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1.
Regulation of actin dynamics in rapidly moving cells: a quantitative analysis 总被引:11,自引:0,他引:11
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We develop a mathematical model that describes key details of actin dynamics in protrusion associated with cell motility. The model is based on the dendritic-nucleation hypothesis for lamellipodial protrusion in nonmuscle cells such as keratocytes. We consider a set of partial differential equations for diffusion and reactions of sequestered actin complexes, nucleation, and growth by polymerization of barbed ends of actin filaments, as well as capping and depolymerization of the filaments. The mechanical aspect of protrusion is based on an elastic polymerization ratchet mechanism. An output of the model is a relationship between the protrusion velocity and the number of filament barbed ends pushing the membrane. Significantly, this relationship has a local maximum: too many barbed ends deplete the available monomer pool, too few are insufficient to generate protrusive force, so motility is stalled at either extreme. Our results suggest that to achieve rapid motility, some tuning of parameters affecting actin dynamics must be operating in the cell. 相似文献
2.
Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility 总被引:42,自引:0,他引:42
Bear JE Svitkina TM Krause M Schafer DA Loureiro JJ Strasser GA Maly IV Chaga OY Cooper JA Borisy GG Gertler FB 《Cell》2002,109(4):509-521
Cell motility requires lamellipodial protrusion, a process driven by actin polymerization. Ena/VASP proteins accumulate in protruding lamellipodia and promote the rapid actin-driven motility of the pathogen Listeria. In contrast, Ena/VASP negatively regulate cell translocation. To resolve this paradox, we analyzed the function of Ena/VASP during lamellipodial protrusion. Ena/VASP-deficient lamellipodia protruded slower but more persistently, consistent with their increased cell translocation rates. Actin networks in Ena/VASP-deficient lamellipodia contained shorter, more highly branched filaments compared to controls. Lamellipodia with excess Ena/VASP contained longer, less branched filaments. In vitro, Ena/VASP promoted actin filament elongation by interacting with barbed ends, shielding them from capping protein. We conclude that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within lamellipodia. 相似文献
3.
Longhua Hu 《Biophysical journal》2010,98(8):1375-1384
During cell motion on a substratum, eukaryotic cells project sheetlike lamellipodia which contain a dynamically remodeling three-dimensional actin mesh. A number of regulatory proteins and subtle mechano-chemical couplings determine the lamellipodial protrusion dynamics. To study these processes, we constructed a microscopic physico-chemical computational model, which incorporates a number of fundamental reaction and diffusion processes, treated in a fully stochastic manner. Our work sheds light on the way lamellipodial protrusion dynamics is affected by the concentrations of actin and actin-binding proteins. In particular, we found that protrusion speed saturates at very high actin concentrations, where filament nucleation does not keep up with protrusion. This results in sparse filamentous networks, and, consequently, high resistance forces on individual filaments. We also observed maxima in lamellipodial growth rates as a function of Arp2/3, a nucleating protein, and capping proteins. We provide detailed physical explanations behind these effects. In particular, our work supports the actin-funneling-hypothesis explanation of protrusion speed enhancement at low capping protein concentrations. Our computational results are in agreement with a number of related experiments. Overall, our work emphasizes that elongation and nucleation processes work highly cooperatively in determining the optimal protrusion speed for the actin mesh in lamellipodia. 相似文献
4.
There has been a great deal of interest in the mechanism of lamellipodial protrusion (Pollard, T., and G. Borisy. 2003. Cell. 112:453-465). However, one of this mechanism's endpoints, the force of protrusion, has never been directly measured. We place an atomic force microscopy cantilever in the path of a migrating keratocyte. The deflection of the cantilever, which occurs over a period of approximately 10 s, provides a direct measure of the force exerted by the lamellipodial leading edge. Stall forces are consistent with approximately 100 polymerizing actin filaments per micrometer of the leading edge, each working as an elastic Brownian ratchet and generating a force of several piconewtons. However, the force-velocity curves obtained from this measurement, in which velocity drops sharply under very small loads, is not sensitive to low loading forces, and finally stalls rapidly at large loads, are not consistent with current theoretical models for the actin polymerization force. Rather, the curves indicate that the protrusive force generation is a complex multiphase process involving actin and adhesion dynamics. 相似文献
5.
Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches. Unexpectedly, mDia2 depletion by short interfering RNA (siRNA) severely inhibited lamellipodia. Structural analysis of the actin network in the few remaining lamellipodia suggested an mDia2 role in generation of long filaments. Consistently, constitutively active mDia2 (ΔGBD-mDia2) induced accumulation of long actin filaments in lamellipodia and increased persistence of lamellipodial protrusion. Depletion of mDia2 also inhibited filopodia, whereas expression of ΔGBD-mDia2 promoted their formation. Correlative light and electron microscopy showed that ΔGBD-mDia2–induced filopodia were formed from lamellipodial network through gradual convergence of long lamellipodial filaments into bundles. Efficient filopodia induction required mDia2 targeting to the membrane, likely through a scaffolding protein Abi1. Furthermore, mDia2 and Abi1 interacted through the N-terminal regulatory sequences of mDia2 and the SH3-containing Abi1 sequences. We propose that mDia2 plays an important role in formation of lamellipodia by nucleating and/or protecting from capping lamellipodial actin filaments, which subsequently exhibit high tendency to converge into filopodia. 相似文献
6.
Steffen A Faix J Resch GP Linkner J Wehland J Small JV Rottner K Stradal TE 《Molecular biology of the cell》2006,17(6):2581-2591
Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable. 相似文献
7.
Cytoskeletal dynamics underlying collateral membrane protrusions induced by neurotrophins in cultured Xenopus embryonic neurons 总被引:1,自引:0,他引:1
The establishment and refinement of neuronal connections depend on dynamic modification of the morphology and physiology of developing axons in response to extrinsic factors. In embryonic cultures of Xenopus spinal neurons, acute application of brain-derived neurotrophic factor (BDNF) induced rapid collateral protrusion of filopodium-like microspikes and lamellipodia along the neurite processes, leading to a morphologic alternation of the neuron. Both types of membrane protrusions contained high concentrations of actin filaments and depended on the polymerization of the actin cytoskeleton. Immunofluorescent staining, however, revealed the presence of microtubules (MTs) in lamellipodia induced by BDNF. These MTs appeared to have arisen from debundling of MTs in the neurite shaft at the protrusion sites, splaying and extending in the rapidly protruding lamellipodia. Inhibition of microtubule polymerization by nocodazole largely abolished the formation of lamellipodia but not of microspikes. Taken together, our results suggest that collateral sprouting of microspikes and lamellipodia involve distinctly different cytoskeletal mechanisms. Although the actin cytoskeleton is solely responsible for microspike formation, cooperative efforts by microtubules and actin filaments are essential for lamellipodial protrusion in response to extrinsic factors. 相似文献
8.
Block J Breitsprecher D Kühn S Winterhoff M Kage F Geffers R Duwe P Rohn JL Baum B Brakebusch C Geyer M Stradal TE Faix J Rottner K 《Current biology : CB》2012,22(11):1005-1012
Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2, accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia. 相似文献
9.
Many cell movements appear to be driven by the polymerization of actin. Here we show how the force of polymerization can
be generated by the thermal motions of the actin filaments near the sites of polymerization. We apply the model to explain
the observations that the lamellipodial cytoskeleton is organized into an orthogonal network interspersed with filopodial
protrusions, and that the protrusion of lamellipodia generally proceeds in the presence of a rearward cytoskeletal flow.
Received: 9 May 1996 / Accepted: 28 May 1996 相似文献
10.
Microtubules are involved in actin-based protrusion at the leading-edge lamellipodia of migrating fibroblasts. Here we show that the growth of microtubules induced in fibroblasts by removal of the microtubule destabilizer nocodazole activates Rac1 GTPase, leading to the polymerization of actin in lamellipodial protrusions. Lamellipodial protrusions are also activated by the rapid growth of a disorganized array of very short microtubules induced by the microtubule-stabilizing drug taxol. Thus, neither microtubule shortening nor long-range microtubule-based intracellular transport is required for activating protrusion. We suggest that the growth phase of microtubule dynamic instability at leading-edge lamellipodia locally activates Rac1 to drive actin polymerization and lamellipodial protrusion required for cell migration. 相似文献
11.
Hilpelä P Oberbanscheidt P Hahne P Hund M Kalhammer G Small JV Bähler M 《Molecular biology of the cell》2003,14(8):3242-3253
Functionally different subsets of actin filament arrays contribute to cellular organization and motility. We report the identification of a novel subset of loose actin filament arrays through regulated association with the widely expressed protein SWAP-70. These loose actin filament arrays were commonly located behind protruding lamellipodia and membrane ruffles. Visualization of these loose actin filament arrays was dependent on lamellipodial protrusion and the binding of the SWAP-70 PH-domain to a 3'-phosphoinositide. SWAP-70 with a functional pleckstrin homology-domain lacking the C-terminal 60 residues was targeted to the area of the loose actin filament arrays, but it did not associate with actin filaments. The C-terminal 60 residues were sufficient for actin filament association, but they provided no specificity for the subset of loose actin filament arrays. These results identify SWAP-70 as a phosphoinositide 3-kinase signaling-dependent marker for a distinct, hitherto unrecognized, array of actin filaments. Overexpression of SWAP-70 altered the actin organization and lamellipodial morphology. These alterations were dependent on a proper subcellular targeting of SWAP-70. We propose that SWAP-70 regulates the actin cytoskeleton as an effector or adaptor protein in response to agonist stimulated phosphatidylinositol (3,4)-bisphosphate production and cell protrusion. 相似文献
12.
Polymerization of actin filaments is necessary for both protrusion of the leading edge of crawling cells and propulsion of certain intracellular pathogens, and it is sufficient for generating force for bacterial motility in vitro. Motile intracellular pathogens are associated with actin-rich comet tails containing many of the same molecular components present in lamellipodia, and this suggests that these two systems use a similar mechanism for motility. However, available structural evidence suggests that the organization of comet tails differs from that of lamellipodia. Actin filaments in lamellipodia form branched arrays, which are thought to arise by dendritic nucleation mediated by the Arp2/3 complex. In contrast, comet tails have been variously described as consisting of short, randomly oriented filaments, with a higher degree of alignment at the periphery, or as containing long, straight axial filaments with a small number of oblique filaments. Because the assembly of pathogen-associated comet tails has been used as a model system for lamellipodial protrusion, it is important to resolve this apparent discrepancy. Here, using a platinum replica approach, we show that actin filament arrays in comet tails in fact have a dendritic organization with the Arp2/3 complex localizing to Y-junctions as in lamellipodia. Thus, comet tails and lamellipodia appear to share a common dendritic nucleation mechanism for protrusive motility. However, comet tails differ from lamellipodia in that their actin filaments are usually twisted and appear to be under significant torsional stress. 相似文献
13.
The motion of many intracellular pathogens is driven by the polymerization of actin filaments. The propulsive force developed by the polymerization process is thought to arise from the thermal motions of the polymerizing filament tips. Recent experiments suggest that the nucleation of actin filaments involves a phase when the filaments are attached to the pathogen surface by a protein complex. Here we extend the "elastic ratchet model" of Mogilner and Oster to incorporate these new findings. We apply this "tethered ratchet" model to derive the force-velocity relation for Listeria and discuss relations of our theoretical predictions to experimental measurements. We also discuss "symmetry breaking" dynamics observed in ActA-coated bead experiments, and the implications of the model for lamellipodial protrusion in migrating cells. 相似文献
14.
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. 相似文献
15.
RalA activation at nascent lamellipodia of epidermal growth factor-stimulated Cos7 cells and migrating Madin-Darby canine kidney cells
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RalA, a member of the Ras-family GTPases, regulates various cellular functions such as filopodia formation, endocytosis, and exocytosis. On epidermal growth factor (EGF) stimulation, activated Ras recruits guanine nucleotide exchange factors (GEFs) for RalA, followed by RalA activation. By using fluorescence resonance energy transfer-based probes for RalA activity, we found that the EGF-induced RalA activation in Cos7 cells was restricted at the EGF-induced nascent lamellipodia, whereas under a similar condition both Ras activation and Ras-dependent translocation of Ral GEFs occurred more diffusely at the plasma membrane. This EGF-induced RalA activation was not observed when lamellipodial protrusion was suppressed by a dominant negative mutant of Rac1, a GTPase-activating protein for Cdc42, inhibitors of phosphatidylinositol 3-kinase, or inhibitors of actin polymerization. On the other hand, EGF-induced lamellipodial protrusion was inhibited by microinjection of the RalA-binding domains of RalBP1 and Sec5. Furthermore, we found that RalA activity was high at the lamellipodia of migrating Madin-Darby canine kidney cells and that the migration of Madin-Darby canine kidney cells was perturbed by the microinjection of RalBP1-RalA-binding domain. Thus, RalA activation is required for the induction of lamellipodia, and conversely, lamellipodial protrusion seems to be required for the RalA activation, suggesting the presence of a positive feedback loop between RalA activation and lamellipodial protrusion. Our observation also demonstrates that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral guanine nucleotide exchange factors. 相似文献
16.
We used optical tweezers to analyze the effect of jasplakinolide and cyclodextrin on the force exerted by lamellipodia from developing growth cones (GCs) of isolated dorsal root ganglia (DRG) neurons. We found that 25 nM of jasplakinolide, which is known to inhibit actin filament turnover, reduced both the maximal exerted force and maximal velocity during lamellipodia leading-edge protrusion. By using atomic force microscopy, we verified that cyclodextrin, which is known to remove cholesterol from membranes, decreased the membrane stiffness of DRG neurons. Lamellipodia treated with 2.5 mM of cyclodextrin exerted a larger force, and their leading edge could advance with a higher velocity. Neither jasplakinolide nor cyclodextrin affected force or velocity during lamellipodia retraction. The amplitude and frequency of elementary jumps underlying force generation were reduced by jasplakinolide but not by cyclodextrin. The action of both drugs at the used concentration was fully reversible. These results support the notion that membrane stiffness provides a selective pressure that shapes force generation, and confirm the pivotal role of actin turnover during protrusion. 相似文献
17.
Krause M Leslie JD Stewart M Lafuente EM Valderrama F Jagannathan R Strasser GA Rubinson DA Liu H Way M Yaffe MB Boussiotis VA Gertler FB 《Developmental cell》2004,7(4):571-583
Lamellipodial protrusion is regulated by Ena/VASP proteins. We identified Lamellipodin (Lpd) as an Ena/VASP binding protein. Both proteins colocalize at the tips of lamellipodia and filopodia. Lpd is recruited to EPEC and Vaccinia, pathogens that exploit the actin cytoskeleton for their own motility. Lpd contains a PH domain that binds specifically to PI(3,4)P2, an asymmetrically localized signal in chemotactic cells. Lpd's PH domain can localize to ruffles in PDGF-treated fibroblasts. Lpd overexpression increases lamellipodial protrusion velocity, an effect observed when Ena/VASP proteins are overexpressed or artificially targeted to the plasma membrane. Conversely, knockdown of Lpd expression impairs lamellipodia formation, reduces velocity of residual lamellipodial protrusion, and decreases F-actin content. These phenotypes are more severe than loss of Ena/VASP, suggesting that Lpd regulates other effectors of the actin cytoskeleton in addition to Ena/VASP. 相似文献
18.
Mechanical work in cells is performed by specialized motor proteins that operate in a continuous mechanochemical cycle. Less complex, but still efficient, 'one-shot' motors evolved based on the assembly and disassembly of polymers. We review the mechanisms of pushing and pulling by actin and microtubule filaments and the organizational principles of actin networks. We show how these polymer force generators are used for the propulsion of intracellular pathogens, protrusion of lamellipodia and mitotic movements. We discuss several examples of cellular forces generated by the assembly and disassembly of polymer gels. 相似文献
19.
We report on the characterization of actin driven lamellipodial protrusion forces and velocities in keratocytes. A vertically mounted glass fiber acted as a flexible barrier positioned in front of migrating keratocytes with parallel phase contrast microscopy. A laser beam was coupled into the fiber and allowed detecting the position of the fiber by a segmented photodiode. Calibration of the fiber was carried out with the thermal oscillation method. Deflection and force signals were measured during lamellipodial protrusion. Velocity was constant during initial contact whereas loading force increased until finally the cell was stalled at higher forces. Stall forces were on the order of 2.9 ± 0.6 nN, which corresponds to a stall pressure of 2.7 ± 1.6 nN/μm2. Assuming a density of actin filaments of 240 filaments per μm, we can estimate a stall force per actin filament of 1.7 ± 0.8 pN. To check for adaption of the cell against an external force, we let the cell push toward the glass fiber several times. On the timescale of the experiment (∼1 min), however, the cell did not adapt to previous loading events. 相似文献
20.
Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity 总被引:23,自引:11,他引:12
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The organization and polarity of actin filaments in neuronal growth cones was studied with negative stain and freeze-etch EM using a permeabilization protocol that caused little detectable change in morphology when cultured nerve growth cones were observed by video-enhanced differential interference contrast microscopy. The lamellipodial actin cytoskeleton was composed of two distinct subpopulations: a population of 40-100-nm-wide filament bundles radiated from the leading edge, and a second population of branching short filaments filled the volume between the dorsal and ventral membrane surfaces. Together, the two populations formed the three-dimensional structural network seen within expanding lamellipodia. Interaction of the actin filaments with the ventral membrane surface occurred along the length of the filaments via membrane associated proteins. The long bundled filament population was primarily involved in these interactions. The filament tips of either population appeared to interact with the membrane only at the leading edge; this interaction was mediated by a globular Triton-insoluble material. Actin filament polarity was determined by decoration with myosin S1 or heavy meromyosin. Previous reports have suggested that the polarity of the actin filaments in motile cells is uniform, with the barbed ends toward the leading edge. We observed that the actin filament polarity within growth cone lamellipodia is not uniform; although the predominant orientation was with the barbed end toward the leading edge (47-56%), 22-25% of the filaments had the opposite orientation with their pointed ends toward the leading edge, and 19-31% ran parallel to the leading edge. The two actin filament populations display distinct polarity profiles: the longer filaments appear to be oriented predominantly with their barbed ends toward the leading edge, whereas the short filaments appear to be randomly oriented. The different length, organization and polarity of the two filament populations suggest that they differ in stability and function. The population of bundled long filaments, which appeared to be more ventrally located and in contact with membrane proteins, may be more stable than the population of short branched filaments. The location, organization, and polarity of the long bundled filaments suggest that they may be necessary for the expansion of lamellipodia and for the production of tension mediated by receptors to substrate adhesion molecules. 相似文献