Marlene Vinzenz

Differentially oriented populations of actin filaments generated in lamellipodia collaborate in pushing and pausing at the cell front

Eukaryotic cells advance in phases of protrusion, pause and withdrawal. Protrusion occurs in lamellipodia, which are composed of diagonal networks of actin filaments, and withdrawal terminates with the formation of actin bundles parallel to the cell edge. Using correlated live-cell imaging and electron microscopy, we have shown that actin filaments in protruding lamellipodia subtend angles from 15–90° to the front, and that transitions from protrusion to pause are associated with a proportional increase in filaments oriented more parallel to the cell edge. Microspike bundles of actin filaments also showed a wide angular distribution and correspondingly variable bilateral polymerization rates along the cell front. We propose that the angular shift of filaments in lamellipodia serves in adapting to slower protrusion rates while maintaining the filament densities required for structural support; further, we suggest that single filaments and microspike bundles contribute to the construction of the lamella behind and to the formation of the cell edge when protrusion ceases. Our findings provide an explanation for the variable turnover dynamics of actin filaments in lamellipodia observed by fluorescence speckle microscopy and are inconsistent with a current model of lamellipodia structure that features actin filaments branching at 70° in a dendritic array.

Molecular mechanism of Ena/VASP-mediated actin-filament elongation

Ena/VASP proteins are implicated in a variety of fundamental cellular processes including axon guidance and cell migration. In vitro, they enhance elongation of actin filaments, but at rates differing in nearly an order of magnitude according to species, raising questions about the molecular determinants of rate control. Chimeras from fast and slow elongating VASP proteins were generated and their ability to promote actin polymerization and to bind G‐actin was assessed. By in vitro TIRF microscopy as well as thermodynamic and kinetic analyses, we show that the velocity of VASP‐mediated filament elongation depends on G‐actin recruitment by the WASP homology 2 motif. Comparison of the experimentally observed elongation rates with a quantitative mathematical model moreover revealed that Ena/VASP‐mediated filament elongation displays a saturation dependence on the actin monomer concentration, implying that Ena/VASP proteins, independent of species, are fully saturated with actin in vivo and generally act as potent filament elongators. Moreover, our data showed that spontaneous addition of monomers does not occur during processive VASP‐mediated filament elongation on surfaces, suggesting that most filament formation in cells is actively controlled.

F- and G-Actin Concentrations in Lamellipodia of Moving Cells

Cells protrude by polymerizing monomeric (G) into polymeric (F) actin at the tip of the lamellipodium. Actin filaments are depolymerized towards the rear of the lamellipodium in a treadmilling process, thereby supplementing a G-actin pool for a new round of polymerization. In this scenario the concentrations of F- and G-actin are principal parameters, but have hitherto not been directly determined. By comparing fluorescence intensities of bleached and unbleached regions of lamellipodia in B16-F1 mouse melanoma cells expressing EGFP-actin, before and after extraction with Triton X-100, we show that the ratio of F- to G-actin is 3.2+/−0.9. Using electron microscopy to determine the F-actin content, this ratio translates into F- and G-actin concentrations in lamellipodia of approximately 500 µM and 150 µM, respectively. The excess of G-actin, at several orders of magnitude above the critical concentrations at filament ends indicates that the polymerization rate is not limited by diffusion and is tightly controlled by polymerization/depolymerization modulators.

Actin branching in the initiation and maintenance of lamellipodia

Using correlated live-cell imaging and electron tomography we found that actin branch junctions in protruding and treadmilling lamellipodia are not concentrated at the front as previously supposed, but link actin filament subsets in which there is a continuum of distances from a junction to the filament plus ends, for up to at least 1 μm. When branch sites were observed closely spaced on the same filament their separation was commonly a multiple of the actin helical repeat of 36 nm. Image averaging of branch junctions in the tomograms yielded a model for the in vivo branch at 2.9 nm resolution, which was comparable with that derived for the in vitro actin–Arp2/3 complex. Lamellipodium initiation was monitored in an intracellular wound-healing model and was found to involve branching from the sides of actin filaments oriented parallel to the plasmalemma. Many filament plus ends, presumably capped, terminated behind the lamellipodium tip and localized on the dorsal and ventral surfaces of the actin network. These findings reveal how branching events initiate and maintain a network of actin filaments of variable length, and provide the first structural model of the branch junction in vivo. A possible role of filament capping in generating the lamellipodium leaflet is discussed and a mathematical model of protrusion is also presented.

Arp2/3 complex is essential for actin network treadmilling as well as for targeting of capping protein and cofilin.

Acute suppression of Arp2/3 complex activity in lamellipodia demonstrates its essential role in actin network treadmilling and filament organization and geometry. Arp2/3 complex activity also defines the recruitment of crucial independent factors, including capping protein and cofilin, and is essential for lamellipodia-based keratocyte migration.

Direct Determination of Actin Polarity in the Cell

Actin filaments are polar structures that exhibit a fast growing plus end and a slow growing minus end. According to their organization in cells, in parallel or antiparallel arrays, they can serve, respectively, in protrusions or in contractions. The determination of actin filament polarity in subcellular compartments is therefore required to establish their local function. Myosin binding has previously been the sole method of polarity determination. Here, we report the first direct determination of actin filament polarity in the cell without myosin binding. Negatively stained cytoskeletons of lamellipodia were analyzed by adapting electron tomography and a single particle analysis for filamentous complexes. The results of the stained cytoskeletons confirmed that all actin filament ends facing the cell membrane were the barbed ends. In general, this approach should be applicable to the analysis of actin polarity in tomograms of the actin cytoskeleton.

Reply: Visualizing branched actin filaments in lamellipodia by electron tomography

colleagues have questioned the dendritic nucleation model in a recent paper1. In their study, they challenge key evidence supporting the dendritic nucleation model; namely, visualization of branched actin filaments in lamellipodia by platinum-replica electron microscopy4,5. Urban et al. argue1 that the branched configuration of the actin filaments in these samples are an artefact of critical-point drying, a part of the sample preparation. To prove their hypothesis that the actin filaments in lamellipodia are long and unbranched, they analysed the structure of lamellipodia by cryo-electron microscopy, and did not detect branched actin filaments in lamellipodia1. Because the data from platinum-replica electron microscopy that demonstrate branched actin filaments in lamellipodia have been reported primarily by our group, and because we have received multiple requests from the scientific community to comment on the results of Urban et al., we would like to share our opinion with a broader audience through this commentary. Several points, including comparison of potential artefacts produced by the two electron microscopy techniques and evaluation of evidence for and against the dendritic nucleation model, have already been addressed6,7. However, one critical point has not been discussed; namely, the fundamental consistency between our results and those of Urban et al. in spite of the different interpretations. We have carefully analysed the primary data provided as Supplementary Information by Urban et al. Based on this analysis, we argue that a subset of their results provides strong supporting evidence for the dendritic nucleation model by showing branched actin filaments in lamellipodia. We have found that Supplementary Movie S6, showing an electron tomogram of the lamellipodium in a 3T3 cell, is of particularly good quality to illustrate this point. Several examples of branched actin filaments from this movie are shown in Fig. 1a as a montage of z planes. In these examples, one of two actin filaments never re-emerges on the other side of the second filament in adjacent movie frames, despite the fact that both filaments are in the same z plane in at least one frame of the series. These features demonstrate that the end of the former filament makes a contact with the side of the latter filament, but does not cross it, which is the definition of a branch. In the zoomed region of the movie that shows a cell area of ~0.53 μm2, we have found a total of 147 branches (Fig. 1b), which is in contrast to the authors’ statement that “branches at the sides of actin filaments were extremely rare”. Branched actin filaments can be also found in other Supplementary movies, but fewer branches can be clearly detected in these movies owing to their insufficient quality and selection of regions corresponding to filopodia or filopodial precursors, which contain long unbranched filaments. Remarkably, all branches identified in our analysis invariably contained a blob at the branch point that probably corresponds to the Arp2/3 complex. Furthermore, the angle between branched filaments was 70 ± 7 degrees (n = 57), consistent with the conventional angle of ~70° produced by the Arp2/3 complex in vitro8. The actin filament branches found in data from the Urban et al. paper are virtually indistinguishable by appearance from those obtained by Hanein using cryo-electron microscopy of actin branches reconstituted in vitro from the Arp2/3 complex and actin, and could even be fitted into the three-dimensional branch model developed in these studies9. In conclusion, existence of branched actin filaments in lamellipodia is supported by two different electron microscopy techniques that probably counterbalance their potential artefacts. We are not sure why Urban et al. failed to detect branches, but this is definitely an issue of seeing or not seeing, rather than a problem with the techniques. Thus, in our opinion there should be no controversy regarding the structural organization of actin filaments in lamellipodia, and the dendritic nucleation model can serve as a conceptual framework for subsequent studies in the field.

Actin filament tracking in electron tomograms of negatively stained lamellipodia using the localized radon transform

The aim of this work was to develop a protocol for automated tracking of actin filaments in electron tomograms of lamellipodia embedded in negative stain. We show that a localized version of the Radon transform for the detection of filament directions enables three-dimensional visualizations of filament network architecture, facilitating extraction of statistical information including orientation profiles. We discuss the requirements for parameter selection set by the raw image data in the context of other, similar tracking protocols.

Microtubules as Platforms for Assaying Actin Polymerization In Vivo

The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol. Although nucleation has been extensively studied using purified proteins in vitro, dissection of the process in cells is complicated by the abundance and molecular complexity of actin filament arrays. We here describe the ectopic nucleation of actin filaments on the surface of microtubules, free of endogenous actin and interfering membrane or lipid. All major mechanisms of actin filament nucleation were recapitulated, including filament assembly induced by Arp2/3 complex, formin and Spir. This novel approach allows systematic dissection of actin nucleation in the cytosol of live cells, its genetic re-engineering as well as screening for new modifiers of the process.