Abstract
Protrusion of lamellipodia during cell migration depends on the assembly of actin network. The assembly mechanism, based on dendritic filament branching, has been investigated in reconstituted in vitro systems, but little is known about the dynamical and structural properties of the actin network in the lamellipodia of migrating cells. The length and orientation of filaments are difficult to measure directly in either optical or electron microscopy images because of the high filament density and overlapping of individual filaments. Here, we use the non-uniformity of optical images of the lamellipodia to extract information about the structural and dynamical properties of the underlying actin network. To determine the relationship between the image features and the properties of the network, we performed simulations of actin network assembly, based on the hypothesis of stochastic branching and capping of filaments, and produced computed ;fluorescence' and ;electron microscopy' images of the simulated network. By varying simulation parameters, in particular the actin filament density, length and orientation, we closely reproduced the contrast and the characteristic diagonal criss-cross pattern observed in the experimental optical images. Thus, matching the images of the simulated network to the experimental images allowed us to estimate parameters of actin filament network in lamellipodia.
Highlights
Actin polymerization is believed to drive protrusion at the leading edge of migrating cells
Biochemical and ultrastructural studies have identified the major components and reactions contributing to the assembly of the actin network, but there is a gap between the knowledge at the molecular level and the understanding of how the structure and dynamics of the lamellipodium are defined at the cellular scale
We created images similar to fluorescence and Electron microscopy (EM) images to study the effects of various simulation parameters on the image pattern and compare to the experimental images
Summary
Actin polymerization is believed to drive protrusion at the leading edge of migrating cells. Dendritic nucleation of filaments mediated by Arp2/3 complex is believed to be a major mechanism of assembly of the lamellipodial actin network (Carlier et al, 2003; Pollard and Borisy, 2003). Subsequent disassembly of filaments is accelerated by ADF/cofilin and serves to replenish the pool of free subunits for continuous assembly Given this sequence of reactions, the capping rate sets the limit for the length of actin filaments in the lamellipodium, whereas the number of growing filaments is defined by the balance between branching and capping rates. The length of actin filaments in the lamellipodia reflects network dynamics: short filaments might indicate high rates of both capping and branching reactions, whereas long filaments suggest persistent filament growth as well as low rates of capping and branching.
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