Simultaneous, Real-Time Imaging of Intracellular Calcium and Cellular Traction Force Production

Doyle, Andrew D.; Lee, Juliet

doi:10.2144/02332rr02

Cited by 30 publications

(33 citation statements)

References 25 publications

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“…Axenically grown Dictyostelium WT and mutant cells were prepared for chemotaxis as described (33). The cells were seeded onto a gelatin gel as described in SI Appendix A (34). Time-lapse sequences of chemotaxing cells were acquired on an inverted microscope controlled by Metamorph software (Molecular Devices, Downington, PA).…”

Section: Methodsmentioning

confidence: 99%

Spatio-temporal analysis of eukaryotic cell motility by improved force cytometry

Álamo

Meili

Alonso-Latorre

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

195

309

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Cell motility plays an essential role in many biological systems, but precise quantitative knowledge of the biophysical processes involved in cell migration is limited. Better measurements are needed to ultimately build models with predictive capabilities. We present an improved force cytometry method and apply it to the analysis of the dynamics of the chemotactic migration of the amoeboid form of Dictyostelium discoideum. Our explicit calculation of the force field takes into account the finite thickness of the elastic substrate and improves the accuracy and resolution compared with previous methods. This approach enables us to quantitatively study the differences in the mechanics of the migration of wildtype (WT) and mutant cell lines. The time evolution of the strain energy exerted by the migrating cells on their substrate is quasiperiodic and can be used as a simple indicator of the stages of the cell motility cycle. We have found that the mean velocity of migration v and the period of the strain energy T cycle are related through a hyperbolic law v ‫؍‬ L/T, where L is a constant step length that remains unchanged in mutants with adhesion or contraction defects. Furthermore, when cells adhere to the substrate, they exert opposing pole forces that are orders of magnitude higher than required to overcome the resistance from their environment.Dictyostelium ͉ myosin ͉ traction forces ͉ cytoskeleton ͉ chemotaxis

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Section: Methodsmentioning

confidence: 99%

Spatio-temporal analysis of eukaryotic cell motility by improved force cytometry

Álamo

Meili

Alonso-Latorre

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

195

309

View full text Add to dashboard Cite

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“…Gelatin substrata were made as described previously (Doyle and Lee, 2002;Doyle and Lee, 2004) but with several modifications. Briefly, stock gels were made with 4.0% gelatin (90-110 Bloom, G6144-100G, Sigma) dissolved in BSS containing 3 mM Mes, pH 6.3 with KOH.…”

Section: Preparation Of Elastic Substratamentioning

confidence: 99%

“…After solidification, fluorescent orange microspheres (200 nm in diameter, F-8809, Molecular Probes) were attached to the surface of the substrata. The subsequent procedure was identical to that described previously (Doyle and Lee, 2002;Doyle and Lee, 2004).…”

Section: Preparation Of Elastic Substratamentioning

confidence: 99%

Actin-based propulsive forces and myosin-II-based contractile forces in migratingDictyosteliumcells

Iwadate

Yumura

2008

Journal of Cell Science

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It has been suggested that myosin II exerts traction forces at the posterior ends and retracting pseudopodia of migrating cells, but there is no direct evidence. Here, using a combination of total internal reflection fluorescence (TIRF) microscopy and force microscopy with a high spatial resolution of ∼400 nm, we simultaneously recorded GFP-myosin II dynamics and traction forces under migrating Dictyostelium cells. Accumulation of filamentous myosin II and a subsequent increase in traction forces were detected in pseudopodia just before retraction. In the case of motorless myosin II, traction forces did not increase after accumulation, suggesting that the source of the retraction force is the motor activity of accumulated myosin II. Simultaneous recording of F-actin and traction forces revealed that traction forces were exerted under spot-like regions where F-actin accumulated. Cells migrated in a direction counter to the sum of the force vectors exerted at each spot, suggesting that the stress spots act as scaffolds to transmit the propulsive forces at the leading edge generated by actin polymerization.

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“…The gelatin substrata were manufactured and calibrated as previously described (Doyle and Lee, 2002). Stock gels were made with 2.5% gelatin (Nabisco, Parsippany, NJ) dissolved in HL-5 medium.…”

Section: Manufacture and Calibration Of Gelatin Substratamentioning

confidence: 99%

“…Examples of these include weakly crosslinked films of silicone (Burton et al, 1999;Lee et al, 1994;Oliver et al, 1998) and gelatin (Doyle and Lee, 2002). Recently, both wild-type and myosin II null Dictyostelium cells were found to deform weakly crosslinked silicone substrata (Uchida et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Traction force microscopy in Dictyostelium reveals distinct roles for myosin II motor and actin-crosslinking activity in polarized cell movement

Lombardi

Knecht

Dembo

et al. 2007

Journal of Cell Science

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Continuous cell movement requires the coordination of protrusive forces at the leading edge with contractile forces at the rear of the cell. Myosin II is required to generate the necessary contractile force to facilitate retraction; however, Dictyostelium cells that lack myosin II (mhcA–) are still motile. To directly investigate the role of myosin II in contractility we used a gelatin traction force assay to measure the magnitude and dynamic redistribution of traction stresses generated by randomly moving wild-type, myosin II essential light chain null (mlcE–) and mhcA– cells. Our data show that for each cell type, periods of rapid, directed cell movement occur when an asymmetrical distribution of traction stress is present, in which traction stresses at the rear are significantly higher than those at the front. We found that the major determinants of cell speed are the rate and frequency at which traction stress asymmetry develops, not the absolute magnitude of traction stress. We conclude that traction stress asymmetry is important for rapid, polarized cell movement because high traction stresses at the rear promote retraction, whereas low traction at the front allows protrusion. We propose that myosin II motor activity increases the rate and frequency at which traction stress asymmetry develops, whereas actin crosslinking activity is important for stabilizing it.

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Simultaneous, Real-Time Imaging of Intracellular Calcium and Cellular Traction Force Production

Cited by 30 publications

References 25 publications

Spatio-temporal analysis of eukaryotic cell motility by improved force cytometry

Spatio-temporal analysis of eukaryotic cell motility by improved force cytometry

Actin-based propulsive forces and myosin-II-based contractile forces in migratingDictyosteliumcells

Traction force microscopy in Dictyostelium reveals distinct roles for myosin II motor and actin-crosslinking activity in polarized cell movement

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