Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells.
Lamellipodium protrusion is linked to actin filament disassembly in migrating fibroblasts [Cramer, 1999: Curr. Biol. 9:1095-1105]. To further study this relationship, we have identified a method to specifically and sensitively detect G-actin in distinct spatial locations in motile cells using deoxyr...
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| Format: | Journal article |
| Language: | English |
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2002
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| _version_ | 1797092781889421312 |
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| author | Cramer, L Briggs, L Dawe, H |
| author_facet | Cramer, L Briggs, L Dawe, H |
| author_sort | Cramer, L |
| collection | OXFORD |
| description | Lamellipodium protrusion is linked to actin filament disassembly in migrating fibroblasts [Cramer, 1999: Curr. Biol. 9:1095-1105]. To further study this relationship, we have identified a method to specifically and sensitively detect G-actin in distinct spatial locations in motile cells using deoxyribonuclease I (DNase I). Although DNase I can bind both G- and F-actin in vitro [Mannherz et al., 1980: Eur. J. Biochem. 95:377-385], when cells were fixed in formaldehyde and permeabilized in detergent, fluorescently-labelled DNase I specifically stained G-actin and not F-actin. 92-98% of actin molecules were stably retained in cells during fixation and permeabilization. Further, increasing or decreasing cellular G-actin concentration by treating live cells with latrunculin-A or jasplakinolide, respectively, caused a respective increase and decrease in DNase I cell-staining intensity as expected. These changes in DNase I fluorescence intensity accurately reflected increases and decreases in cellular G-actin concentration independently measured in lysates prepared from drug-treated live cells (regression coefficient = 0.98). This shows that DNase I cell-staining is very sensitive using this method. Applying this method, we found that the ratio of G-/F-actin is lower in both the lamellipodium and in a broad band immediately behind the lamellipodium in migrating compared to non-migrating fibroblasts. Thus, we predict that protrusion of the lamellipodium in migrating fibroblasts requires tight coupling to filament disassembly at least in part because G-actin is relatively limited within and behind the lamellipodium. This is the first report to directly demonstrate high sensitivity of cell-staining for any G-actin probe and this, together with the ready commercial accessibility of fluorescently-labelled DNase I, make it a simple, convenient, and sensitive tool for cell-staining of G-actin. |
| first_indexed | 2024-03-07T03:50:56Z |
| format | Journal article |
| id | oxford-uuid:c13b4dcb-1329-41e3-993c-0f185a5af77c |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T03:50:56Z |
| publishDate | 2002 |
| record_format | dspace |
| spelling | oxford-uuid:c13b4dcb-1329-41e3-993c-0f185a5af77c2022-03-27T05:59:59ZUse of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:c13b4dcb-1329-41e3-993c-0f185a5af77cEnglishSymplectic Elements at Oxford2002Cramer, LBriggs, LDawe, HLamellipodium protrusion is linked to actin filament disassembly in migrating fibroblasts [Cramer, 1999: Curr. Biol. 9:1095-1105]. To further study this relationship, we have identified a method to specifically and sensitively detect G-actin in distinct spatial locations in motile cells using deoxyribonuclease I (DNase I). Although DNase I can bind both G- and F-actin in vitro [Mannherz et al., 1980: Eur. J. Biochem. 95:377-385], when cells were fixed in formaldehyde and permeabilized in detergent, fluorescently-labelled DNase I specifically stained G-actin and not F-actin. 92-98% of actin molecules were stably retained in cells during fixation and permeabilization. Further, increasing or decreasing cellular G-actin concentration by treating live cells with latrunculin-A or jasplakinolide, respectively, caused a respective increase and decrease in DNase I cell-staining intensity as expected. These changes in DNase I fluorescence intensity accurately reflected increases and decreases in cellular G-actin concentration independently measured in lysates prepared from drug-treated live cells (regression coefficient = 0.98). This shows that DNase I cell-staining is very sensitive using this method. Applying this method, we found that the ratio of G-/F-actin is lower in both the lamellipodium and in a broad band immediately behind the lamellipodium in migrating compared to non-migrating fibroblasts. Thus, we predict that protrusion of the lamellipodium in migrating fibroblasts requires tight coupling to filament disassembly at least in part because G-actin is relatively limited within and behind the lamellipodium. This is the first report to directly demonstrate high sensitivity of cell-staining for any G-actin probe and this, together with the ready commercial accessibility of fluorescently-labelled DNase I, make it a simple, convenient, and sensitive tool for cell-staining of G-actin. |
| spellingShingle | Cramer, L Briggs, L Dawe, H Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells. |
| title | Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells. |
| title_full | Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells. |
| title_fullStr | Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells. |
| title_full_unstemmed | Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells. |
| title_short | Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells. |
| title_sort | use of fluorescently labelled deoxyribonuclease i to spatially measure g actin levels in migrating and non migrating cells |
| work_keys_str_mv | AT cramerl useoffluorescentlylabelleddeoxyribonucleaseitospatiallymeasuregactinlevelsinmigratingandnonmigratingcells AT briggsl useoffluorescentlylabelleddeoxyribonucleaseitospatiallymeasuregactinlevelsinmigratingandnonmigratingcells AT daweh useoffluorescentlylabelleddeoxyribonucleaseitospatiallymeasuregactinlevelsinmigratingandnonmigratingcells |