Maria Nemethova

Building the actin cytoskeleton: filopodia contribute to the construction of contractile bundles in the lamella

Filopodia are rodlike extensions generally attributed with a guidance role in cell migration. We now show in fish fibroblasts that filopodia play a major role in generating contractile bundles in the lamella region behind the migrating front. Filopodia that developed adhesion to the substrate via paxillin containing focal complexes contributed their proximal part to stress fiber assembly, and filopodia that folded laterally contributed to the construction of contractile bundles parallel to the cell edge. Correlated light and electron microscopy of cells labeled for actin and fascin confirmed integration of filopodia bundles into the lamella network. Inhibition of myosin II did not subdue the waving and folding motions of filopodia or their entry into the lamella, but filopodia were not then integrated into contractile arrays. Comparable results were obtained with B16 melanoma cells. These and other findings support the idea that filaments generated in filopodia and lamellipodia for protrusion are recycled for seeding actomyosin arrays for use in retraction.

Inhibitory signalling to the Arp2/3 complex steers cell migration

Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott–Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac–Arpin–Arp2/3 inhibitory circuit with the Rac–WAVE–Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.

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.

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.

Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion

Electron tomography reveals the structural organization of actin comet tails generated by a baculovirus, providing an understanding of how this pathogen hijacks host machinery to propel itself between cells.

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.

Spectrally coded optical nanosectioning (SpecON) with biocompatible metal–dielectric-coated substrates

Significance In this paper we describe a high-resolution light microscopy technique that translates spatial (position) information of fluorescent markers into spectral (color) information for improved biological imaging. By designing a thin biocompatible nanostructure on a microscope slide, we show how the distance-dependent spectral “fingerprint” of fluorophores can be used to monitor their relative distance from the nanostructure with an accuracy far beyond the resolution power of a conventional light microscope. We demonstrate the technique by studying the positions and dynamics of key proteins that play a role in cell motility. Fluorescence nanosectioning within a submicron region above an interface is desirable for many disciplines in the life sciences. A drawback, however, to most current approaches is the a priori need to physically scan a sculptured point spread function in the axial dimension, which can be undesirable for optically sensitive or highly dynamic samples. Here we demonstrate a fluorescence imaging approach that can overcome the need for scanning by exploiting the position-dependent emission spectrum of fluorophores above a simple biocompatible nanostructure. To achieve this we have designed a thin metal–dielectric-coated substrate, where the spectral modification to the total measured fluorescence can be used to estimate the axial fluorophore distribution within distances of 10–150 nm above the substrate with an accuracy of up to 5–10 nm. The modeling and feasibility of the approach are verified and successfully applied to elucidate nanoscale adhesion protein and filopodia dynamics in migrating cells. It is likely that the general principle can find broader applications in, for example, single-molecule studies, biosensing, and studying fast dynamic processes.

Fluorescence enhancements and spectral modifications near the cut-off frequency of plasmonic structure

The effect of a population of fluorophores coupling to weakly bound surface plasmons in dielectric/metal/dielectric structures is investigated for the purpose of fluorescence enhancement near interfaces and live cell fluorescence surface imaging. We show theoretically and experimentally that for sufficient fluorophore concentrations near such SPP supporting structures significant enhancements in the radiative emission intensity can be observed, with a spectral modification that can be correlated to the average separation of the fluorophores from the substrate. We will discuss the theory behind the effect and some experimental results on imaging labeled proteins in the focal adhesion sites of cells.