Gupton, S.L. et al. Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin. J. Cell Biol. 168, 619-631

Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
The Journal of Cell Biology (Impact Factor: 9.69). 03/2005; 168(4):619-31. DOI: 10.1083/jcb.200406063
Source: PubMed

ABSTRACT The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin-binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle alphaTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.

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    • "FSM study: correlation of actin assembly with a green fluorescent protein (GFP)–p34 signal In biological systems, Arp2/3 is a protein complex that is thought to promote polymerization of networking filaments by aiding new filament creation from preexisting ones. This complex has been described in the literature as a mediator of actin, helping to regulate processes of polymerization and depolymerization in tandem [15] [16] [17]. It has been shown that Arp2/3 is present along the lamellipodium, the cytoskeletal actin at the mobile edge of cells. "
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    ABSTRACT: Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin-ABP interactions in these processes. In this article, we describe the basic concepts of fluorescence speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryo-electron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs.
    Analytical Biochemistry 09/2013; DOI:10.1016/j.ab.2013.09.008 · 2.22 Impact Factor
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    • "Because engagement of F-actin flow at FA is thought to limit the width of the lamellipodium and to establish a border between lamellipodium and lamellum (Ponti et al., 2004; Alexandrova et al., 2008; Shemesh et al., 2009), we sought to test whether vinculin affected the spatial organization of lamellipodium and lamellum. We localized F-actin and the lamellipodial protein cortactin or the lamellum protein phosphoserine19 myosin regulatory light chain-2 (pS19MLC2) in control and Vcl-KO MEF (Wu and Parsons, 1993; Ponti et al., 2004; Gupton et al., 2005; Lai et al., 2008). Line scans of staining intensity across the leading edge of control MEF revealed a sharply defined, narrow band of cortactin that colocalized with dense lamellipodial F-actin (Fig. 2, A [arrow], D, and E). "
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    ABSTRACT: In migrating cells, integrin-based focal adhesions (FAs) assemble in protruding lamellipodia in association with rapid filamentous actin (F-actin) assembly and retrograde flow. How dynamic F-actin is coupled to FA is not known. We analyzed the role of vinculin in integrating F-actin and FA dynamics by vinculin gene disruption in primary fibroblasts. Vinculin slowed F-actin flow in maturing FA to establish a lamellipodium-lamellum border and generate high extracellular matrix (ECM) traction forces. In addition, vinculin promoted nascent FA formation and turnover in lamellipodia and inhibited the frequency and rate of FA maturation. Characterization of a vinculin point mutant that specifically disrupts F-actin binding showed that vinculin-F-actin interaction is critical for these functions. However, FA growth rate correlated with F-actin flow speed independently of vinculin. Thus, vinculin functions as a molecular clutch, organizing leading edge F-actin, generating ECM traction, and promoting FA formation and turnover, but vinculin is dispensible for FA growth.
    The Journal of Cell Biology 07/2013; 202(1):163-77. DOI:10.1083/jcb.201303129 · 9.69 Impact Factor
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    • "Tropomyosin inhibits both the rate of polymerization and depolymerization of actin, regulates the actin-myosin interaction, and by the temporal and spatial regulation of its expression is involved with functional contributions to the actin cytoskeleton (Gunning et al. 2008). In addition, tropomyosin is a major regulator of F-actin functional specialization in migrating cells and is important for regionally defining the properties of the actin cytoskeleton in mediating changes in cell morphology (Gupton et al. 2005). Tropomyosin and troponin bind to actin and block myosin binding. "
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    ABSTRACT: Changes in cellular and synaptic plasticity related to learning and memory are accompanied by both upregulation and downregulation of the expression levels of proteins. Both de novo protein synthesis and post-translational modification of existing proteins have been proposed to support the induction and maintenance of memory underlying learning. However, little is known regarding the identity of proteins regulated by learning that are associated with the early stages supporting the formation of memory over time. In this study we have examined changes in protein abundance at two different times following one-trial in vitro conditioning of Hermissenda using two-dimensional difference gel electrophoresis (2D-DIGE), quantification of differences in protein abundance between conditioned and unpaired controls, and protein identification with tandem mass spectrometry. Significant regulation of protein abundance following one-trial in vitro conditioning was detected 30 min and 3 h post-conditioning. Proteins were identified that exhibited statistically significant increased or decreased abundance at both 30 min and 3 h post-conditioning. Proteins were also identified that exhibited a significant increase in abundance only at 30 min, or only at 3 h post-conditioning. A few proteins were identified that expressed a significant decrease in abundance detected at both 30 min and 3 h post-conditioning, or a significant decrease in abundance only at 3 h post-conditioning. The proteomic analysis indicates that proteins involved in diverse cellular functions such as translational regulation, cell signaling, cytoskeletal regulation, metabolic activity, and protein degradation contribute to the formation of memory produced by one-trial in vitro conditioning. These findings support the view that changes in protein abundance over time following one-trial in vitro conditioning involve dynamic and complex interactions of the proteome.
    Neuroscience 06/2011; 192:102-11. DOI:10.1016/j.neuroscience.2011.06.063 · 3.33 Impact Factor
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