Source:http://linkedlifedata.com/resource/pubmed/id/11810694
Switch to
| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
1
|
| pubmed:dateCreated |
2002-1-25
|
| pubmed:abstractText |
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.
|
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical | |
| pubmed:status |
MEDLINE
|
| pubmed:month |
Jan
|
| pubmed:issn |
0886-1544
|
| pubmed:author | |
| pubmed:copyrightInfo |
Copyright 2002 Wiley-Liss, Inc.
|
| pubmed:issnType |
Print
|
| pubmed:volume |
51
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
27-38
|
| pubmed:dateRevised |
2006-11-15
|
| pubmed:meshHeading |
pubmed-meshheading:11810694-3T3 Cells,
pubmed-meshheading:11810694-Actins,
pubmed-meshheading:11810694-Animals,
pubmed-meshheading:11810694-Cell Line,
pubmed-meshheading:11810694-Cell Movement,
pubmed-meshheading:11810694-Cells, Cultured,
pubmed-meshheading:11810694-Chick Embryo,
pubmed-meshheading:11810694-Cytoskeleton,
pubmed-meshheading:11810694-Deoxyribonuclease I,
pubmed-meshheading:11810694-Dipodomys,
pubmed-meshheading:11810694-Fibroblasts,
pubmed-meshheading:11810694-Fluorescent Antibody Technique, Indirect,
pubmed-meshheading:11810694-Fluorescent Dyes,
pubmed-meshheading:11810694-Mice,
pubmed-meshheading:11810694-Microscopy, Fluorescence,
pubmed-meshheading:11810694-Sensitivity and Specificity,
pubmed-meshheading:11810694-Staining and Labeling,
pubmed-meshheading:11810694-Tissue Fixation
|
| pubmed:year |
2002
|
| pubmed:articleTitle |
Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells.
|
| pubmed:affiliation |
MRC, Laboratory for Molecular Cell Biology and Department of Biology, University College London, London, United Kingdom. l.cramer@ucl.ac.uk
|
| pubmed:publicationType |
Journal Article,
Comparative Study,
Research Support, Non-U.S. Gov't
|