Real-time monitoring of cell protrusion dynamics by impedance responses

Gagliardi, Paolo Armando; Blasio, Laura di; Chianale, Federica; Somale, Desiana; Seano, Giorgio; Bussolino, Federico; Primo, Luca

doi:10.1038/srep10206

Cited by 32 publications

(19 citation statements)

References 56 publications

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“…S7) and transfected into 293T cells for live cell imaging. The formation of filopodia and lamellipodia, and their dynamic activities, such as filopodial motility or retrograde transport of ligand-activated EGFR clusters, are characteristic of EGF-stimulated cells [29, 30]. Thus, the transport of fluorescent EGFR-GFP-V96 vs. EGFR-GFP-A96 was monitored following a temperature ramp over 20 min (Fig.…”

Section: Resultsmentioning

confidence: 99%

A new temperature-dependent strategy to modulate the epidermal growth factor receptor

Tyrpak

Park

et al. 2018

Biomaterials

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The dynamic manipulation of kinases remains a major obstacle to unraveling cell-signaling networks responsible for the activation of biological systems. For example, epidermal growth factor (EGF) stimulates the epidermal growth factor receptor (EGFR/ErbB1); however, EGF also recruits other kinases (HER2/ErbB2) involved with various signaling pathways. To better study EGFR we report a new strategy to selectively activate receptor tyrosine kinases fused to elastin-like polypeptides (ELPs), which can be visualized inside mammalian cells using fixed and live-cell fluorescence microscopy. ELPs are high molecular weight polypeptides that phase separate abruptly upon heating. When an EGFR-ELP fusion is heated, it clusters, initiates receptor internalization, phosphorylates, initiates downstream kinase signaling, and undergoes retrograde transport towards the cell body. Unlike other strategies to block EGFR (small molecule inhibitors, RNAi, or transcriptional regulators), EGFR-ELP clustering can be specifically switched on or off within minutes. Live-cell imaging suggests that EGFR-ELPs assemble in most cells with only a 3 °C increase in temperature. This strategy was found reversible and able to dynamically control the downstream phosphorylation/activation of the ERK1/2 pathway. For the first time, this strategy enables the rational engineering of specific temperature-sensitive receptors that may have broad applications in the study and manipulation of biological processes.

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Section: Resultsmentioning

confidence: 99%

A new temperature-dependent strategy to modulate the epidermal growth factor receptor

Tyrpak

Park

et al. 2018

Biomaterials

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“… 27 In this study, we assessed whether SYK mediates actin leading edge protrusion using Total Internal Reflection Fluorescence (TIRF) microscopy to image ovarian cancer cells transfected with LifeAct-RFP as a biosensor for F-actin. 28 , 29 This technique allows visualization of cytoskeletal dynamics on the basal plasma membrane of the cell. We analyzed fluorescence images and calculated the protrusion dynamics in isogenic pairs of OVISE SYK KO and SYK WT cells in the presence of the motility stimulant, EGF ( Supplementary video 1–3 ).…”

Section: Resultsmentioning

confidence: 99%

Inhibition of ovarian tumor cell invasiveness by targeting SYK in the tyrosine kinase signaling pathway

Rahmanto

Lee

et al. 2018

Oncogene

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Ovarian cancer cell motility and invasiveness are prerequisites for dissemination, and largely account for cancer mortality. We have identified an actionable kinase, spleen tyrosine kinase (SYK), which is keenly associated with tumor progression in ovarian cancer. Here, we report that active recombinant SYK directly phosphorylates cortactin and cofilin, which are critically involved in assembly and dynamics of actin filament through phosphorylation signaling. Enhancing SYK activity by inducing expression of a constitutively active SYK mutant, SYK130E, increased growth factor-stimulated migration and invasion of ovarian cancer cells, which was abrogated by cortactin knockdown. Similarly, SYK inhibitors significantly decreased invasion of ovarian cancer cells through basement membrane matrix in a real-time transwell assays and in a 3-D tumor spheroid model. SYK inactivation by gene knockout or by small molecule inhibition reduced actin polymerization. Collectively, the results reported here identify a new mechanism by which SYK signaling regulates ovarian cancer cell motility and invasiveness, and pinpoint a target-based strategy to prevent or suppress the advancement of ovarian malignancies.

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“…In this way, actin binding protein mediated-actin dynamics regulate cellular homeostasis and maintain cytoskeletal structure (17, 21, 22, 24–31). During metastasis, actin cytoskeletal remodeling at the leading edges of cancer cells facilitate invasive organelle (lamelipodia, filopodia and invadopodia) formation and matrix degradation (15, 32–34). …”

Section: Introductionmentioning

confidence: 99%

Alterative Expression and Localization of Profilin 1/VASPpS157 and Cofilin 1/VASPpS239 Regulates Metastatic Growth and Is Modified by DHA Supplementation

Ali

Heyob

Jacob

et al. 2016

Molecular Cancer Therapeutics

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Profilin 1, cofilin 1, and vasodialator stimulated phosphoprotein (VASP) are actin binding proteins (ABP) which regulate actin remodelling and facilitate cancer cell metastases. MiR~17–92 is highly expressed in metastatic tumors and profilin1 and cofilin1 are predicted targets. Docosahexaenoic acid (DHA) inhibits cancer cell proliferation and adhesion. These studies tested the hypothesis that the metastatic phenotype is driven by changes in ABPs including alternative phosphorylation and/or changes in subcellular localization. Additionally, we tested the efficacy of DHA supplementation to attenuate or inhibit these changes. Human lung cancer tissue sections were analyzed for F-actin content and expression and cellular localization of profilin1, cofilin1 and VASP (S157 or S239 phosphorylation). The metastatic phenotype was investigated in A549 and MLE12 cells lines using 8 Br-cAMP as a metastasis inducer and DHA as a therapeutic agent. Migration was assessed by wound assay and expression measured by western blot and confocal analysis. MiR~17–92 expression was measured by qRT-PCR. Results indicated increased expression and altered cellular distribution of profilin1/VASPpS157 but no changes in cofilin1/VASPpS239 in the human malignant tissues compared to normal tissues. In A549 and MLE12 cells, the expression patterns of profilin1/VASPpS157 or cofilin1/VASPpS239 suggested an interaction in regulation of actin dynamics. Furthermore, DHA inhibited cancer cell migration and viability, ABP expression and cellular localization, and modulated expression of miR~17–92 in A549 cells with minimal effects in MLE12 cells. Further investigations are warranted to understand ABP interactions, changes in cellular localization, regulation by miR~17–92, and DHA as a novel therapeutic.

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Real-time monitoring of cell protrusion dynamics by impedance responses

Cited by 32 publications

References 56 publications

A new temperature-dependent strategy to modulate the epidermal growth factor receptor

A new temperature-dependent strategy to modulate the epidermal growth factor receptor

Inhibition of ovarian tumor cell invasiveness by targeting SYK in the tyrosine kinase signaling pathway

Alterative Expression and Localization of Profilin 1/VASPpS157 and Cofilin 1/VASPpS239 Regulates Metastatic Growth and Is Modified by DHA Supplementation

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