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Journal articles on the topic 'Filopodia'
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1
Krndija, Denis, and Michael Fairhead. "IGF1R undergoes active and directed centripetal transport on filopodia upon receptor activation." Biochemical Journal 476, no. 23 (December 3, 2019): 3583–93. http://dx.doi.org/10.1042/bcj20190665.
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Filopodia are thin, actin-based membrane protrusions with roles in sensing external mechanical and chemical cues, such as growth factor gradients in tissues. It was proposed that the chemical sensing role of filopodia is achieved through clearance of activated signaling receptors from filopodia. Type I insulin-like growth factor receptor (IGF1R) is a key regulator of normal development and growth, as well as tumor development and progression. Its biological roles depend on its activation upon IGF1 binding at the cell membrane. IGF1R behavior at the cell membrane and in particular in filopodia, has not been established. We found that IGF1 activation led to a gradual reduction in IGF1R puncta in filopodia, and that this clearance depended on actin, non-muscle myosin II, and IGF1R kinase activity. Using single particle tracking of filopodial IGF1R, we established that ligand-free IGF1R undergoes non-directional unidimensional diffusion along the filopodium. Moreover, after initial diffusion, the ligand-bound IGF1R is actively transported along the filopodium towards the filopodium base, and consequently cleared from the filopodium. Our results show that IGF1R can move directionally on the plasma membrane protrusions, supporting a sensory role for filopodia in interpreting local IGF1 gradients.
2
Leijnse, Natascha, Lene B. Oddershede, and Poul M. Bendix. "Helical buckling of actin inside filopodia generates traction." Proceedings of the National Academy of Sciences 112, no. 1 (December 22, 2014): 136–41. http://dx.doi.org/10.1073/pnas.1411761112.
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Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell–cell communication. Filopodia contain cross-linked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle.
3
Lau, Pak-ming, Robert S. Zucker, and David Bentley. "Induction of Filopodia by Direct Local Elevation of Intracellular Calcium Ion Concentration." Journal of Cell Biology 145, no. 6 (June 14, 1999): 1265–76. http://dx.doi.org/10.1083/jcb.145.6.1265.
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In neuronal growth cones, cycles of filopodial protrusion and retraction are important in growth cone translocation and steering. Alteration in intracellular calcium ion concentration has been shown by several indirect methods to be critically involved in the regulation of filopodial activity. Here, we investigate whether direct elevation of [Ca2+]i, which is restricted in time and space and is isolated from earlier steps in intracellular signaling pathways, can initiate filopodial protrusion. We raised [Ca2+]i level transiently in small areas of nascent axons near growth cones in situ by localized photolysis of caged Ca2+ compounds. After photolysis, [Ca2+]i increased from ∼60 nM to ∼1 μM within the illuminated zone, and then returned to resting level in ∼10–15 s. New filopodia arose in this area within 1–5 min, and persisted for ∼15 min. Elevation of calcium concentration within a single filopodium induced new branch filopodia. In neurons coinjected with rhodamine-phalloidin, F-actin was observed in dynamic cortical patches along nascent axons; after photolysis, new filopodia often emerged from these patches. These results indicate that local transient [Ca2+]i elevation is sufficient to induce new filopodia from nascent axons or from existing filopodia.
4
Marchenko, Olena O., Sulagna Das, Ji Yu, Igor L. Novak, Vladimir I. Rodionov, Nadia Efimova, Tatyana Svitkina, Charles W. Wolgemuth, and Leslie M. Loew. "A minimal actomyosin-based model predicts the dynamics of filopodia on neuronal dendrites." Molecular Biology of the Cell 28, no. 8 (April 15, 2017): 1021–33. http://dx.doi.org/10.1091/mbc.e16-06-0461.
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Dendritic filopodia are actin-filled dynamic subcellular structures that sprout on neuronal dendrites during neurogenesis. The exploratory motion of the filopodia is crucial for synaptogenesis, but the underlying mechanisms are poorly understood. To study filopodial motility, we collected and analyzed image data on filopodia in cultured rat hippocampal neurons. We hypothesized that mechanical feedback among the actin retrograde flow, myosin activity, and substrate adhesion gives rise to various filopodial behaviors. We formulated a minimal one-dimensional partial differential equation model that reproduced the range of observed motility. To validate our model, we systematically manipulated experimental correlates of parameters in the model: substrate adhesion strength, actin polymerization rate, myosin contractility, and the integrity of the putative microtubule-based barrier at the filopodium base. The model predicts the response of the system to each of these experimental perturbations, supporting the hypothesis that our actomyosin-driven mechanism controls dendritic filopodia dynamics.
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Saha, Tanumoy, Isabel Rathmann, Abhiyan Viplav, Sadhana Panzade, Isabell Begemann, Christiane Rasch, Jürgen Klingauf, Maja Matis, and Milos Galic. "Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking." Molecular Biology of the Cell 27, no. 22 (November 7, 2016): 3616–26. http://dx.doi.org/10.1091/mbc.e16-06-0406.
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Filopodia are dynamic, actin-rich structures that transiently form on a variety of cell types. To understand the underlying control mechanisms requires precise monitoring of localization and concentration of individual regulatory and structural proteins as filopodia elongate and subsequently retract. Although several methods exist that analyze changes in filopodial shape, a software solution to reliably correlate growth dynamics with spatially resolved protein concentration along the filopodium independent of bending, lateral shift, or tilting is missing. Here we introduce a novel approach based on the convex-hull algorithm for parallel analysis of growth dynamics and relative spatiotemporal protein concentration along flexible filopodial protrusions. Detailed in silico tests using various geometries confirm that our technique accurately tracks growth dynamics and relative protein concentration along the filopodial length for a broad range of signal distributions. To validate our technique in living cells, we measure filopodial dynamics and quantify spatiotemporal localization of filopodia-associated proteins during the filopodial extension–retraction cycle in a variety of cell types in vitro and in vivo. Together these results show that the technique is suitable for simultaneous analysis of growth dynamics and spatiotemporal protein enrichment along filopodia. To allow readily application by other laboratories, we share source code and instructions for software handling.
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Xue, Fei, Deanna M. Janzen, and David A. Knecht. "Contribution of Filopodia to Cell Migration: A Mechanical Link between Protrusion and Contraction." International Journal of Cell Biology 2010 (2010): 1–13. http://dx.doi.org/10.1155/2010/507821.
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Numerous F-actin containing structures are involved in regulating protrusion of membrane at the leading edge of motile cells. We have investigated the structure and dynamics of filopodia as they relate to events at the leading edge and the function of the trailing actin networks. We have found that although filopodia contain parallel bundles of actin, they contain a surprisingly nonuniform spatial and temporal distribution of actin binding proteins. Along the length of the actin filaments in a single filopodium, the most distal portion contains primarily T-plastin, while the proximal portion is primarily bound byα-actinin and coronin. Some filopodia are stationary, but lateral filopodia move with respect to the leading edge. They appear to form a mechanical link between the actin polymerization network at the front of the cell and the myosin motor activity in the cell body. The direction of lateral filopodial movement is associated with the direction of cell migration. When lateral filopodia initiate from and move toward only one side of a cell, the cell will turn opposite to the direction of filopodial flow. Therefore, this filopodia-myosin II system allows actin polymerization driven protrusion forces and myosin II mediated contractile force to be mechanically coordinated.
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Kim, Min-Cheol, Yaron R. Silberberg, Rohan Abeyaratne, Roger D. Kamm, and H. Harry Asada. "Computational modeling of three-dimensional ECM-rigidity sensing to guide directed cell migration." Proceedings of the National Academy of Sciences 115, no. 3 (January 2, 2018): E390—E399. http://dx.doi.org/10.1073/pnas.1717230115.
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Filopodia have a key role in sensing both chemical and mechanical cues in surrounding extracellular matrix (ECM). However, quantitative understanding is still missing in the filopodial mechanosensing of local ECM stiffness, resulting from dynamic interactions between filopodia and the surrounding 3D ECM fibers. Here we present a method for characterizing the stiffness of ECM that is sensed by filopodia based on the theory of elasticity and discrete ECM fiber. We have applied this method to a filopodial mechanosensing model for predicting directed cell migration toward stiffer ECM. This model provides us with a distribution of force and displacement as well as their time rate of changes near the tip of a filopodium when it is bound to the surrounding ECM fibers. Aggregating these effects in each local region of 3D ECM, we express the local ECM stiffness sensed by the cell and explain polarity in the cellular durotaxis mechanism.
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Young, Lorna E., Ernest G. Heimsath, and Henry N. Higgs. "Cell type–dependent mechanisms for formin-mediated assembly of filopodia." Molecular Biology of the Cell 26, no. 25 (December 15, 2015): 4646–59. http://dx.doi.org/10.1091/mbc.e15-09-0626.
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Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex–nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 complex inhibition in adherent cells. In adherent cells only, mDia1 and VASP also contribute to filopodial assembly, and filopodia are disproportionately associated with focal adhesions. We propose an extension of the existing models for filopodial assembly in which any cluster of actin filament barbed ends in proximity to the plasma membrane, either Arp2/3 complex dependent or independent, can initiate filopodial assembly by specific formins.
9
Urbančič, Vasja, Richard Butler, Benjamin Richier, Manuel Peter, Julia Mason, Frederick J. Livesey, Christine E. Holt, and Jennifer L. Gallop. "Filopodyan: An open-source pipeline for the analysis of filopodia." Journal of Cell Biology 216, no. 10 (July 31, 2017): 3405–22. http://dx.doi.org/10.1083/jcb.201705113.
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Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time.
10
Steketee, Michael, Kenneth Balazovich, and Kathryn W. Tosney. "Filopodial Initiation and a Novel Filament-organizing Center, the Focal Ring." Molecular Biology of the Cell 12, no. 8 (August 2001): 2378–95. http://dx.doi.org/10.1091/mbc.12.8.2378.
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This study examines filopodial initiation and implicates a putative actin filament organizer, the focal ring. Filopodia were optically recorded as they emerged from veils, the active lamellar extensions of growth cones. Motile histories revealed three events that consistently preceded filopodial emergence: an influx of cytoplasm into adjacent filopodia, a focal increase in phase density at veil margins, and protrusion of nubs that transform into filopodia. The cytoplasmic influx probably supplies materials needed for initiation. In correlated time lapse-immunocytochemistry, these focal phase densities corresponded to adhesions. These adhesions persisted at filopodial bases, regardless of subsequent movements. In correlated time lapse-electron microscopy, these adhesion sites contained a focal ring (an oblate, donut-shaped structure ∼120 nm in diameter) with radiating actin filaments. Filament geometry may explain filopodial emergence at 30 degree angles relative to adjacent filopodia. A model is proposed in which focal rings play a vital role in initiating and stabilizing filopodia: 1) they anchor actin filaments at adhesions, thereby facilitating tension development and filopodial emergence; 2) “axial” filaments connect focal rings to nub tips, thereby organizing filament bundling and ensuring the bundle intersects an adhesion; and 3) “lateral” filaments interconnect focal rings and filament bundles, thereby helping stabilize lamellar margins and filopodia.
11
Wu, D. Y., and D. J. Goldberg. "Regulated tyrosine phosphorylation at the tips of growth cone filopodia." Journal of Cell Biology 123, no. 3 (November 1, 1993): 653–64. http://dx.doi.org/10.1083/jcb.123.3.653.
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Several types of evidence suggest that protein-tyrosine phosphorylation is important during the growth of neuronal processes, but few specific roles, or subcellular localizations suggestive of such roles, have been defined. We report here a localization of tyrosine-phosphorylated protein at the tips of growth cone filopodia. Immunocytochemistry using a mAb to phosphorylated tyrosine residues revealed intense staining of the tips of most filopodia of Aplysia axons growing slowly on a polylysine substrate, but of few filopodia of axons growing rapidly on a substrate coated with Aplysia hemolymph, which has growth-promoting material. Cytochalasin D, which causes F-actin to withdraw rapidly from the growth cone, caused the tyrosine-phosphorylated protein to withdraw rapidly from filopodia, suggesting that the protein associates or interacts with actin filaments. Phosphotyrosine has previously been found concentrated at adherens junctions, where bundles of actin filaments terminate, but video-enhanced contrast-differential interference contrast and confocal interference reflection microscopy demonstrated that the filopodial tips were not adherent to the substrate. Acute application of either hemolymph or inhibitors of protein-tyrosine kinases to neurons on polylysine resulted in a rapid loss of intense staining at filopodial tips concomitant with a lengthening of the filopodia (and their core bundles of actin filaments). These results demonstrate that tyrosine-phosphorylated protein can be concentrated at the barbed ends of actin filaments in a context other than an adherens junction, indicate an association between changes in phosphorylation and filament dynamics, and provide evidence for tyrosine phosphorylation as a signaling mechanism in the filopodium that can respond to environmental cues controlling growth cone dynamics.
12
Vignjevic, Danijela, Shin-ichiro Kojima, Yvonne Aratyn, Oana Danciu, Tatyana Svitkina, and Gary G. Borisy. "Role of fascin in filopodial protrusion." Journal of Cell Biology 174, no. 6 (September 11, 2006): 863–75. http://dx.doi.org/10.1083/jcb.200603013.
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In this study, the mechanisms of actin-bundling in filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of filopodia, and remaining filopodia had abnormal morphology with wavy and loosely bundled actin organization. Dephosphorylation of serine 39 likely determined cellular filopodia frequency. The constitutively active fascin mutant S39A increased the number and length of filopodia, whereas the inactive fascin mutant S39E reduced filopodia frequency. Fluorescence recovery after photobleaching of GFP-tagged wild-type and S39A fascin showed that dephosphorylated fascin underwent rapid cycles of association to and dissociation from actin filaments in filopodia, with t1/2 < 10 s. We propose that fascin is a key specific actin cross-linker, providing stiffness for filopodial bundles, and that its dynamic behavior allows for efficient coordination between elongation and bundling of filopodial actin filaments.
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Tamada, Atsushi, Satoshi Kawase, Fujio Murakami, and Hiroyuki Kamiguchi. "Autonomous right-screw rotation of growth cone filopodia drives neurite turning." Journal of Cell Biology 188, no. 3 (February 1, 2010): 429–41. http://dx.doi.org/10.1083/jcb.200906043.
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The direction of neurite elongation is controlled by various environmental cues. However, it has been reported that even in the absence of any extrinsic directional signals, neurites turn clockwise on two-dimensional substrates. In this study, we have discovered autonomous rotational motility of the growth cone, which provides a cellular basis for inherent neurite turning. We have developed a technique for monitoring three-dimensional motility of growth cone filopodia and demonstrate that an individual filopodium rotates on its own longitudinal axis in the right-screw direction from the viewpoint of the growth cone body. We also show that the filopodial rotation involves myosins Va and Vb and may be driven by their spiral interactions with filamentous actin. Furthermore, we provide evidence that the unidirectional rotation of filopodia causes deflected neurite elongation, most likely via asymmetric positioning of the filopodia onto the substrate. Although the growth cone itself has been regarded as functionally symmetric, our study reveals the asymmetric nature of growth cone motility.
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Svitkina, Tatyana M., Elena A. Bulanova, Oleg Y. Chaga, Danijela M. Vignjevic, Shin-ichiro Kojima, Jury M. Vasiliev, and Gary G. Borisy. "Mechanism of filopodia initiation by reorganization of a dendritic network." Journal of Cell Biology 160, no. 3 (February 3, 2003): 409–21. http://dx.doi.org/10.1083/jcb.200210174.
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Afilopodium protrudes by elongation of bundled actin filaments in its core. However, the mechanism of filopodia initiation remains unknown. Using live-cell imaging with GFP-tagged proteins and correlative electron microscopy, we performed a kinetic-structural analysis of filopodial initiation in B16F1 melanoma cells. Filopodial bundles arose not by a specific nucleation event, but by reorganization of the lamellipodial dendritic network analogous to fusion of established filopodia but occurring at the level of individual filaments. Subsets of independently nucleated lamellipodial filaments elongated and gradually associated with each other at their barbed ends, leading to formation of cone-shaped structures that we term Λ-precursors. An early marker of initiation was the gradual coalescence of GFP-vasodilator-stimulated phosphoprotein (GFP-VASP) fluorescence at the leading edge into discrete foci. The GFP-VASP foci were associated with Λ-precursors, whereas Arp2/3 was not. Subsequent recruitment of fascin to the clustered barbed ends of Λ-precursors initiated filament bundling and completed formation of the nascent filopodium. We propose a convergent elongation model of filopodia initiation, stipulating that filaments within the lamellipodial dendritic network acquire privileged status by binding a set of molecules (including VASP) to their barbed ends, which protect them from capping and mediate association of barbed ends with each other.
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McConnell, Russell E., J. Edward van Veen, Marina Vidaki, Adam V. Kwiatkowski, Aaron S. Meyer, and Frank B. Gertler. "A requirement for filopodia extension toward Slit during Robo-mediated axon repulsion." Journal of Cell Biology 213, no. 2 (April 18, 2016): 261–74. http://dx.doi.org/10.1083/jcb.201509062.
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Axons navigate long distances through complex 3D environments to interconnect the nervous system during development. Although the precise spatiotemporal effects of most axon guidance cues remain poorly characterized, a prevailing model posits that attractive guidance cues stimulate actin polymerization in neuronal growth cones whereas repulsive cues induce actin disassembly. Contrary to this model, we find that the repulsive guidance cue Slit stimulates the formation and elongation of actin-based filopodia from mouse dorsal root ganglion growth cones. Surprisingly, filopodia form and elongate toward sources of Slit, a response that we find is required for subsequent axonal repulsion away from Slit. Mechanistically, Slit evokes changes in filopodium dynamics by increasing direct binding of its receptor, Robo, to members of the actin-regulatory Ena/VASP family. Perturbing filopodium dynamics pharmacologically or genetically disrupts Slit-mediated repulsion and produces severe axon guidance defects in vivo. Thus, Slit locally stimulates directional filopodial extension, a process that is required for subsequent axonal repulsion downstream of the Robo receptor.
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Sinnar, Shamim A., Susumu Antoku, Jean-Michel Saffin, Jon A. Cooper, and Shelley Halpain. "Capping protein is essential for cell migration in vivo and for filopodial morphology and dynamics." Molecular Biology of the Cell 25, no. 14 (July 15, 2014): 2152–60. http://dx.doi.org/10.1091/mbc.e13-12-0749.
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Capping protein (CP) binds to barbed ends of growing actin filaments and inhibits elongation. CP is essential for actin-based motility in cell-free systems and in Dictyostelium. Even though CP is believed to be critical for creating the lamellipodial actin structure necessary for protrusion and migration, CP's role in mammalian cell migration has not been directly tested. Moreover, recent studies have suggested that structures besides lamellipodia, including lamella and filopodia, may have unappreciated roles in cell migration. CP has been postulated to be absent from filopodia, and thus its role in filopodial activity has remained unexplored. We report that silencing CP in both cultured mammalian B16F10 cells and in neurons of developing neocortex impaired cell migration. Moreover, we unexpectedly observed that low levels of CP were detectable in the majority of filopodia. CP depletion decreased filopodial length, altered filopodial shape, and reduced filopodial dynamics. Our results support an expansion of the potential roles that CP plays in cell motility by implicating CP in filopodia as well as in lamellipodia, both of which are important for locomotion in many types of migrating cells.
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Lee, Kwonmoo, Jennifer L. Gallop, Komal Rambani, and Marc W. Kirschner. "Self-Assembly of Filopodia-Like Structures on Supported Lipid Bilayers." Science 329, no. 5997 (September 9, 2010): 1341–45. http://dx.doi.org/10.1126/science.1191710.
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Filopodia are finger-like protrusive structures, containing actin bundles. By incubating frog egg extracts with supported lipid bilayers containing phosphatidylinositol 4,5 bisphosphate, we have reconstituted the assembly of filopodia-like structures (FLSs). The actin assembles into parallel bundles, and known filopodial components localize to the tip and shaft. The filopodia tip complexes self-organize—they are not templated by preexisting membrane microdomains. The F-BAR domain protein toca-1 recruits N-WASP, followed by the Arp2/3 complex and actin. Elongation proteins, Diaphanous-related formin, VASP, and fascin are recruited subsequently. Although the Arp2/3 complex is required for FLS initiation, it is not essential for elongation, which involves formins. We propose that filopodia form via clustering of Arp2/3 complex activators, self-assembly of filopodial tip complexes on the membrane, and outgrowth of actin bundles.
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Passey, S., S. Pellegrin, and H. Mellor. "What is in a filopodium? Starfish versus hedgehogs." Biochemical Society Transactions 32, no. 6 (October 26, 2004): 1115–17. http://dx.doi.org/10.1042/bst0321115.
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Many cell types can generate thin actin-based protrusive structures, which are often classified under the general term of ‘filopodia’. However, a range of filopodia-like structures exists that differ both morphologically and functionally. In this brief review, we discuss the different types of filopodial structures, together with the actin-binding proteins and signalling pathways involved in their formation. Specifically, we highlight the differences between the filopodial extensions induced by the Rho GTPases Cdc42 and Rif.
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Aratyn, Yvonne S., Thomas E. Schaus, Edwin W. Taylor, and Gary G. Borisy. "Intrinsic Dynamic Behavior of Fascin in Filopodia." Molecular Biology of the Cell 18, no. 10 (October 2007): 3928–40. http://dx.doi.org/10.1091/mbc.e07-04-0346.
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Recent studies showed that the actin cross-linking protein, fascin, undergoes rapid cycling between filopodial filaments. Here, we used an experimental and computational approach to dissect features of fascin exchange and incorporation in filopodia. Using expression of phosphomimetic fascin mutants, we determined that fascin in the phosphorylated state is primarily freely diffusing, whereas actin bundling in filopodia is accomplished by fascin dephosphorylated at serine 39. Fluorescence recovery after photobleaching analysis revealed that fascin rapidly dissociates from filopodial filaments with a kinetic off-rate of 0.12 s−1 and that it undergoes diffusion at moderate rates with a coefficient of 6 μm2s−1. This kinetic off-rate was recapitulated in vitro, indicating that dynamic behavior is intrinsic to the fascin cross-linker. A computational reaction–diffusion model showed that reversible cross-linking is required for the delivery of fascin to growing filopodial tips at sufficient rates. Analysis of fascin bundling indicated that filopodia are semiordered bundles with one bound fascin per 25–60 actin monomers.
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Les Erickson, F., Amoreena C. Corsa, Andréa C. Dosé, and Beth Burnside. "Localization of a Class III Myosin to Filopodia Tips in Transfected HeLa Cells Requires an Actin-binding Site in its Tail Domain." Molecular Biology of the Cell 14, no. 10 (October 2003): 4173–80. http://dx.doi.org/10.1091/mbc.e02-10-0656.
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Bass Myo3A, a class III myosin, was expressed in HeLa cells as a GFP fusion in order to study its cellular localization. GFP-Myo3A localized to the cytoplasm and to the tips of F-actin bundles in filopodia, a localization that is consistent with the observed concentration toward the distal ends of F-actin bundles in photoreceptor cells. A mutation in the motor active site resulted in a loss of filopodia localization, suggesting that Myo3A motor activity is required for filopodial tip localization. Deletion analyses showed that the NH2-terminal kinase domain is not required but the CO2H-terminal 22 amino acids of the Myo3A tail are required for filopodial localization. Expression of this tail fragment alone produced fluorescence associated with F-actin throughout the cytoplasm and filopodia and a recombinant tail fragment bound to F-actin in vitro. An actin-binding motif was identified within this tail fragment, and a mutation within this motif abolished both filopodia localization by Myo3A and F-actin binding by the tail fragment alone. Calmodulin localized to filopodial tips when coexpressed with Myo3A but not in the absence of Myo3A, an observation consistent with the previous proposal that class III myosins bind calmodulin and thereby localize it in certain cell types.
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Applewhite, Derek A., Melanie Barzik, Shin-ichiro Kojima, Tatyana M. Svitkina, Frank B. Gertler, and Gary G. Borisy. "Ena/VASP Proteins Have an Anti-Capping Independent Function in Filopodia Formation." Molecular Biology of the Cell 18, no. 7 (July 2007): 2579–91. http://dx.doi.org/10.1091/mbc.e06-11-0990.
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Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at filopodial tips. Mutation of the G-actin–binding motif (GAB) partially compromised stabilization of Ena/VASP at filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends.
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Gauthier-Campbell, Catherine, David S. Bredt, Timothy H. Murphy, and Alaa El-Din El-Husseini. "Regulation of Dendritic Branching and Filopodia Formation in Hippocampal Neurons by Specific Acylated Protein Motifs." Molecular Biology of the Cell 15, no. 5 (May 2004): 2205–17. http://dx.doi.org/10.1091/mbc.e03-07-0493.
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Although neuronal axons and dendrites with their associated filopodia and spines exhibit a profound cell polarity, the mechanism by which they develop is largely unknown. Here, we demonstrate that specific palmitoylated protein motifs, characterized by two adjacent cysteines and nearby basic residues, are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and the branching of dendrites and axons in neurons. Such motifs are present at the N-terminus of GAP-43 and the C-terminus of paralemmin, two neuronal proteins implicated in cytoskeletal organization and filopodial outgrowth. Filopodia induction is blocked by mutations of the palmitoylated sites or by treatment with 2-bromopalmitate, an agent that inhibits protein palmitoylation. Moreover, overexpression of a constitutively active form of ARF6, a GTPase that regulates membrane cycling and dendritic branching reversed the effects of the acylated protein motifs. Filopodia induction by the specific palmitoylated motifs was also reduced upon overexpression of a dominant negative form of the GTPase cdc42. These results demonstrate that select dually lipidated protein motifs trigger changes in the development and growth of neuronal processes.
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Barzik, Melanie, Leslie M. McClain, Stephanie L. Gupton, and Frank B. Gertler. "Ena/VASP regulates mDia2-initiated filopodial length, dynamics, and function." Molecular Biology of the Cell 25, no. 17 (September 2014): 2604–19. http://dx.doi.org/10.1091/mbc.e14-02-0712.
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Filopodia are long plasma membrane extensions involved in the formation of adhesive, contractile, and protrusive actin-based structures in spreading and migrating cells. Whether filopodia formed by different molecular mechanisms equally support these cellular functions is unresolved. We used Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP)–deficient MVD7 fibroblasts, which are also devoid of endogenous mDia2, as a model system to investigate how these different actin regulatory proteins affect filopodia morphology and dynamics independently of one another. Filopodia initiated by either Ena/VASP or mDia2 contained similar molecular inventory but differed significantly in parameters such as number, length, F-actin organization, lifetime, and protrusive persistence. Moreover, in the absence of Ena/VASP, filopodia generated by mDia2 did not support initiation of integrin-dependent signaling cascades required for adhesion and subsequent lamellipodial extension, thereby causing a defect in early cell spreading. Coexpression of VASP with constitutively active mDia2M/A rescued these early adhesion defects. We conclude that Ena/VASP and mDia2 support the formation of filopodia with significantly distinct properties and that Ena/VASP regulates mDia2-initiated filopodial morphology, dynamics, and function.
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Mallavarapu, Aneil, and Tim Mitchison. "Regulated Actin Cytoskeleton Assembly at Filopodium Tips Controls Their Extension and Retraction." Journal of Cell Biology 146, no. 5 (September 6, 1999): 1097–106. http://dx.doi.org/10.1083/jcb.146.5.1097.
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The extension and retraction of filopodia in response to extracellular cues is thought to be an important initial step that determines the direction of growth cone advance. We sought to understand how the dynamic behavior of the actin cytoskeleton is regulated to produce extension or retraction. By observing the movement of fiduciary marks on actin filaments in growth cones of a neuroblastoma cell line, we found that filopodium extension and retraction are governed by a balance between the rate of actin cytoskeleton assembly at the tip and retrograde flow. Both assembly and flow rate can vary with time in a single filopodium and between filopodia in a single growth cone. Regulation of assembly rate is the dominant factor in controlling filopodia behavior in our system.
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Hardin, J. "The role of secondary mesenchyme cells during sea urchin gastrulation studied by laser ablation." Development 103, no. 2 (June 1, 1988): 317–24. http://dx.doi.org/10.1242/dev.103.2.317.
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It has long been thought that traction exerted by filopodia of secondary mesenchyme cells (SMCs) is a sufficient mechanism to account for elongation of the archenteron during sea urchin gastrulation. The filopodial traction hypothesis has been directly tested here by laser ablation of SMCs in gastrulae of the sea urchin, Lytechinus pictus. When SMCs are ablated at the onset of secondary invagination, the archenteron doubles in length at the normal rate of elongation, but advance of the tip of the archenteron stops at the 2/3 gastrula stage. In contrast, when all SMCs are ablated at or following the 2/3 gastrula stage, further elongation does not occur. However, if a few SMCs are allowed to remain in 2/3-3/4 gastrulae, elongation continues, although more slowly than in controls. The final length of archenterons in embryos ablated at the 1/3-1/2 gastrula stage is virtually identical to the final length of everted archenterons in LiCl-induced exogastrulae; since filopodial traction is not exerted in either case, an alternate, common mechanism of elongation probably operates in both cases. These results suggest that archenteron elongation involves two processes: (1) active, filopodia-independent elongation, which depends on active cell rearrangement and (2) filopodia-dependent elongation, which depends on mechanical tension exerted by the filopodia.
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Sydor, A. M., A. L. Su, F. S. Wang, A. Xu, and D. G. Jay. "Talin and vinculin play distinct roles in filopodial motility in the neuronal growth cone." Journal of Cell Biology 134, no. 5 (September 1, 1996): 1197–207. http://dx.doi.org/10.1083/jcb.134.5.1197.
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Filopodial motility is critical for many biological processes, particularly for axon guidance. This motility is based on altering the F-actin-based cytoskeleton, but the mechanisms of how this occurs and the actin-associated proteins that function in this process remain unclear. We investigated two of these proteins found in filopodia, talin and vinculin, by inactivating them in subregions of chick dorsal root ganglia neuronal growth cones and by observing subsequent behavior by video-enhanced microscopy and quantitative morphometry. Microscale chromophore-assisted laser inactivation of talin resulted in the temporary cessation of filopodial extension and retraction. Inactivation of vinculin caused an increased incidence of filopodial bending and buckling within the laser spot but had no effect on extension or retraction. These findings show that talin acts in filopodial motility and may couple both extension and retraction to actin dynamics. They also suggest that vinculin is not required for filopodial extension and retraction but plays a role in the structural integrity of filopodia.
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Horev, Melanie B., Yishaia Zabary, Revital Zarka, Simona Sorrentino, Ohad Medalia, Assaf Zaritsky, and Benjamin Geiger. "Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces." F1000Research 9 (May 27, 2020): 449. http://dx.doi.org/10.12688/f1000research.23598.1.
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Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet’s periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.
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Horev, Melanie B., Yishaia Zabary, Revital Zarka, Simona Sorrentino, Ohad Medalia, Assaf Zaritsky, and Benjamin Geiger. "Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces." F1000Research 9 (July 3, 2020): 449. http://dx.doi.org/10.12688/f1000research.23598.2.
Full textAbstract:
Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet’s periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.
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Lu, Mei, Walter Witke, David J. Kwiatkowski, and Kenneth S. Kosik. "Delayed Retraction of Filopodia in Gelsolin Null Mice." Journal of Cell Biology 138, no. 6 (September 22, 1997): 1279–87. http://dx.doi.org/10.1083/jcb.138.6.1279.
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Growth cones extend dynamic protrusions called filopodia and lamellipodia as exploratory probes that signal the direction of neurite growth. Gelsolin, as an actin filament-severing protein, may serve an important role in the rapid shape changes associated with growth cone structures. In wild-type (wt) hippocampal neurons, antibodies against gelsolin labeled the neurite shaft and growth cone. The behavior of filopodia in cultured hippocampal neurons from embryonic day 17 wt and gelsolin null (Gsn−) mice (Witke, W., A.H. Sharpe, J.H. Hartwig, T. Azuma, T.P. Stossel, and D.J. Kwiatkowski. 1995. Cell. 81:41–51.) was recorded with time-lapse video microscopy. The number of filopodia along the neurites was significantly greater in Gsn− mice and gave the neurites a studded appearance. Dynamic studies suggested that most of these filopodia were formed from the region of the growth cone and remained as protrusions from the newly consolidated shaft after the growth cone advanced. Histories of individual filopodia in Gsn− mice revealed elongation rates that did not differ from controls but an impaired retraction phase that probably accounted for the increased number of filopodia long the neutrite shaft. Gelsolin appears to function in the initiation of filopodial retraction and in its smooth progression.
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Kyykallio, Heikki, Sanna Oikari, María Bueno Álvez, Carlos José Gallardo Dodd, Janne Capra, and Kirsi Rilla. "The Density and Length of Filopodia Associate with the Activity of Hyaluronan Synthesis in Tumor Cells." Cancers 12, no. 7 (July 15, 2020): 1908. http://dx.doi.org/10.3390/cancers12071908.
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Filopodia are multifunctional finger-like plasma membrane protrusions with bundles of actin filaments that exist in virtually all cell types. It has been known for some time that hyaluronan synthesis activity induces filopodial growth. However, because of technical challenges in the studies of these slender and fragile structures, no quantitative analyses have been performed so far to indicate their association with hyaluronan synthesis. In this work we comprehensively address the direct quantification of filopodial traits, covering for the first time length and density measurements in a series of human cancer cell lines with variable levels of hyaluronan synthesis. The synthesis and plasma membrane binding of hyaluronan were manipulated with hyaluronan synthase 3 (HAS3) and hyaluronan receptor CD44 overexpression, and treatments with mannose, 4-methylumbelliferone (4-MU), and glucosamine. The results of this work show that the growth of filopodia was associated with the levels of hyaluronan synthesis but was not dependent on CD44 expression. The results confirm the hypothesis that abundance and length of filopodia in cancer cells is associated with the activity of hyaluronan synthesis.
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Choi, A. H., and C. H. Siu. "Filopodia are enriched in a cell cohesion molecule of Mr 80,000 and participate in cell-cell contact formation in Dictyostelium discoideum." Journal of Cell Biology 104, no. 5 (May 1, 1987): 1375–87. http://dx.doi.org/10.1083/jcb.104.5.1375.
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During the early phase of Dictyostelium discoideum development, cells undergo chemotactic migration to form tight aggregates. A developmentally regulated surface glycoprotein of Mr 80,000 (gp80) has been implicated in mediating the EDTA-resistant type of cell cohesion at this stage. We have used a monoclonal antibody directed against gp80 to study the topographical distribution of gp80 on the cell surface. Indirect immunofluorescence studies showed that gp80 was primarily localized on the cell surface, with a higher concentration at contact areas. Immunoelectron microscopy was carried out by indirect labeling using protein A-gold, and a nonrandom distribution of gp80 was revealed. In addition to contact regions, gold particles were found preferentially localized on filopodia. Quantitative analysis using transmission electron microscopy (TEM) showed that approximately 60% more gold particles were localized in contact regions in comparison with the noncontact regions, and the filopodial surfaces had a twofold higher gold density. Both TEM and scanning electron microscopy showed that contact areas were enriched in filopodial structures. Filopodia often appeared to adhere to either smooth surfaces or similar filopodial structures of an adjacent cell. These observations suggest that the formation of stable cell-cell contacts involves at least four sequential steps in which filopodia and gp80 probably play an important role in the initial stages of recognition and cohesion among cells.
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Miller, J., S. E. Fraser, and D. McClay. "Dynamics of thin filopodia during sea urchin gastrulation." Development 121, no. 8 (August 1, 1995): 2501–11. http://dx.doi.org/10.1242/dev.121.8.2501.
Full textAbstract:
At gastrulation in the sea urchin embryo, a dramatic rearrangement of cells establishes the three germ layers of the organism. Experiments have revealed a number of cell interactions at this stage that transfer patterning information from cell to cell. Of particular significance, primary mesenchyme cells, which are responsible for production of the embryonic skeleton, have been shown to obtain extensive positional information from the embryonic ectoderm. In the present study, high resolution Nomarski imaging reveals the presence of very thin filopodia (02-0.4 micron in diameter) extending from primary mesenchyme cells as well as from ectodermal and secondary mesenchyme cells. These thin filopodia sometimes extend to more than 80 microns in length and show average growth and retraction rates of nearly 10 microns/minute. The filopodia are highly dynamic, rapidly changing from extension to resorption; frequently, the resorption changes to resumption of assembly. The behavior, location and timing of active thin filopodial movements does not correlate with cell locomotion; instead, there is a strong correlation suggesting their involvement in cell-cell interactions associated with signaling and patterning at gastrulation. Nickel-treatment, which is known to create a patterning defect in skeletogenesis due to alterations in the ectoderm, alters the normal position-dependent differences in the thin filopodia. The effect is present in recombinant embryos in which the ectoderm alone was treated with nickel, and is absent in recombinant embryos in which only the primary mesenchyme cells were treated, suggesting that the filopodial length is substratum dependent rather than being primary mesenchyme cell autonomous. The thin filopodia provide a means by which cells can contact others several cell diameters away, suggesting that some of the signaling previously thought to be mediated by diffusible signals may instead by the result of direct receptor-ligand interactions between cell membranes.
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Pi, Xinchun, Rongqin Ren, Russell Kelley, Chunlian Zhang, Martin Moser, Aparna B. Bohil, Melinda DiVito, Richard E. Cheney, and Cam Patterson. "Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors." Journal of Cell Biology 179, no. 7 (December 24, 2007): 1569–82. http://dx.doi.org/10.1083/jcb.200704010.
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Endothelial cell migration is an important step during angiogenesis, and its dysregulation contributes to aberrant neovascularization. The bone morphogenetic proteins (BMPs) are potent stimulators of cell migration and angiogenesis. Using microarray analyses, we find that myosin-X (Myo10) is a BMP target gene. In endothelial cells, BMP6-induced Myo10 localizes in filopodia, and BMP-dependent filopodial assembly decreases when Myo10 expression is reduced. Likewise, cellular alignment and directional migration induced by BMP6 are Myo10 dependent. Surprisingly, we find that Myo10 and BMP6 receptor ALK6 colocalize in a BMP6-dependent fashion. ALK6 translocates into filopodia after BMP6 stimulation, and both ALK6 and Myo10 possess intrafilopodial motility. Additionally, Myo10 is required for BMP6-dependent Smad activation, indicating that in addition to its function in filopodial assembly, Myo10 also participates in a requisite amplification loop for BMP signaling. Our data indicate that Myo10 is required to guide endothelial migration toward BMP6 gradients via the regulation of filopodial function and amplification of BMP signals.
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Pertz, Olivier. "Filopodia." Communicative & Integrative Biology 4, no. 4 (July 2011): 436–39. http://dx.doi.org/10.4161/cib.15325.
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Jacinto, Antonio, and Lewis Wolpert. "Filopodia." Current Biology 11, no. 16 (August 2001): R634. http://dx.doi.org/10.1016/s0960-9822(01)00378-5.
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Petersen, Karl J., Holly V. Goodson, Ashley L. Arthur, G. W. Gant Luxton, Anne Houdusse, and Margaret A. Titus. "MyTH4-FERM myosins have an ancient and conserved role in filopod formation." Proceedings of the National Academy of Sciences 113, no. 50 (November 23, 2016): E8059—E8068. http://dx.doi.org/10.1073/pnas.1615392113.
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The formation of filopodia in Metazoa and Amoebozoa requires the activity of myosin 10 (Myo10) in mammalian cells and of Dictyostelium unconventional myosin 7 (DdMyo7) in the social amoeba Dictyostelium. However, the exact roles of these MyTH4-FERM myosins (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in the initiation and elongation of filopodia are not well defined and may reflect conserved functions among phylogenetically diverse MF myosins. Phylogenetic analysis of MF myosin domains suggests that a single ancestral MF myosin existed with a structure similar to DdMyo7, which has two MF domains, and that subsequent duplications in the metazoan lineage produced its functional homolog Myo10. The essential functional features of the DdMyo7 myosin were identified using quantitative live-cell imaging to characterize the ability of various mutants to rescue filopod formation in myo7-null cells. The two MF domains were found to function redundantly in filopod formation with the C-terminal FERM domain regulating both the number of filopodia and their elongation velocity. DdMyo7 mutants consisting solely of the motor plus a single MyTH4 domain were found to be capable of rescuing the formation of filopodia, establishing the minimal elements necessary for the function of this myosin. Interestingly, a chimeric myosin with the Myo10 MF domain fused to the DdMyo7 motor also was capable of rescuing filopod formation in the myo7-null mutant, supporting fundamental functional conservation between these two distant myosins. Together, these findings reveal that MF myosins have an ancient and conserved role in filopod formation.
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Hoelzle, Matthew K., and Tatyana Svitkina. "The cytoskeletal mechanisms of cell–cell junction formation in endothelial cells." Molecular Biology of the Cell 23, no. 2 (January 15, 2012): 310–23. http://dx.doi.org/10.1091/mbc.e11-08-0719.
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The actin cytoskeleton and associated proteins play a vital role in cell–cell adhesion. However, the procedure by which cells establish adherens junctions remains unclear. We investigated the dynamics of cell–cell junction formation and the corresponding architecture of the underlying cytoskeleton in cultured human umbilical vein endothelial cells. We show that the initial interaction between cells is mediated by protruding lamellipodia. On their retraction, cells maintain contact through thin bridges formed by filopodia-like protrusions connected by VE-cadherin–rich junctions. Bridges share multiple features with conventional filopodia, such as an internal actin bundle associated with fascin along the length and vasodilator-stimulated phosphoprotein at the tip. It is striking that, unlike conventional filopodia, transformation of actin organization from the lamellipodial network to filopodial bundle during bridge formation occurs in a proximal-to-distal direction and is accompanied by recruitment of fascin in the same direction. Subsequently, bridge bundles recruit nonmuscle myosin II and mature into stress fibers. Myosin II activity is important for bridge formation and accumulation of VE-cadherin in nascent adherens junctions. Our data reveal a mechanism of cell–cell junction formation in endothelial cells using lamellipodia as the initial protrusive contact, subsequently transforming into filopodia-like bridges connected through adherens junctions. Moreover, a novel lamellipodia-to-filopodia transition is used in this context.
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Vignjevic, Danijela, Defne Yarar, Matthew D. Welch, John Peloquin, Tatyana Svitkina, and Gary G. Borisy. "Formation of filopodia-like bundles in vitro from a dendritic network." Journal of Cell Biology 160, no. 6 (March 17, 2003): 951–62. http://dx.doi.org/10.1083/jcb.200208059.
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We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)–activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)–coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.
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Li, Pan P., Cheng Chen, Chi-Wai Lee, Raghavan Madhavan, and H. Benjamin Peng. "Axonal filopodial asymmetry induced by synaptic target." Molecular Biology of the Cell 22, no. 14 (July 15, 2011): 2480–90. http://dx.doi.org/10.1091/mbc.e11-03-0198.
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During vertebrate neuromuscular junction (NMJ) assembly, motor axons and their muscle targets exchange short-range signals that regulate the subsequent steps of presynaptic and postsynaptic specialization. We report here that this interaction is in part mediated by axonal filopodia extended preferentially by cultured Xenopus spinal neurons toward their muscle targets. Immunoblotting and labeling experiments showed that basic fibroblast growth factor (bFGF) was expressed by muscle and associated with the cell surface, and treatment of cultured spinal neurons with recombinant bFGF nearly doubled the normal density of filopodia in neurites. This effect of bFGF was abolished by SU5402, a selective inhibitor of FGF-receptor 1 (FGFR1), and forced expression of wild-type or dominant-negative FGFR1 in neurons enhanced or suppressed the assembly of filopodia, respectively. Significantly, in nerve–muscle cocultures, knocking down bFGF in muscle decreased both the asymmetric extension of filopodia by axons toward muscle and the assembly of NMJs. In addition, neurons expressing dominant-negative FGFR1 less effectively triggered the aggregation of muscle acetylcholine receptors at innervation sites than did control neurons. These results suggest that bFGF activation of neuronal FGFR1 generates filopodial processes in neurons that promote nerve–muscle interaction and facilitate NMJ establishment.
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Rabinovitz, Isaac, and Arthur M. Mercurio. "The Integrin α6β4 Functions in Carcinoma Cell Migration on Laminin-1 by Mediating the Formation and Stabilization of Actin-containing Motility Structures." Journal of Cell Biology 139, no. 7 (December 29, 1997): 1873–84. http://dx.doi.org/10.1083/jcb.139.7.1873.
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Functional studies on the α6β4 integrin have focused primarily on its role in the organization of hemidesmosomes, stable adhesive structures that associate with the intermediate filament cytoskeleton. In this study, we examined the function of the α6β4 integrin in clone A cells, a colon carcinoma cell line that expresses α6β4 but no α6β1 integrin and exhibits dynamic adhesion and motility on laminin-1. Time-lapse videomicroscopy of clone A cells on laminin-1 revealed that their migration is characterized by filopodial extension and stabilization followed by lamellae that extend in the direction of stabilized filopodia. A function-blocking mAb specific for the α6β4 integrin inhibited clone A migration on laminin-1. This mAb also inhibited filopodial formation and stabilization and lamella formation. Indirect immunofluorescence microscopy revealed that the α6β4 integrin is localized as discrete clusters in filopodia, lamellae, and retraction fibers. Although β1 integrins were also localized in the same structures, a spatial separation of these two integrin populations was evident. In filopodia and lamellae, a striking colocalization of the α6β4 integrin and F-actin was seen. An association between α6β4 and F-actin is supported by the fact that α6β4 integrin and actin were released from clone A cells by treatment with the F-actin– severing protein gelsolin and that α6β4 immunostaining at the marginal edges of clone A cells on laminin-1 was resistant to solubilization with Triton X-100. Cytokeratins were not observed in filopodia and lamellipodia. Moreover, α6β4 was extracted from these marginal edges with a Tween-40/deoxycholate buffer that solubilizes the actin cytoskeleton but not cytokeratins. Three other carcinoma cell lines (MIP-101, CCL-228, and MDA-MB-231) exhibited α6β4 colocalized with actin in filopodia and lamellae. Formation of lamellae in these cells was inhibited with an α6-specific antibody. Together, these results indicate that the α6β4 integrin functions in carcinoma migration on laminin-1 through its ability to promote the formation and stabilization of actin-containing motility structures.
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Chitu, Violeta, Fiona J. Pixley, Frank Macaluso, Daniel R. Larson, John Condeelis, Yee-Guide Yeung, and E. Richard Stanley. "The PCH Family Member MAYP/PSTPIP2 Directly Regulates F-Actin Bundling and Enhances Filopodia Formation and Motility in Macrophages." Molecular Biology of the Cell 16, no. 6 (June 2005): 2947–59. http://dx.doi.org/10.1091/mbc.e04-10-0914.
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Macrophage actin-associated tyrosine phosphorylated protein (MAYP) belongs to the Pombe Cdc15 homology (PCH) family of proteins involved in the regulation of actin-based functions including cell adhesion and motility. In mouse macrophages, MAYP is tyrosine phosphorylated after activation of the colony-stimulating factor-1 receptor (CSF-1R), which also induces actin reorganization, membrane ruffling, cell spreading, polarization, and migration. Because MAYP associates with F-actin, we investigated the function of MAYP in regulating actin organization in macrophages. Overexpression of MAYP decreased CSF-1–induced membrane ruffling and increased filopodia formation, motility and CSF-1-mediated chemotaxis. The opposite phenotype was observed with reduced expression of MAYP, indicating that MAYP is a negative regulator of CSF-1–induced membrane ruffling and positively regulates formation of filopodia and directional migration. Overexpression of MAYP led to a reduction in total macrophage F-actin content but was associated with increased actin bundling. Consistent with this, purified MAYP bundled F-actin and regulated its turnover in vitro. In addition, MAYP colocalized with cortical and filopodial F-actin in vivo. Because filopodia are postulated to increase directional motility by acting as environmental sensors, the MAYP-stimulated increase in directional movement may be at least partly explained by enhancement of filopodia formation.
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He, Yingpei, Yuan Ren, Bingbing Wu, Boris Decourt, Aih Cheun Lee, Aaron Taylor, and Daniel M. Suter. "Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones." Molecular Biology of the Cell 26, no. 18 (September 15, 2015): 3229–44. http://dx.doi.org/10.1091/mbc.e15-03-0142.
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Src tyrosine kinases have been implicated in axonal growth and guidance; however, the underlying cellular mechanisms are not well understood. Specifically, it is unclear which aspects of actin organization and dynamics are regulated by Src in neuronal growth cones. Here, we investigated the function of Src2 and one of its substrates, cortactin, in lamellipodia and filopodia of Aplysia growth cones. We found that up-regulation of Src2 activation state or cortactin increased lamellipodial length, protrusion time, and actin network density, whereas down-regulation had opposite effects. Furthermore, Src2 or cortactin up-regulation increased filopodial density, length, and protrusion time, whereas down-regulation promoted lateral movements of filopodia. Fluorescent speckle microscopy revealed that rates of actin assembly and retrograde flow were not affected in either case. In summary, our results support a model in which Src and cortactin regulate growth cone motility by increasing actin network density and protrusion persistence of lamellipodia by controlling the state of actin-driven protrusion versus retraction. In addition, both proteins promote the formation and stability of actin bundles in filopodia.
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Clarke, Akanni, Philip G. McQueen, Hsiao Yu Fang, Ramakrishnan Kannan, Victor Wang, Evan McCreedy, Tyler Buckley, Erika Johannessen, Stephen Wincovitch, and Edward Giniger. "Dynamic morphogenesis of a pioneer axon in Drosophila and its regulation by Abl tyrosine kinase." Molecular Biology of the Cell 31, no. 6 (March 15, 2020): 452–65. http://dx.doi.org/10.1091/mbc.e19-10-0563.
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Live imaging of an axon in its native tissue shows that its growth is protrusive and occurs by stabilization of selected filopodia. Guidance signaling, however, for example, via Abl tyrosine kinase, does not control these morphological properties directly but rather controls actin distribution to determine where filopodial dynamics can occur.
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Yang, Changsong, and Tatyana Svitkina. "Filopodia initiation." Cell Adhesion & Migration 5, no. 5 (September 2011): 402–8. http://dx.doi.org/10.4161/cam.5.5.16971.
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Korobova, Farida, and Tatyana Svitkina. "Molecular Architecture of Synaptic Actin Cytoskeleton in Hippocampal Neurons Reveals a Mechanism of Dendritic Spine Morphogenesis." Molecular Biology of the Cell 21, no. 1 (January 2010): 165–76. http://dx.doi.org/10.1091/mbc.e09-07-0596.
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Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility.
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Robens, Jeffrey M., Lee Yeow-Fong, Elsa Ng, Christine Hall, and Ed Manser. "Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization." Molecular and Cellular Biology 30, no. 3 (November 23, 2009): 829–44. http://dx.doi.org/10.1128/mcb.01574-08.
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ABSTRACT Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.
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Goldberg, D. J., and D. W. Burmeister. "Stages in axon formation: observations of growth of Aplysia axons in culture using video-enhanced contrast-differential interference contrast microscopy." Journal of Cell Biology 103, no. 5 (November 1, 1986): 1921–31. http://dx.doi.org/10.1083/jcb.103.5.1921.
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The regenerative growth in culture of the axons of two giant identified neurons from the central nervous system of Aplysia californica was observed using video-enhanced contrast-differential interference contrast microscopy. This technique allowed the visualization in living cells of the membranous organelles of the growth cone. Elongation of axonal branches always occurred through the same sequence of events: A flat organelle-free veil protruded from the front of the growth cone, gradually filled with vesicles that entered by fast axonal transport and Brownian motion from the main body of the growth cone, became more voluminous and engorged with organelles (vesicles, mitochondria, and one or two large, irregular, refractile bodies), and, finally, assumed the cylindrical shape of the axon branch with the organelles predominantly moving by bidirectional fast axonal transport. The veil is thus the nascent axon. Because veils appear to be initially free of membranous organelles, addition of membrane to the plasmalemma by exocytosis is likely to occur in the main body of the growth cone rather than at the leading edge. Veils almost always formed with filopodial borders, protruding between either fully extended or growing filopodia. Therefore, one function of the filopodia is to direct elongation by demarcating the pathway along which axolemma flows. Models of axon growth in which the body of the growth cone is pulled forward, or in which advance of the leading edge is achieved by filopodial shortening or contraction against an adhesion to the substrate, are inconsistent with our observations. We suggest that, during the elongation phase of growth, filopodia may act as structural supports.
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Hoffmann, Bernd, and Claudia Schäfer. "Filopodial focal complexes direct adhesion and force generation towards filopodia outgrowth." Cell Adhesion & Migration 4, no. 2 (April 2010): 190–93. http://dx.doi.org/10.4161/cam.4.2.10899.
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Almagro, Sébastien, Claire Durmort, Adeline Chervin-Pétinot, Stephanie Heyraud, Mathilde Dubois, Olivier Lambert, Camille Maillefaud, et al. "The Motor Protein Myosin-X Transports VE-Cadherin along Filopodia To Allow the Formation of Early Endothelial Cell-Cell Contacts." Molecular and Cellular Biology 30, no. 7 (February 1, 2010): 1703–17. http://dx.doi.org/10.1128/mcb.01226-09.
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ABSTRACT Vascular endothelium (VE), the monolayer of endothelial cells that lines the vascular tree, undergoes damage at the basis of some vascular diseases. Its integrity is maintained by VE-cadherin, an adhesive receptor localized at cell-cell junctions. Here, we show that VE-cadherin is also located at the tip and along filopodia in sparse or subconfluent endothelial cells. We observed that VE-cadherin navigates along intrafilopodial actin filaments. We found that the actin motor protein myosin-X is colocalized and moves synchronously with filopodial VE-cadherin. Immunoprecipitation and pulldown assays confirmed that myosin-X is directly associated with the VE-cadherin complex. Furthermore, expression of a dominant-negative mutant of myosin-X revealed that myosin-X is required for VE-cadherin export to cell edges and filopodia. These features indicate that myosin-X establishes a link between the actin cytoskeleton and VE-cadherin, thereby allowing VE-cadherin transportation along intrafilopodial actin cables. In conclusion, we propose that VE-cadherin trafficking along filopodia using myosin-X motor protein is a prerequisite for cell-cell junction formation. This mechanism may have functional consequences for endothelium repair in pathological settings.
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Tsui, H. C., D. Schubert, and W. L. Klein. "Molecular basis of growth cone adhesion: anchoring of adheron-containing filaments at adhesive loci." Journal of Cell Biology 106, no. 6 (June 1, 1988): 2095–108. http://dx.doi.org/10.1083/jcb.106.6.2095.
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Adhesive contacts made by filopodia of neuronal growth cones are essential for proper neurite elongation and may have a role in the formation of synaptic junctions. Previously we described the appearance of filamentous materials extending from growth cone surfaces that seem to be associated with the strongly adhesive behavior of filopodia (Tsui, H.-C., K. L. Lankford, and W. L. Klein. 1985. Proc. Natl. Acad. Sci. USA. 82:8256-8260). Here, we have used immunogold labeling to determine whether known adhesive molecules might be localized at points of adhesion and possibly be constituents of the filamentous material. Antibodies to an adhesive molecule (neural cell adhesion molecule [N-CAM]) and to an adhesive macromolecular complex of proteins and proteoglycans (adheron) were localized at the EM level in whole mounts of cultured avian retina cells. Labeling of fixed cells showed that N-CAM and adheron molecules were both present on growth cones and on filopodia. However, filamentous materials extending from the cell surface were labeled with anti-adheron but not with anti-N-CAM. If cells were labeled before fixation, patches of anti-N-CAM labeling occurred in random areas over the growth cones, but adheron antibodies concentrated at points of apparent adhesion. Particularly dense clustering of anti-adheron occurred at individual filopodial tips and at points of contact between pairs of filopodia. The different patterns of labeling imply that N-CAMS do not associate with the main antigenic components of adheron on the membrane surface. Most importantly, the data indicate the N-CAMs were mobile in the membrane but that constituents of adherons were anchored at adhesive loci. An appealing hypothesis is that molecules found in adheron preparations have an important role in establishing the adhesive junctions formed by growth cone filopodia.