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PMID: 15548591 Published · ppublish English Comparative Study Journal Article Research Support, U.S. Gov't, Non-P.H.S.

Slipping or gripping? Fluorescent speckle microscopy in fish keratocytes reveals two different mechanisms for generating a retrograde flow of actin.

Molecular biology of the cell ·Vol. 16 ·No. 2 ·2005-02-00 ·Pages 507-18

Jurado C, Haserick JR, Lee J

Abstract

Fish keratocytes can generate rearward directed traction forces within front portions of the lamellipodium, suggesting that a retrograde flow of actin may also occur here but this was not detected by previous photoactivation experiments. To investigate the relationship between retrograde flow and traction force generation, we have transfected keratocytes with GFP-actin and used fluorescent speckle microscopy, to observe speckle flow. We detected a retrograde flow of actin within the leading lamellipodium that is inversely proportional to both protrusion rate and cell speed. To observe the effect of reducing contractility, we treated transfected cells with ML7, a potent inhibitor of myosin II. Surprisingly, ML7 treatment led to an increase in retrograde flow rate, together with a decrease in protrusion and cell speed, but only in rapidly moving cells. In slower moving cells, retrograde flow decreased, whereas protrusion rate and cell speed increased. These results suggest that there are two mechanisms for producing retrograde flow. One involves slippage between the cytoskeleton and adhesions, that decreases traction force production. The other involves slippage between adhesions and the substratum, which increases traction force production. We conclude that a biphasic relationship exists between retrograde actin flow and adhesiveness in moving keratocytes.

MeSH Terms
Actins/metabolism Animals Azepines/pharmacology Cell Adhesion Cell Movement/drug effects,physiology Cells, Cultured Cornea/cytology Enzyme Inhibitors/pharmacology Epithelial Cells/drug effects,physiology Extracellular Matrix/metabolism Fluorescent Antibody Technique Fluorescent Dyes Gelatin/metabolism Green Fluorescent Proteins/metabolism Image Processing, Computer-Assisted Kinetics Microscopy, Fluorescence Models, Biological Myosin Type II/drug effects Myosin-Light-Chain Kinase/antagonists & inhibitors Naphthalenes/pharmacology Phalloidine Poecilia Pseudopodia/physiology Transfection
Chemicals
Actins Azepines Enzyme Inhibitors Fluorescent Dyes Naphthalenes ML 7 Green Fluorescent Proteins Phalloidine Gelatin Myosin-Light-Chain Kinase Myosin Type II
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Jurado Carlos
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA.
Haserick John R
Lee Juliet
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.e04-10-0860
Published
2005-02-00
Epub
2004-00-17
Pages
507-18
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC545886
Subset
IM
External Links
PubMed source DOI: 10.1091/mbc.e04-10-0860 PMC Full Text View journal details
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