Live Cell Imaging of Src/FAK Signaling by FRET

Lu, Shaoying; Wang, Yingxiao

doi:10.1007/s12195-011-0161-3

Cited by 16 publications

(17 citation statements)

References 57 publications

(90 reference statements)

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“…The Src and FAK family kinases were shown to regulate actin cytoskeleton reorganization (58,59); therefore, the importance of these molecules in R. montanensis invasion of tick cells was examined in this study. As shown in Fig.…”

Section: Fig 3 Arp2/3 Complex Is Important For Rickettsial Internalizmentioning

confidence: 99%

Identification of Host Proteins Involved in Rickettsial Invasion of Tick Cells

et al. 2015

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Tick-borne spotted fever group (SFG) Rickettsia species are obligate intracellular bacteria capable of infecting both vertebrate and invertebrate host cells, an essential process for subsequent bacterial survival in distinct hosts. The host cell signaling molecules involved in the uptake of Rickettsia into mammalian and Drosophila cells have been identified; however, invasion into tick cells is understudied. Considering the movement of SFG Rickettsia between vertebrate and invertebrate hosts, the hypothesis is that conserved mechanisms are utilized for host cell invasion. The current study employed biochemical inhibition assays to determine the tick proteins involved in Rickettsia montanensis infection of tick-derived cells from a natural host, Dermacentor variabilis. The results revealed several tick proteins important for rickettsial invasion, including actin filaments, actin-related protein 2/3 complex, phosphatidylinositol-3=-kinase, protein tyrosine kinases (PTKs), Src family PTK, focal adhesion kinase, Rho GTPase Rac1, and neural Wiskott-Aldrich syndrome protein. Delineating the molecular mechanisms of rickettsial infection is critical to a thorough understanding of rickettsial transmission in tick populations and the ecology of tick-borne rickettsial diseases.T ick-borne rickettsial diseases caused by pathogenic bacteria belonging to the genera Rickettsia, Anaplasma, and Ehrlichia are steadily increasing in the United States (1). Infection associated with these obligate intracellular bacteria ranges from mild, self-limiting to severe, including death (2). In the typical transmission cycle, spotted fever group (SFG) rickettsiae transmit vertically during the tick life cycle stages and between vectors and vertebrate hosts during tick acquisition of a blood meal. In vertebrate hosts, mechanisms of SFG Rickettsia infection, including invasion, escape from the phagosome, intracellular growth, intracellular movement, and cell-to-cell spread, have been studied (3). Tick acquisition of SFG Rickettsia from rickettsemic hosts is poorly described, and despite the essential role for tick hosts in maintenance and transmission of SFG Rickettsia to vertebrate hosts, the biology of the tick cell-SFG Rickettsia interaction is understudied.The obligate intracellular nature of Rickettsia requires bacteria to invade both vertebrate and invertebrate host cells, and this process, host cell invasion by SFG Rickettsia, has been studied in mammalian and Drosophila cell lines. Mammalian ligands Ku70 and ␣ 2 ␤ 1 integrin interact with rickettsial outer membrane proteins B and A (OmpB and OmpA), respectively, and are involved in Rickettsia conorii invasion of mammalian host cells (4, 5). After binding to rickettsial OmpB, R. conorii induces signaling cascades in which Ku70 is initially ubiquitinated by c-Cbl ubiquitin ligase. Subsequently, signaling molecules, including Cdc42, protein tyrosine kinases (PTKs), phosphatidylinositol-3=-kinase (PI3-kinase), Src family tyrosine kinases (Src), focal adhesion kinase (FAK), and cortactin, c...

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Section: Fig 3 Arp2/3 Complex Is Important For Rickettsial Internalizmentioning

confidence: 99%

Identification of Host Proteins Involved in Rickettsial Invasion of Tick Cells

et al. 2015

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“…The authors demonstrated that Src was activated upon integrin ligation using fibronectin‐coated beads and that this depended upon intact actin and microtubule cytoskeletons. This probe has subsequently been used in other studies to analyse activation of Src during cell adhesion and migration in a number of contexts (reviewed in Seong et al , ).…”

Section: Use Of Single‐chain Fret Biosensors To Study Signalling Durimentioning

confidence: 99%

Using FRET to analyse signals controlling cell adhesion and migration

Kemp-O'Brien

Parsons

2012

Journal of Microscopy

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Summary Cell adhesion and migration are dynamic, complex processes that require tight temporal control of signalling cascades at defined subcellular sites to occur. Decades of research have used biochemical methods to identify numerous signalling pathways that are involved in the coordination of adhesion, membrane protrusion and contractility that all contribute to migration. However, understanding the way in which these signals are controlled within discrete sites in individual cells undergoing migration is essential to enable a clearer understanding of how these pathways are integrated during motility. Recent advances in the generation of fluorescent protein variants have enabled the development of probes to analyse localized changes in protein conformation, activation or interactions in single cells using microscopy. Many of these probes are based on the use of fluorescence resonance energy transfer that permits precise determination of interacting species based on the emission or lifetime properties of the fluorophores involved. Here, we provide an overview of some of the recent studies that have developed such probes and examples of how these biosensors have been used to further our understanding of cell migration signalling.

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“…Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase consisting of a FERM domain (protein 4.1, ezrin, radixin and moesin homology), a kinase domain, and a FAT domain (targeting focal adhesion (Seong et al, 2011a). Phosphorylation of FAK at Tyr397 leads its binding to Src kinase, and then regulates many signaling molecules such as PI3K, Grb7 and Rho GTPases (Zhao et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

“…The activity of FAK is directly related to its subcellular localization (Seong et al, 2011a). It not only exists in the cytoplasm, but also targets focal adhesion through its carboxyl-terminal FAT region (Lee et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…For instance, disruption of the lipid caveolin protein prevents integrin-mediated 46 FEI XIE et al (Seong et al, 2011b). A fluorescence resonance energy transfer (FRET) study found that the degree of FAK activation in lipid rafts is higher than in non-rafts domain during cell adhesion and platelet-derived growth factor (PDGF) stimulation (Guan, 2004;Seong et al, 2011a;Seong et al, 2011b). The fluid shear stress can also regulate the localization and activity of FAK in endothelial cells, thereby regulating different processes (Li et al, 2002;Andersen et al, 2017;Lee et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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Membrane fluidity regulates high shear stress-induced FAK activation at different subcellular compartments

Xie¹,

Zhang²,

Xu³

et al. 2018

Biocell

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Focal adhesion kinase (FAK) plays a vital role in mediating the adaptability of tumor cells under mechanical stimuli. Previous studies revealed that FAK can locate to different cell compartments, and its regulation is highly dependent on its subcellular localization. However, the local FAK activities and its regulation mechanism in different cell compartments of tumor cells in response to fluid shear stress are still unclear. In this study, 5 dyn/cm 2 and 20 dyn/cm 2 of shear stress was applied to HeLa cells for 30 min. The activities of FAK targeting different subcellular compartments (lipids rafts, non-rafts, focal adhesions and cytoplasm) were investigated with fluorescence resonance energy transfer (FRET) technology. Results showed that the activity of FAK in response to high shear stress at focal adhesion sites was lower than that of other three areas, while no difference among four areas was observed in response to low shear stress. Furthermore, high shear stress-induced distinct FAK activation at different compartments was inhibited by decreasing membrane fluidity, but Src inhibition prevented high shear stress-induced FAK activation only in the cytoplasm. This study revealed the spatiotemporal characteristics of FAK under the different magnitude of shear stress, which provides a deeper understanding of mechanotransduction in tumor cells.

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Live Cell Imaging of Src/FAK Signaling by FRET

Cited by 16 publications

References 57 publications

Identification of Host Proteins Involved in Rickettsial Invasion of Tick Cells

Identification of Host Proteins Involved in Rickettsial Invasion of Tick Cells

Using FRET to analyse signals controlling cell adhesion and migration

Membrane fluidity regulates high shear stress-induced FAK activation at different subcellular compartments

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