Spatial association of signaling proteins and F-actin effects on cluster assembly analyzed via photoactivation localization microscopy in T cells.
Recognition of antigens by T cell receptors (TCRs) triggers cellular signaling cascades initiated by recruitment to the plasma membrane of numerous effector molecules to form signaling microclusters (MCs). Here we show that the method of high-resolution photoactivation localization microscopy (PALM)...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2011-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3160965?pdf=render |
| _version_ | 1811275027402719232 |
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| author | Chih-Jung Hsu Tobias Baumgart |
| author_facet | Chih-Jung Hsu Tobias Baumgart |
| author_sort | Chih-Jung Hsu |
| collection | DOAJ |
| description | Recognition of antigens by T cell receptors (TCRs) triggers cellular signaling cascades initiated by recruitment to the plasma membrane of numerous effector molecules to form signaling microclusters (MCs). Here we show that the method of high-resolution photoactivation localization microscopy (PALM) imaging can be used to analyze the spatial correlation between kinase ZAP70 and adaptor SLP76 MCs at the cell periphery and the effects of F-actin on MC assembly. We first determined the photophysical rate constants of Dronpa and tdEos fluorescence probes, which allowed us to optimize our dual-color PALM imaging method. We next analyzed the degrees of spatial association through determination of Mander's colocalization coefficients from PALM images, which revealed increasing spatial segregation of ZAP70 and SLP76 MCs at the cell periphery after initiation of signaling. We showed that this spatial segregation at the cell periphery occurred in parallel with the reduction of MC phosphorylation levels. Furthermore, we used Ripley's K function to analyze spatial randomness, and determined average radii of clusters as a function of activation time. The average radii of SLP76 and LAT MCs were found to decrease, whereas ZAP70 MC radii remained relatively constant. Finally, effects of F-actin depolymerization on MC morphology were studied by determining radial distributions of cluster circularity. Our data suggest that MC morphology is affected by actin polymerization. The quantitative analysis of sub-diffraction PALM images may provide a starting point for a molecular interpretation of cluster-cluster interactions and of the regulation of T cell signaling MCs by the cytoskeleton. |
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| format | Article |
| id | doaj.art-b8f2a21e7e6b4cd28cb356ac91846fb4 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-12T23:30:44Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-b8f2a21e7e6b4cd28cb356ac91846fb42022-12-22T03:12:17ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0168e2358610.1371/journal.pone.0023586Spatial association of signaling proteins and F-actin effects on cluster assembly analyzed via photoactivation localization microscopy in T cells.Chih-Jung HsuTobias BaumgartRecognition of antigens by T cell receptors (TCRs) triggers cellular signaling cascades initiated by recruitment to the plasma membrane of numerous effector molecules to form signaling microclusters (MCs). Here we show that the method of high-resolution photoactivation localization microscopy (PALM) imaging can be used to analyze the spatial correlation between kinase ZAP70 and adaptor SLP76 MCs at the cell periphery and the effects of F-actin on MC assembly. We first determined the photophysical rate constants of Dronpa and tdEos fluorescence probes, which allowed us to optimize our dual-color PALM imaging method. We next analyzed the degrees of spatial association through determination of Mander's colocalization coefficients from PALM images, which revealed increasing spatial segregation of ZAP70 and SLP76 MCs at the cell periphery after initiation of signaling. We showed that this spatial segregation at the cell periphery occurred in parallel with the reduction of MC phosphorylation levels. Furthermore, we used Ripley's K function to analyze spatial randomness, and determined average radii of clusters as a function of activation time. The average radii of SLP76 and LAT MCs were found to decrease, whereas ZAP70 MC radii remained relatively constant. Finally, effects of F-actin depolymerization on MC morphology were studied by determining radial distributions of cluster circularity. Our data suggest that MC morphology is affected by actin polymerization. The quantitative analysis of sub-diffraction PALM images may provide a starting point for a molecular interpretation of cluster-cluster interactions and of the regulation of T cell signaling MCs by the cytoskeleton.http://europepmc.org/articles/PMC3160965?pdf=render |
| spellingShingle | Chih-Jung Hsu Tobias Baumgart Spatial association of signaling proteins and F-actin effects on cluster assembly analyzed via photoactivation localization microscopy in T cells. PLoS ONE |
| title | Spatial association of signaling proteins and F-actin effects on cluster assembly analyzed via photoactivation localization microscopy in T cells. |
| title_full | Spatial association of signaling proteins and F-actin effects on cluster assembly analyzed via photoactivation localization microscopy in T cells. |
| title_fullStr | Spatial association of signaling proteins and F-actin effects on cluster assembly analyzed via photoactivation localization microscopy in T cells. |
| title_full_unstemmed | Spatial association of signaling proteins and F-actin effects on cluster assembly analyzed via photoactivation localization microscopy in T cells. |
| title_short | Spatial association of signaling proteins and F-actin effects on cluster assembly analyzed via photoactivation localization microscopy in T cells. |
| title_sort | spatial association of signaling proteins and f actin effects on cluster assembly analyzed via photoactivation localization microscopy in t cells |
| url | http://europepmc.org/articles/PMC3160965?pdf=render |
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