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Properties of the force exerted by filopodia and lamellipodia and the involvement of cytoskeletal components.

ABSTRACT: During neuronal differentiation, lamellipodia and filopodia explore the environment in search for the correct path to the axon's final destination. Although the motion of lamellipodia and filopodia has been characterized to an extent, little is known about the force they exert. In this study, we used optical tweezers to measure the force exerted by filopodia and lamellipodia with a millisecond temporal resolution. We found that a single filopodium exerts a force not exceeding 3 pN, whereas lamellipodia can exert a force up to 20 pN. Using metabolic inhibitors, we showed that no force is produced in the absence of actin polymerization and that development of forces larger than 3 pN requires microtubule polymerization. These results show that actin polymerization is necessary for force production and demonstrate that not only do neurons process information, but they also act on their environment exerting forces varying from tenths pN to tens of pN.

SUBMITTER: Cojoc D

PROVIDER: S-EPMC2034605 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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Properties of the force exerted by filopodia and lamellipodia and the involvement of cytoskeletal components.

Cojoc Dan D Difato Francesco F Ferrari Enrico E Shahapure Rajesh B RB Laishram Jummi J Righi Massimo M Di Fabrizio Enzo M EM Torre Vincent V

PloS one 20071024 10

During neuronal differentiation, lamellipodia and filopodia explore the environment in search for the correct path to the axon's final destination. Although the motion of lamellipodia and filopodia has been characterized to an extent, little is known about the force they exert. In this study, we used optical tweezers to measure the force exerted by filopodia and lamellipodia with a millisecond temporal resolution. We found that a single filopodium exerts a force not exceeding 3 pN, whereas lamel ...[more]

PMID: 17957254

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Project description:Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

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Properties of the force exerted by filopodia and lamellipodia and the involvement of cytoskeletal components.

Publications

Properties of the force exerted by filopodia and lamellipodia and the involvement of cytoskeletal components.

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