtsushi Hidekazu eTsutsui Hidekazu eTsutsui Hidekazu eTsutsui |
| author_sort | Yuka eJinno |
| collection | DOAJ |
| description | 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 -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 (~7.3º) from the membrane normal. Genetically-encoded SHG chromophores could serve as a molecular platform for imaging membrane potential. |
| first_indexed | 2024-04-13T07:00:14Z |
| format | Article |
| id | doaj.art-bebbce596950480ab5c13ff7f70006d7 |
| institution | Directory Open Access Journal |
| issn | 1662-5099 |
| language | English |
| last_indexed | 2024-04-13T07:00:14Z |
| publishDate | 2014-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Neuroscience |
| spelling | doaj.art-bebbce596950480ab5c13ff7f70006d72022-12-22T02:57:09ZengFrontiers Media S.A.Frontiers in Molecular Neuroscience1662-50992014-11-01710.3389/fnmol.2014.00093112244Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membraneYuka eJinno0Keiko eShoda1Emiliano eRial-Verde2Rafael eYuste3Miyawaki eAtsushi4Hidekazu eTsutsui5Hidekazu eTsutsui6Hidekazu eTsutsui7Osaka UniversityRIKENColumbia UniversityColumbia UniversityRIKENJAISTRIKENJapan Science and Technology AgencyAlthough 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 -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 (~7.3º) from the membrane normal. Genetically-encoded SHG chromophores could serve as a molecular platform for imaging membrane potential.http://journal.frontiersin.org/Journal/10.3389/fnmol.2014.00093/fullMutagenesiselectrostatic potentialsecond harmonic generationfluoresence proteinKras4B |
| spellingShingle | Yuka eJinno Keiko eShoda Emiliano eRial-Verde Rafael eYuste Miyawaki eAtsushi Hidekazu eTsutsui Hidekazu eTsutsui Hidekazu eTsutsui Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membrane Frontiers in Molecular Neuroscience Mutagenesis electrostatic potential second harmonic generation fluoresence protein Kras4B |
| title | Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membrane |
| title_full | Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membrane |
| title_fullStr | Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membrane |
| title_full_unstemmed | Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membrane |
| title_short | Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membrane |
| title_sort | engineering a genetically encoded shg chromophore by electrostatic targeting to the membrane |
| topic | Mutagenesis electrostatic potential second harmonic generation fluoresence protein Kras4B |
| url | http://journal.frontiersin.org/Journal/10.3389/fnmol.2014.00093/full |
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