High-Resolution Quantification of Focal Adhesion Spatiotemporal Dynamics in Living Cells

Berginski, Mathew E.; Vitriol, Eric A.; Hahn, Klaus M.; Gomez, Shawn M.

doi:10.1371/journal.pone.0022025

Cited by 153 publications

(156 citation statements)

References 30 publications

(44 reference statements)

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“…To further investigate the role of paxillin in HGF-mediated motility, we monitored paxillin turnover in HeLa cells by total internal reflection fluorescence (TIRF) microscopy. When analysing focal adhesions containing GFP-tagged paxillin over time as described (Berginski et al, 2011), we saw an increase of focal adhesion disassembly rates upon HGF stimulation (Fig. 4C).…”

Section: Paxillin Is Required For Hgf-mediated Migrationmentioning

confidence: 81%

ERK2 but not ERK1 mediates HGF-induced motility in non small cell lung carcinoma cell lines

Radtke

Milanovic

Rossé

et al. 2013

Journal of Cell Science

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SummaryAberrant signalling of receptor tyrosine kinases (RTKs), such as c-Met, the receptor for hepatocyte growth factor (HGF), has been implicated in the oncogenesis of various tumours including non-small cell lung carcinoma (NSCLC). Through its pro-migratory properties, c-Met has been implicated specifically in the process of tumour metastasis, demanding a better understanding of the underlying signalling pathways. Various players downstream of c-Met have been well characterised, including the extracellular-signalregulated kinases (ERKs) 1 and 2. In a small interfering RNA (siRNA)-based high-throughput wound healing screen performed in A549 lung carcinoma cells, we identified ERK2 but not ERK1 as a strong mediator of HGF-induced motility. This finding was confirmed in several NSCLC cell lines as well as in HeLa cells. One known substrate for ERK kinases in cell migration, the focal adhesion protein paxillin, was also one of the hits identified in the screen. We demonstrate that HGF stimulation results in a time-dependent phosphorylation of paxillin on serine 126, a process that can be blocked by inhibition of the ERK1/2 upstream kinase mitogen-activated protein kinase/ERK kinase 1 (MEK1) or inhibition of glycogen synthase kinase 3 (GSK3). Further, we show that paxillin turnover at focal adhesions is increased upon stimulation by HGF, an effect that is dependent on serine residues 126 (GSK3 site) and 130 (ERK site) within paxillin. In line with the isoform-specific requirement of ERK2 for HGF-mediated migration in lung tumour cell models, ERK2 but not ERK1 is shown to be responsible for paxillin serine 126 phosphorylation and its increased turnover at focal adhesions.

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Section: Paxillin Is Required For Hgf-mediated Migrationmentioning

confidence: 81%

ERK2 but not ERK1 mediates HGF-induced motility in non small cell lung carcinoma cell lines

Radtke

Milanovic

Rossé

et al. 2013

Journal of Cell Science

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“…Cell areas were estimated by the cut-and-weigh method using images of cells stained by Alexa-Fluor 488 phalloidin. Focal adhesions were analyzed using the Focal Adhesion Analysis Server to quantify images of cells stained by immunofluorescence for vinculin (56,57).…”

Section: Methodsmentioning

confidence: 99%

Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts

Liu

Chang

Grinnell

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The most common genetic alterations for familial thoracic aortic aneurysms and dissections (TAAD) are missense mutations in vascular smooth muscle (SM) α-actin encoded by ACTA2. We focus here on ACTA2-R258C, a recurrent mutation associated with early onset of TAAD and occlusive moyamoya-like cerebrovascular disease. Recent biochemical results with SM α-actin-R258C predicted that this variant will compromise multiple actin-dependent functions in intact cells and tissues, but a model system to measure R258C-induced effects was lacking. We describe the development of an approach to interrogate functional consequences of actin mutations in affected patient-derived cells. Primary dermal fibroblasts from R258C patients exhibited increased proliferative capacity compared with controls, consistent with inhibition of growth suppression attributed to SM α-actin. Telomeraseimmortalized lines of control and R258C human dermal fibroblasts were established and SM α-actin expression induced with adenovirus encoding myocardin-related transcription factor A, a potent coactivator of ACTA2. Two-dimensional Western blotting confirmed induction of both wild-type and mutant SM α-actin in heterozygous ACTA2-R258C cells. Expression of mutant SM α-actin in heterozygous ACTA2-R258C fibroblasts abrogated the significant effects of SM α-actin induction on formation of stress fibers and focal adhesions, filamentous to soluble actin ratio, matrix contraction, and cell migration. These results demonstrate that R258C dominantly disrupts cytoskeletal functions attributed to SM α-actin in fibroblasts and are consistent with deficiencies in multiple cytoskeletal functions. Thus, cellular defects due to this ACTA2 mutation in both aortic smooth muscle cells and adventitial fibroblasts may contribute to development of TAAD and proliferative occlusive vascular disease.thoracic aortic aneurysms | ACTA2 mutation | vascular disease | cell migration | human fibroblasts

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“…6A,B). FA parameters were extracted using an automated image analysis platform (Berginski et al, 2011). Compared with the control cells, the Fmn2 siRNA-treated cells showed reduced FA size (P<0.0001), ) and phalloidin to mark the F-actin structures.…”

Section: Fmn2 Regulates Focal Adhesion Stability In Fibroblastsmentioning

confidence: 99%

Formin 2 regulates the stabilization of filopodial tip adhesions in growth cones and affects neuronal outgrowth and pathfinding in vivo

Sahasrabudhe¹,

Ghate²,

Mutalik³

et al. 2016

Journal of Cell Science

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Growth cone filopodia are actin-based mechanosensory structures that are essential for chemoreception and the generation of contractile forces necessary for directional motility. However, little is known about the influence of filopodial actin structures on substrate adhesion and filopodial contractility. Formin 2 (Fmn2) localizes along filopodial actin bundles and its depletion does not affect filopodia initiation or elongation. However, Fmn2 activity is required for filopodial tip adhesion maturation and the ability of filopodia to generate traction forces. Dysregulation of filopodia in Fmn2-depleted neurons leads to compromised growth cone motility. Additionally, in mouse fibroblasts, Fmn2 regulates ventral stress fiber assembly and affects the stability of focal adhesions. In the developing chick spinal cord, Fmn2 activity is required cellautonomously for the outgrowth and pathfinding of spinal commissural neurons. Our results reveal an unanticipated function for Fmn2 in neural development. Fmn2 regulates structurally diverse bundled actin structures, parallel filopodial bundles in growth cones and anti-parallel stress fibers in fibroblasts, in turn modulating the stability of substrate adhesions. We propose Fmn2 as a mediator of actin bundle integrity, enabling efficient force transmission to the adhesion sites.

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High-Resolution Quantification of Focal Adhesion Spatiotemporal Dynamics in Living Cells

Cited by 153 publications

References 30 publications

ERK2 but not ERK1 mediates HGF-induced motility in non small cell lung carcinoma cell lines

ERK2 but not ERK1 mediates HGF-induced motility in non small cell lung carcinoma cell lines

Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts

Formin 2 regulates the stabilization of filopodial tip adhesions in growth cones and affects neuronal outgrowth and pathfinding in vivo

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