CitationDifferentially oriented populations of actin filaments generated in lamellipodia collaborate in pushing and pausing at the cell front. 2008, 10 (3):306-13 Nat. Cell Biol. This is a postprint of an article published in Koestler, S.A., Auinger, S., Vinzenz, M., Rottner, K., Small, J.V. Differentially oriented populations of actin filaments generated in lamellipodia collaborate in pushing and pausing at the cell front (2008) Nature Cell Biology, 10 (3), pp. 306-313. NCB-LetterDifferentially 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 1 . Protrusion occurs in lamellipodia 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 show that actin filaments in protruding lamellipodia subtend angles from 15-90 deg 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 show a wide angular distribution and correspondingly variable bilateral polymerisation 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, 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 suggest an explanation for the variable turnover dynamics of actin filaments in lamellipodia observed by fluorescence speckle microscopy 2 and are inconsistent with a current model of lamellipodia structure that features actin filaments branching at 70deg in a dendritic array 3 .Migrating cells exploit two properties of actin filaments to move: the property to polymerise and push, to effect protrusion and the ability to slide with myosin II, to drive retraction. Protrusion is effected by lamellipodia 1,4 , thin sheets of cytoplasm composed of networks of actin filaments that have their fast growing, plus ends abutting the leading membrane 5 . Current ideas of how protruding lamellipodia are organized have come mainly from electron microscopy of cells that show constant motility, in particular the epidermal keratocyte 3,6 . And from images obtained using a critical point drying procedure for specimen preparation, a model of lamellipodium organization has been proposed that features a dendritic network of actin filaments with the Arp2/3 complex situated at 70degree branch points 3,7,8 . In migrating cells lamellipodia not only protrude, they undergo phases of protrusion, pause and withdrawal, the latter often associated with ruffling 1 . Filopodia and related bundles embedded in the lamellipodia mesh, also referred to as microspikes 4 , contribute to thes...

SummaryUsing 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 mm. 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 actinArp2/3 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.

Molecular mechanism of Ena/VASP-mediated actin-filament elongationEna/VASP proteins have important functions in actin-dependent processes. A model for the actin elongation activity of Ena/VASP based on the affinity and saturation state of WH2-domain-mediated actin monomer binding is presented.

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.

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.

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.

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