Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion
The fluorescence microscopy methods presently used to characterize protein motion in cells infer protein motion from indirect observables, rather than measuring protein motion directly. Operationalizing these methods requires expertise that can constitute a barrier to their broad utilization. Here,...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2017-03-01
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| Series: | Cell Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2211124717302784 |
| _version_ | 1818031812693196800 |
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| author | Rotem Gura Sadovsky Shlomi Brielle Daniel Kaganovich Jeremy L. England |
| author_facet | Rotem Gura Sadovsky Shlomi Brielle Daniel Kaganovich Jeremy L. England |
| author_sort | Rotem Gura Sadovsky |
| collection | DOAJ |
| description | The fluorescence microscopy methods presently used to characterize protein motion in cells infer protein motion from indirect observables, rather than measuring protein motion directly. Operationalizing these methods requires expertise that can constitute a barrier to their broad utilization. Here, we have developed PIPE (photo-converted intensity profile expansion) to directly measure the motion of tagged proteins and quantify it using an effective diffusion coefficient. PIPE works by pulsing photo-convertible fluorescent proteins, generating a peaked fluorescence signal at the pulsed region, and analyzing the spatial expansion of the signal. We demonstrate PIPE’s success in measuring accurate diffusion coefficients in silico and in vitro and compare effective diffusion coefficients of native cellular proteins and free fluorophores in vivo. We apply PIPE to measure diffusion anomality in the cell and use it to distinguish free fluorophores from native cellular proteins. PIPE’s direct measurement and ease of use make it appealing for cell biologists. |
| first_indexed | 2024-12-10T05:57:26Z |
| format | Article |
| id | doaj.art-0ce092e64ca5477c9f88bc134e6c2b4f |
| institution | Directory Open Access Journal |
| issn | 2211-1247 |
| language | English |
| last_indexed | 2024-12-10T05:57:26Z |
| publishDate | 2017-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Reports |
| spelling | doaj.art-0ce092e64ca5477c9f88bc134e6c2b4f2022-12-22T01:59:53ZengElsevierCell Reports2211-12472017-03-0118112795280610.1016/j.celrep.2017.02.063Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile ExpansionRotem Gura Sadovsky0Shlomi Brielle1Daniel Kaganovich2Jeremy L. England3Physics of Living Systems Group, Massachusetts Institute of Technology, Cambridge, MA 02138, USADepartment of Cell and Developmental Biology, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, IsraelDepartment of Cell and Developmental Biology, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, IsraelPhysics of Living Systems Group, Massachusetts Institute of Technology, Cambridge, MA 02138, USAThe fluorescence microscopy methods presently used to characterize protein motion in cells infer protein motion from indirect observables, rather than measuring protein motion directly. Operationalizing these methods requires expertise that can constitute a barrier to their broad utilization. Here, we have developed PIPE (photo-converted intensity profile expansion) to directly measure the motion of tagged proteins and quantify it using an effective diffusion coefficient. PIPE works by pulsing photo-convertible fluorescent proteins, generating a peaked fluorescence signal at the pulsed region, and analyzing the spatial expansion of the signal. We demonstrate PIPE’s success in measuring accurate diffusion coefficients in silico and in vitro and compare effective diffusion coefficients of native cellular proteins and free fluorophores in vivo. We apply PIPE to measure diffusion anomality in the cell and use it to distinguish free fluorophores from native cellular proteins. PIPE’s direct measurement and ease of use make it appealing for cell biologists.http://www.sciencedirect.com/science/article/pii/S2211124717302784fluorescence-based techniquesmolecular diffusionprotein mobilityphoto-conversionanomalous diffusionmolecular crowdingprotein oligomerizationprotein quality controlprotein aggregationSod1 |
| spellingShingle | Rotem Gura Sadovsky Shlomi Brielle Daniel Kaganovich Jeremy L. England Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion Cell Reports fluorescence-based techniques molecular diffusion protein mobility photo-conversion anomalous diffusion molecular crowding protein oligomerization protein quality control protein aggregation Sod1 |
| title | Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion |
| title_full | Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion |
| title_fullStr | Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion |
| title_full_unstemmed | Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion |
| title_short | Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion |
| title_sort | measurement of rapid protein diffusion in the cytoplasm by photo converted intensity profile expansion |
| topic | fluorescence-based techniques molecular diffusion protein mobility photo-conversion anomalous diffusion molecular crowding protein oligomerization protein quality control protein aggregation Sod1 |
| url | http://www.sciencedirect.com/science/article/pii/S2211124717302784 |
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