Electron tomography reveals unbranched networks of actin filaments in lamellipodia

Abstract

Eukaryotic cells can initiate movement using the forces exerted by polymerizing actin filaments to extend lamellipodial and filopodial protrusions. In the current model, actin filaments in lamellipodia are organized in a branched, dendritic network. We applied electron tomography to vitreously frozen 'live' cells, fixed cells and cytoskeletons, embedded in vitreous ice or in deep-negative stain. In lamellipodia from four cell types, including rapidly migrating fish keratocytes, we found that actin filaments are almost exclusively unbranched. The vast majority of apparent filament junctions proved to be overlapping filaments, rather than branched end-to-side junctions. Analysis of the tomograms revealed that actin filaments terminate at the membrane interface within a zone several hundred nanometres wide at the lamellipodium front, and yielded the first direct measurements of filament densities. Actin filament pairs were also identified as lamellipodium components and bundle precursors. These data provide a new structural basis for understanding actin-driven protrusion during cell migration.

Actin filament curvature biases branching direction

“…This amount of curvature could result from a lateral force of 1 pN applied perpendicularly to the end of a 0.05-μm-long filament fixed at the other end (44), which reflects the average force per filament due to membrane tension and rigidity (45,46) and the approximate length of free F-actin (47) at the leading edge of the cell. If the length of free F-actin is longer at the leading edge (48,49), the filaments require even less force to bend. Therefore, even modest filament curvature that is caused by the normal force balance of branched actin growth against a membrane can generate a significant bias in the direction of actin branch nucleation.…”

The filamins

“…4). 12,83 FLNa recruitment by one partner could allow it to scaffold additional signaling molecules and enhance the efficiency of signal transduction. In addition, while FLN-binding partners may potentially collect FLN molecules to specific locations within the cell, their function may require the conscription of additional partners by FLNa.…”

Actin branching in the initiation and maintenance of lamellipodia

“…Because actin filaments in lamellipodia are densely packed, the resolution of their spatial organization requires electron tomography, and in the first study using this approach (Urban et al, 2010) a dendritic array of short filaments was not found in lamellipodia of various cell types. Instead, the images revealed only few putative branch sites at the front of lamellipodia and an abundance of long filaments extending to the lamellipodium tip (Urban et al, 2010). One explanation of these observations was that the branch frequency might be much lower than implied in the original model (Pollard and Borisy, 2003;Svitkina and Borisy, 1999), because only one branch is required per filament (Insall, 2011).…”

“…The simultaneous observation that the Arp2/3 complex catalyzed the branching of actin filaments in vitro (Amann and Pollard, 2001) and localized specifically to lamellipodia (Svitkina and Borisy, 1999;Welch et al, 1997) formed the basis of the dendritic nucleation model of lamellipodia protrusion, which presumes that actin filaments must be short and stiff to push (Pollard and Borisy, 2003). Because actin filaments in lamellipodia are densely packed, the resolution of their spatial organization requires electron tomography, and in the first study using this approach (Urban et al, 2010) a dendritic array of short filaments was not found in lamellipodia of various cell types. Instead, the images revealed only few putative branch sites at the front of lamellipodia and an abundance of long filaments extending to the lamellipodium tip (Urban et al, 2010).…”