Stefan A. Koestler

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.

Cofilin cooperates with fascin to disassemble filopodial actin filaments

Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin.

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.

Unravelling the structure of the lamellipodium

Pushing at the cell front is the business of lamellipodia and understanding how lamellipodia function requires knowledge of their structural organization. Analysis of extracted, critical‐point‐dried cells by electron microscopy has led to a current dogma that the lamellipodium pushes as a branched array of actin filaments, with a branching angle of 70°, defined by the Arp2/3 complex. Comparison of different preparative methods indicates that the critical‐point‐drying‐replica technique introduces distortions into actin networks, such that crossing filaments may appear branched. After negative staining and from preliminary studies by cryo‐electron tomography, no clear evidence could be found for actin filament branching in lamellipodia. From recent observations of a sub‐class of actin speckles in lamellipodia that exhibit a dynamic behaviour similar to speckles in the lamella region behind, it has been proposed that the lamellipodium surfs on top of the lamella. Negative stain electron microscopy and cryo‐electron microscopy of fixed cells, which reveal the entire complement of filaments in lamellipodia show, however, that there is no separate, second array of filaments beneath the lamellipodium network. From present data, we conclude that the lamellipodium is a distinct protrusive entity composed of a network of primarily unbranched actin filaments. Cryo‐electron tomography of snap‐frozen intact cells will be required to finally clarify the three‐dimensional arrangement of actin filaments in lamellipodia in vivo.

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.

Requirements for and consequences of Rac-dependent protrusion

Small GTPases of the Rac subfamily exert multiple functions, the most prominent of which includes stimulation of dynamic actin rearrangements at the cell periphery. Frequently, these actin reorganizations cause the protrusion of leaflets of plasma membrane, so-called lamellipodia, which remain anchored at flat surfaces during forward protrusion of migrating cells, or develop into ruffles when lifting up- and backwards. Ruffling membranes are also engaged in fluid and particle uptake during pino- and phagocytosis, respectively. In recent work, we sought to clarify the precise role of Rac GTPases in actin-based protrusion, using a gene disruption approach. Furthermore, we aimed at dissecting the function of its downstream target Arp2/3 complex employing its instantaneous inhibition during simultaneous Rac activation. These complementary approaches allow comparison of the consequences of Rac versus Arp2/3 complex loss of function at the cell periphery, and help to formulate a working hypothesis for how the actin network in lamellipodia is initiated and maintained.

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.

FlyOde - a platform for community curation and interactive visualization of dynamic gene regulatory networks in Drosophila eye development

Motivation: Understanding the regulatory mechanisms governing eye development of the model organism Drosophila melanogaster (D. m.) requires structured knowledge of the involved genes and proteins, their interactions, and dynamic expression patterns. Especially the latter information is however to a large extent scattered throughout the literature. Results: FlyOde is an online platform for the systematic assembly of data on D. m. eye development. It consists of data on eye development obtained from the literature, and a web interface for users to interactively display these data as a gene regulatory network. Our manual curation process provides high standard structured data, following a specifically designed ontology. Visualization of gene interactions provides an overview of network topology, and filtering according to user-defined expression patterns makes it a versatile tool for daily tasks, as demonstrated by usage examples. Users are encouraged to submit additional data via a simple online form.