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Measuring forces at the leading edge: a force assay for cell motility.

ABSTRACT: Cancer cells become mobile by remodelling their cytoskeleton to form migratory structures. This transformation is dominated by actin assembly and disassembly (polymerisation and depolymerisation) in the cytoplasm. Synthesis of filamentous actin produces a force at the leading edge that pushes the plasma membrane forward. We describe an assay to measure the restoring force of the membrane in response to forces generated within the cytoplasm adjacent to the membrane. A laser trap is used to form a long membrane nanotube from a living cell and to measure the axial membrane force at the end of the tube. When the tube, resembling a filopodium, is formed and in a relaxed state the axial membrane force exhibits a positive stationary value. This value reflects the influence of the cytoskeleton that acts to pull the tube back to the cell. A dynamic sawtooth force that rides upon the stationary value is also observed. This force is sensitive to a toxin that affects actin assembly and disassembly, but not affected by agents that influence microtubules and myosin light chain kinase. We deduce from the magnitude and characteristics of dynamic force measurements that it originates from depolymerisation and polymerisation of F-actin. The on- and off-rates, the number of working filaments, and the force per filament (2.5 pN) are determined. We suggest the force-dependent transitions are thermodynamically uncoupled as both the on- and off-rates decrease exponentially with a compressive load. We propose kinetic schemes that require attachment of actin filaments to the membrane during depolymerisation. This demonstrates that actin kinetics can be monitored in a living cell by measuring force at the membrane, and used to probe the mobility of cells including cancer cells.

SUBMITTER: Farrell B

PROVIDER: S-EPMC3529002 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Measuring forces at the leading edge: a force assay for cell motility.

Farrell Brenda B Qian Feng F Kolomeisky Anatoly A Anvari Bahman B Brownell William E WE

Integrative biology : quantitative biosciences from nano to macro 20130101 1

Cancer cells become mobile by remodelling their cytoskeleton to form migratory structures. This transformation is dominated by actin assembly and disassembly (polymerisation and depolymerisation) in the cytoplasm. Synthesis of filamentous actin produces a force at the leading edge that pushes the plasma membrane forward. We describe an assay to measure the restoring force of the membrane in response to forces generated within the cytoplasm adjacent to the membrane. A laser trap is used to form a ...[more]

PMID: 23080534

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Project description:Actin-based cell motility and force generation are central to immune response, tissue development, and cancer metastasis, and understanding actin cytoskeleton regulation is a major goal of cell biologists. Cell spreading is a commonly used model system for motility experiments -- spreading fibroblasts exhibit stereotypic, spatially-isotropic edge dynamics during a reproducible sequence of functional phases: 1) During early spreading, cells form initial contacts with the surface. 2) The middle spreading phase exhibits rapidly increasing attachment area. 3) Late spreading is characterized by periodic contractions and stable adhesions formation. While differences in cytoskeletal regulation between phases are known, a global analysis of the spatial and temporal coordination of motility and force generation is missing. Implementing improved algorithms for analyzing edge dynamics over the entire cell periphery, we observed that a single domain of homogeneous cytoskeletal dynamics dominated each of the three phases of spreading. These domains exhibited a unique combination of biophysical and biochemical parameters -- a motility module. Biophysical characterization of the motility modules revealed that the early phase was dominated by periodic, rapid membrane blebbing; the middle phase exhibited continuous protrusion with very low traction force generation; and the late phase was characterized by global periodic contractions and high force generation. Biochemically, each motility module exhibited a different distribution of the actin-related protein VASP, while inhibition of actin polymerization revealed different dependencies on barbed-end polymerization. In addition, our whole-cell analysis revealed that many cells exhibited heterogeneous combinations of motility modules in neighboring regions of the cell edge. Together, these observations support a model of motility in which regions of the cell edge exhibit one of a limited number of motility modules that, together, determine the overall motility function. Our data and algorithms are publicly available to encourage further exploration.

Dataset Information

Measuring forces at the leading edge: a force assay for cell motility.

Publications

Measuring forces at the leading edge: a force assay for cell motility.

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