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ABSTRACT: Migration of motile cells on flat substrates is usually driven by the polymerization of a flat actin filament network. Theoretical models have made different predictions regarding the distribution of the filament orientation in the lamellipodium with respect to the direction of motion. Here we show how one can automatically reconstruct the orientation distribution of actin filaments in the lamellipodium of migrating keratocytes from electron microscopy tomography data. We use two different image analysis methods, an algorithm which explicitly extracts an abstract network representation and an analysis of the gray scale information based on the structure tensor. We show that the two approaches give similar results, both for simulated data and for electron microscopy tomography data from migrating keratocytes. For the lamellipodium at the leading edge of fast moving cells, we find an orientation distribution that is peaked at +35/-35 degrees. For the lamellipodium at the leading edge of slow moving cells as well as for the lamellipodium at the flanks of fast moving cells, one broad peak around 0 degree dominates the distribution.
Cytometry Part A 04/2012; 81(6):496-507. · 3.73 Impact Factor
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ABSTRACT: 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.
Journal of Molecular Biology 03/2012; 419(5):359-68. · 4.00 Impact Factor
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Marlene Vinzenz,
Maria Nemethova,
Florian Schur,
Jan Mueller,
Akihiro Narita,
Edit Urban,
Christoph Winkler,
Christian Schmeiser,
Stefan A Koestler,
Klemens Rottner,
Guenter P Resch,
Yuichiro Maeda, J Victor Small
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ABSTRACT: 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.
Journal of Cell Science 03/2012; 125(Pt 11):2775-85. · 6.11 Impact Factor
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ABSTRACT: 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.
Journal of Structural Biology 02/2012; 178(1):19-28. · 3.41 Impact Factor
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ABSTRACT: 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.
Journal of Cell Science 10/2011; 124(Pt 19):3305-18. · 6.11 Impact Factor
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Nature Cell Biology 01/2011; 13(9):1013-4. · 19.49 Impact Factor
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J Margit Oelkers,
Marlene Vinzenz,
Maria Nemethova,
Sonja Jacob,
Frank P L Lai,
Jennifer Block,
Malgorzata Szczodrak,
Eugen Kerkhoff,
Steffen Backert,
Kai Schlüter,
Theresia E B Stradal, J Victor Small,
Stefan A Koestler,
Klemens Rottner
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ABSTRACT: 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.
PLoS ONE 01/2011; 6(5):e19931. · 4.09 Impact Factor
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J Victor Small
Trends in cell biology 11/2010; · 12.12 Impact Factor
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J Victor Small
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ABSTRACT: The primary event in the movement of a migrating eukaryotic cell is the extension of cytoplasmic sheets termed lamellipodia composed of networks of actin filaments. Lamellipodia networks are thought to arise through the branching of new filaments from the sides of old filaments, producing a dendritic array. Recent studies by electron tomography have revealed the three dimensional organization of lamellipodia and show, contrary to previous evidence, that actin filaments do not form dendritic arrays in vivo. These findings signal a reconsideration of the structural basis of protrusion and about the roles of the different actin nucleating and elongating complexes involved in the process.
Trends in cell biology 11/2010; 20(11):628-33. · 12.12 Impact Factor
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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.
Nature Cell Biology 04/2010; 12(5):429-35. · 19.49 Impact Factor
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ABSTRACT: Cell migration is initiated by the extension of thin cytoplasmic sheets, termed lamellipodia and finger-like rods, termed
filopodia. Both structures are composed of actin filaments, organized in networks in lamellipodia and bundles in filopodia
and protrusion is driven by the polymerization of actin, with monomer insertion at the tips of these processes, between the
filament plus ends and the membrane. The formation of lamellipodia and filopodia is induced via different but overlapping
pathways, but the extent of their structural and functional interrelationships remains controversial. We propose that lamellipodia
and filopodia are each driven by core machineries, partly overlapping, which are poised for engagement with upstream signalling
pathways. Protrusion in each case is associated with the recruitment of protein complexes to the cell membrane that nucleate
actin filaments, regulate their elongation and tether their ends to the membrane. Other factors are required to stabilise,
crosslink and regulate the turnover of the actin filament assemblies. In addition to their role in protrusion, lamellipodia
and filopodia seed filaments for the construction of the contractile regions of the cytoskeleton required for retraction.
12/2009: pages 3-33;
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Frank P L Lai,
Malgorzata Szczodrak,
J Margit Oelkers,
Markus Ladwein,
Filippo Acconcia,
Stefanie Benesch,
Sonja Auinger,
Jan Faix, J Victor Small,
Simona Polo,
Theresia E B Stradal,
Klemens Rottner
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ABSTRACT: Dynamic actin rearrangements are initiated and maintained by actin filament nucleators, including the Arp2/3-complex. This protein assembly is activated in vitro by distinct nucleation-promoting factors such as Wiskott-Aldrich syndrome protein/Scar family proteins or cortactin, but the relative in vivo functions of each of them remain controversial. Here, we report the conditional genetic disruption of murine cortactin, implicated previously in dynamic actin reorganizations driving lamellipodium protrusion and endocytosis. Unexpectedly, cortactin-deficient cells showed little changes in overall cell morphology and growth. Ultrastructural analyses and live-cell imaging studies revealed unimpaired lamellipodial architecture, Rac-induced protrusion, and actin network turnover, although actin assembly rates in the lamellipodium were modestly increased. In contrast, platelet-derived growth factor-induced actin reorganization and Rac activation were impaired in cortactin null cells. In addition, cortactin deficiency caused reduction of Cdc42 activity and defects in random and directed cell migration. Reduced migration of cortactin null cells could be restored, at least in part, by active Rac and Cdc42 variants. Finally, cortactin removal did not affect the efficiency of receptor-mediated endocytosis. Together, we conclude that cortactin is fully dispensable for Arp2/3-complex activation during lamellipodia protrusion or clathrin pit endocytosis. Furthermore, we propose that cortactin promotes cell migration indirectly, through contributing to activation of selected Rho-GTPases.
Molecular biology of the cell 06/2009; 20(14):3209-23. · 5.98 Impact Factor
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ABSTRACT: 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 microM and 150 microM, 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.
PLoS ONE 02/2009; 4(3):e4810. · 4.09 Impact Factor
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ABSTRACT: Vasodilator-stimulated phosphoprotein (VASP) is a key regulator of dynamic actin structures like filopodia and lamellipodia, but its precise function in their formation is controversial. Using in vitro TIRF microscopy, we show for the first time that both human and Dictyostelium VASP are directly involved in accelerating filament elongation by delivering monomeric actin to the growing barbed end. In solution, DdVASP markedly accelerated actin filament elongation in a concentration-dependent manner but was inhibited by low concentrations of capping protein (CP). In striking contrast, VASP clustered on functionalized beads switched to processive filament elongation that became insensitive even to very high concentrations of CP. Supplemented with the in vivo analysis of VASP mutants and an EM structure of the protein, we propose a mechanism by which membrane-associated VASP oligomers use their WH2 domains to effect both the tethering of actin filaments and their processive elongation in sites of active actin assembly.
The EMBO Journal 11/2008; 27(22):2943-54. · 9.20 Impact Factor
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ABSTRACT: Cell migration is initiated by lamellipodia-membrane-enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin-another prominent Arp2/3 complex regulator-and ADF/cofilin-previously implicated in driving both filament nucleation and disassembly-were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3- and WAVE complex-driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh.
The EMBO Journal 05/2008; 27(7):982-92. · 9.20 Impact Factor
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ABSTRACT: 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 degrees 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 degrees in a dendritic array.
Nature Cell Biology 04/2008; 10(3):306-13. · 19.49 Impact Factor
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ABSTRACT: 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.
The Journal of Cell Biology 04/2008; 180(6):1233-44. · 10.26 Impact Factor
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ABSTRACT: Cell migration is initiated by lamellipodia—membrane-enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin—another prominent Arp2/3 complex regulator—and ADF/cofilin—previously implicated in driving both filament nucleation and disassembly—were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3- and WAVE complex-driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh.
The EMBO Journal 02/2008; 27(7):982-992. · 9.20 Impact Factor
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ABSTRACT: An organized microtubule array is essential for the polarized motility of fibroblasts. Dynamic microtubules closely interact with focal adhesion sites in migrating cells. Here, we examined the effect of focal adhesions on microtubule dynamics. We observed that the probability of microtubule catastrophes (transitions from growth to shrinkage) was seven times higher at focal adhesions than elsewhere. Analysis of the dependence between the microtubule growth rate and catastrophe probability throughout the cytoplasm revealed that a nonspecific (mechanical or spatial) factor provided a minor contribution to the catastrophe induction by decreasing microtubule growth rate at adhesions. Strikingly, at the same growth rate, the probability of catastrophes was significantly higher at adhesions than elsewhere, indicative of a site-specific biochemical trigger. The observed catastrophe induction occurred at adhesion domains containing the scaffolding protein paxillin that has been shown previously to interact with tubulin. Furthermore, replacement of full-length paxillin at adhesion sites by microinjected paxillin LIM2-LIM3 domains suppressed microtubule catastrophes exclusively at adhesions. We suggest that paxillin influences microtubule dynamics at focal adhesions by serving as a scaffold for a putative catastrophe factor and/or regulating its exposure to microtubules.
Journal of Cell Science 02/2008; 121(Pt 2):196-204. · 6.11 Impact Factor
