Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithelial cells analyzed by quantitative Fluorescent Speckle Microscopy.
We measured actin turnover in lamellipodia and lamellae of migrating cells, using quantitative Fluorescent Speckle Microscopy. Lamellae disassembled at low rates from the front to the back. However, the dominant feature in their turnover was a spatially random pattern of periodic polymerization and depolymerization moving with the retrograde flow. Power spectra contained frequencies between 0.5 and 1 cycle/min. The spectra remained unchanged when applying Latrunculin A and Jasplakinolide in low doses, except that additional frequencies occurred beyond 1 cycle/min. Whereas Latrunculin did not change the rate of mean disassembly, Jasplakinolide halted it completely, indicating that the steady state and the dynamics of actin turnover are differentially affected by pharmacological agents. Lamellipodia assembled in recurring bursts at the leading edge and disassembled approximately 2.5 microm behind. Events of polymerization correlated spatially and temporally with transient formation of Arp2/3 clusters. In lamellae, Arp2/3 accumulation and polymerization correlated only spatially, suggesting an Arp2/3-independent mechanism for filament nucleation. To acquire these data we had to enhance the resolution of quantitative Fluorescent Speckle Microscopy to the submicron level. Several algorithmic advances in speckle image processing are described enabling the analysis of kinetic and kinematic activities of polymer networks at the level of single speckles.
Ponti
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Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA.
Matov
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Adams
M
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Gupton
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S
Waterman-Storer
C M
CM
Danuser
G
G
D051377:Actin-Related Protein 2 / Q000378:metabolism; D051378:Actin-Related Protein 3 / Q000378:metabolism; D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D001703:Biophysics / Q000379:methods; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry; D002460:Cell Line; D002465:Cell Movement; D002478:Cells, Cultured; D047630:Depsipeptides / Q000737:chemistry; D004847:Epithelial Cells / Q000166:cytology*; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D008958:Models, Molecular; D015233:Models, Statistical; D011108:Polymers / Q000737:chemistry; D048328:Potoroidae; D011554:Pseudopodia / Q000737:chemistry* / Q000378:metabolism; D013844:Thiazoles / Q000737:chemistry; D053778:Thiazolidines; D013997:Time Factors
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D047630:Depsipeptides; D011108:Polymers; D013844:Thiazoles; D053778:Thiazolidines; C057531:jasplakinolide; C037067:latrunculin A