Abstract
BackgroundThe transport of labeled G-actin from the mid-lamella region to the leading edge in a highly motile malignant rat fibroblast line has been studied using fluorescence localization after photobleaching or FLAP, and the transit times recorded in these experiments were so fast that simple diffusion was deemed an insufficient explanation (see Zicha et al., Science, v. 300, pp. 142–145 [1]).Methodology/Principal FindingsWe re-examine the Zicha FLAP experiments using a two-phase reactive interpenetrating flow formalism to model the cytoplasm and the transport dynamics of bleached and unbleached actin. By allowing an improved treatment of effects related to the retrograde flow of the cytoskeleton and of the geometry and finite thickness of the lamella, this new analysis reveals a mechanism that can realistically explain the timing and the amplitude of all the FLAP signals observed in [1] without invoking special transport modalities.Conclusions/SignificanceWe conclude that simple diffusion is sufficent to explain the observed transport rates, and that variations in the transport of labeled actin through the lamella are minor and not likely to be the cause of the observed physiological variations among different segments of the leading edge. We find that such variations in labeling can easily arise from differences and changes in the microscopic actin dynamics inside the edge compartment, and that the key dynamical parameter in this regard is the so-called “dilatation rate” (the velocity of cytoskeletal retrograde flow divided by a characteristic dimension of the edge compartment where rapid polymerization occurs). If our dilatation hypothesis is correct, the transient kinetics of bleached actin relocalization constitute a novel and very sensitive method for probing the cytoskeletal dynamics in leading edge micro-environments which are otherwise very difficult to directly interrogate.
Highlights
Fluorescence localization after photobleaching (FLAP) is a technique developed in the laboratory of Graham Dunn, whereby the proteins present in a localized region of the cytoplasm are photo-labeled and tracked to ascertain their subsequent transport and fate [2,3]
The investigations we report here were motivated by experiments of Zicha and coworkers, applying this technique to the dynamics of the actin cytoskeleton in the leading lamella of T15 cells, a line of transformed rat fibroblasts [1]
We re-examine the theoretical arguments indicating that diffusion is too slow to explain the reported actin dynamics in T15 cells, and we ask if there is some other mechanism, not involving specialized transport, that could explain the FLAP data. We approach these issues by constructing a computer model of mass transport and FLAP dynamics in a leading lamella that is sufficently detailed to include processes such as channel flow through a porous cytoskeleton as suggested by Zicha et al Based on simulations, we find that this model does produce results that are quite close to what was observed by Zicha et al, but that the reasons for this success have nothing to do with channel flow or with special transport of any kind
Summary
Fluorescence localization after photobleaching (FLAP) is a technique developed in the laboratory of Graham Dunn, whereby the proteins present in a localized region of the cytoplasm are photo-labeled and tracked to ascertain their subsequent transport and fate [2,3]. The investigations we report here were motivated by experiments of Zicha and coworkers, applying this technique to the dynamics of the actin cytoskeleton in the leading lamella of T15 cells, a line of transformed rat fibroblasts [1]. To carry out these studies, actin monomers labeled with yellow fluorophore and monomers labeled with cyan fluorophore were introduced into the cytoplasm via dual transfection with appropriate DNA constructs. The transport of labeled G-actin from the mid-lamella region to the leading edge in a highly motile malignant rat fibroblast line has been studied using fluorescence localization after photobleaching or FLAP, and the transit times recorded in these experiments were so fast that simple diffusion was deemed an insufficient explanation The transport of labeled G-actin from the mid-lamella region to the leading edge in a highly motile malignant rat fibroblast line has been studied using fluorescence localization after photobleaching or FLAP, and the transit times recorded in these experiments were so fast that simple diffusion was deemed an insufficient explanation (see Zicha et al, Science, v. 300, pp. 142–145 [1])
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