Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli
For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach su...
| Prif Awduron: | , , , |
|---|---|
| Fformat: | Journal article |
| Cyhoeddwyd: |
Royal Society
2016
|
| _version_ | 1826274566368919552 |
|---|---|
| author | Di Paolo, D Afanzar, O Armitage, J Berry, R |
| author_facet | Di Paolo, D Afanzar, O Armitage, J Berry, R |
| author_sort | Di Paolo, D |
| collection | OXFORD |
| description | For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time. |
| first_indexed | 2024-03-06T22:45:23Z |
| format | Journal article |
| id | oxford-uuid:5cfe2982-9b4a-4e7d-a1ec-250aecf0eb8f |
| institution | University of Oxford |
| last_indexed | 2024-03-06T22:45:23Z |
| publishDate | 2016 |
| publisher | Royal Society |
| record_format | dspace |
| spelling | oxford-uuid:5cfe2982-9b4a-4e7d-a1ec-250aecf0eb8f2022-03-26T17:31:39ZSingle-molecule imaging of electroporated dye-labelled CheY in live Escherichia coliJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:5cfe2982-9b4a-4e7d-a1ec-250aecf0eb8fSymplectic Elements at OxfordRoyal Society2016Di Paolo, DAfanzar, OArmitage, JBerry, RFor the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time. |
| spellingShingle | Di Paolo, D Afanzar, O Armitage, J Berry, R Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli |
| title | Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli |
| title_full | Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli |
| title_fullStr | Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli |
| title_full_unstemmed | Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli |
| title_short | Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli |
| title_sort | single molecule imaging of electroporated dye labelled chey in live escherichia coli |
| work_keys_str_mv | AT dipaolod singlemoleculeimagingofelectroporateddyelabelledcheyinliveescherichiacoli AT afanzaro singlemoleculeimagingofelectroporateddyelabelledcheyinliveescherichiacoli AT armitagej singlemoleculeimagingofelectroporateddyelabelledcheyinliveescherichiacoli AT berryr singlemoleculeimagingofelectroporateddyelabelledcheyinliveescherichiacoli |