A kinase translocation reporter reveals real-time dynamics of ERK activity in Drosophila

Extracellular Signal-Regulated Kinase (ERK) lies downstream of a core signalling cascade that controls all aspects of development and adult homeostasis. Recent developments have led to new tools to image and manipulate the pathway. However, visualising ERK activity in vivo with high temporal resolution remains a challenge in Drosophila. We adapted a kinase translocation reporter (KTR) for use in Drosophila, which shuttles out of the nucleus when phosphorylated by ERK. We show that ERK-KTR faithfully reports endogenous ERK signalling activity in developing and adult tissues, and that it responds to genetic perturbations upstream of ERK. Using ERK-KTR in time-lapse imaging, we made two novel observations: firstly, sustained hyperactivation of ERK by expression of dominant-active Epidermal Growth Factor Receptor raised the overall level but did not alter the kinetics of ERK activity; secondly, heterogeneity in ERK activity in retinal basal glia correlated with the direction of migration of individual cells. Our results show that KTR technology can be applied in Drosophila to monitor ERK activity in real-time and suggest that this modular tool can be further adapted to study other kinases.

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Real-time analysis of T cell receptors in naive cells in vitro and in vivo reveals flexibility in synapse and signaling dynamics

Journal of Experimental Medicine ◽

10.1084/jem.20091201 ◽

2010 ◽

Vol 207 (12) ◽

pp. 2733-2749 ◽

Cited By ~ 66

Author(s):

Rachel S. Friedman ◽

Peter Beemiller ◽

Caitlin M. Sorensen ◽

Jordan Jacobelli ◽

Matthew F. Krummel

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Real Time ◽

T Cell Activation ◽

Valuable Insight ◽

Time Dynamics ◽

Insight Into

The real-time dynamics of the T cell receptor (TCR) reflect antigen detection and T cell signaling, providing valuable insight into the evolving events of the immune response. Despite considerable advances in studying TCR dynamics in simplified systems in vitro, live imaging of subcellular signaling complexes expressed at physiological densities in intact tissues has been challenging. In this study, we generated a transgenic mouse with a TCR fused to green fluorescent protein to provide insight into the early signaling events of the immune response. To enable imaging of TCR dynamics in naive T cells in the lymph node, we enhanced signal detection of the fluorescent TCR fusion protein and used volumetric masking with a second fluorophore to mark the T cells expressing the fluorescent TCR. These in vivo analyses and parallel experiments in vitro show minimal and transient incorporation of TCRs into a stable central supramolecular activating cluster (cSMAC) structure but strong evidence for rapid, antigen-dependent TCR internalization that was not contingent on T cell motility arrest or cSMAC formation. Short-lived antigen-independent TCR clustering was also occasionally observed. These in vivo observations demonstrate that varied TCR trafficking and cell arrest dynamics occur during early T cell activation.

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Differential Regulation of Tumor Angiogenesis by Distinct ErbB Homo- and Heterodimers

Molecular Biology of the Cell ◽

10.1091/mbc.e02-02-0084 ◽

2002 ◽

Vol 13 (11) ◽

pp. 4029-4044 ◽

Cited By ~ 94

Author(s):

Lily Yen ◽

Naciba Benlimame ◽

Zeng-Rong Nie ◽

Dingzhang Xiao ◽

Taiqi Wang ◽

...

Keyword(s):

Growth Factor ◽

Mutational Analysis ◽

Hypoxia Inducible Factor ◽

Growth Factor Receptor ◽

Vegf Expression ◽

Vegf Mrna ◽

Extracellular Signal ◽

Erbb Receptor Family

Interactions between cancer cells and their microenvironment are critical for the development and progression of solid tumors. This study is the first to examine the role of all members of the ErbB tyrosine kinase receptors (epidermal growth factor receptor [EGFR], ErbB-2, ErbB-3, or ErbB-4), expressed singly or as paired receptor combinations, in the regulation of angiogenesis both in vitro and in vivo. Comparison of all receptor combinations reveals that EGFR/ErbB-2 and ErbB-2/ErbB-3 heterodimers are the most potent inducers of vascular endothelial growth factor (VEGF) mRNA expression compared with EGFR/ErbB-3, EGFR/ErbB-4, ErbB-2/ErbB-4, and ErbB-3/ErbB-4. Immunohistochemistry of tumor xenografts overexpressing these heterodimers shows increased VEGF expression and remarkably enhanced vascularity. Enhanced VEGF expression is associated with increased VEGF transcription. Deletional analysis reveals that ErbB-mediated transcriptional up-regulation of VEGF involves a hypoxia-inducible factor 1-independent responsive region located between nucleotides −88 to −66 of the VEGF promoter. Mutational analysis reveals that the Sp-1 and AP-2 transcription factor binding elements within this region are required for up-regulation of VEGF by heregulin β1 and that this up-regulation is dependent on the activity of extracellular signal-related protein kinases. These results emphasize the biological implications of cell signaling diversity among members of the ErbB receptor family in regulation of the tumor microenvironment.

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A fuzzy-registration approach to track cell divisions in time-lapse fluorescence microscopy

10.1101/419572 ◽

2018 ◽

Author(s):

Saoirse Amarteifio ◽

Todd Fallesen ◽

Gunnar Pruessner ◽

Giovanni Sena

Keyword(s):

Fluorescence Microscopy ◽

Moving Objects ◽

Identity Management ◽

Complex Dynamics ◽

Time Lapse ◽

High Temporal Resolution ◽

Data Sets ◽

Management Problem ◽

3D Tracking

AbstractBackgroundParticle-tracking in 3D is an indispensable computational tool to extract critical information on dynamical processes from raw time-lapse imaging. This is particularly true with in vivo time-lapse fluorescence imaging in cell and developmental biology, where complex dynamics are observed at high temporal resolution. Common tracking algorithms used with time-lapse data in fluorescence microscopy typically assume a continuous signal where background, recognisable keypoints and independently moving objects of interest are permanently visible. Under these conditions, simple registration and identity management algorithms can track the objects of interest over time. In contrast, here we consider the case of transient signals and objects whose movements are constrained within a tissue, where standard algorithms fail to provide robust tracking.ResultsTo optimize 3D tracking in these conditions, we propose the merging of registration and tracking tasks into a fuzzy registration algorithm to solve the identity management problem. We describe the design and application of such an algorithm, illustrated in the domain of plant biology, and make it available as an open-source software implementation. The algorithm is tested on mitotic events in 4D data-sets obtained with light-sheet fluorescence microscopy on growing Arabidopsis thaliana roots expressing CYCB::GFP. We validate the method by comparing the algorithm performance against both surrogate data and manual tracking.ConclusionThis method fills a gap in existing tracking techniques, following mitotic events in challenging data-sets using transient fluorescent markers in unregistered images.

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In Vitro Study of Extracellular Vesicles Migration in Cartilage-Derived Osteoarthritis Samples Using Real-Time Quantitative Multimodal Nonlinear Optics Imaging

Pharmaceutics ◽

10.3390/pharmaceutics12080734 ◽

2020 ◽

Vol 12 (8) ◽

pp. 734

Author(s):

Leonardo Mortati ◽

Laura de Girolamo ◽

Carlotta Perucca Orfei ◽

Marco Viganò ◽

Marco Brayda-Bruno ◽

...

Keyword(s):

Real Time ◽

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Second Harmonic ◽

Time Lapse ◽

Cartilage Explants ◽

Innovative Technology

Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) are promising therapeutic nano-carriers for the treatment of osteoarthritis (OA). The assessment of their uptake in tissues is mandatory but, to date, available technology does not allow to track and quantify incorporation in real-time. To fill this knowledge gap, the present study was intended to develop an innovative technology to determine kinetics of fluorescent MSC-EV uptake by means of time-lapse quantitative microscopy techniques. Adipose-derived mesenchymal stromal cells (ASCs)-EVs were fluorescently labeled and tracked during their uptake into chondrocytes micromasses or cartilage explants, both derived from OA patients. Immunofluorescence and time-lapse coherent anti-Stokes Raman scattering, second harmonic generation and two-photon excited fluorescence were used to follow and quantify incorporation. EVs penetration appeared quickly after few minutes and reached 30–40 μm depth after 5 h in both explants and micromasses. In explants, uptake was slightly faster, with EVs signal overlapping both extracellular matrix and chondrocytes, whereas in micromasses a more homogenous diffusion was observed. The finding of this study demonstrates that this innovative technology is a powerful tool to monitor EVs migration in tissues characterized by a complex extracellular network, and to obtain data resembling in vivo conditions.

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Continuous detection of glucose and insulin in live animals

10.1101/2020.01.22.916106 ◽

2020 ◽

Author(s):

Mahla Poudineh ◽

Caitlin L. Maikawa ◽

Eric Yue Ma ◽

Jing Pan ◽

Dan Mamerow ◽

...

Keyword(s):

Real Time ◽

Temporal Resolution ◽

Simultaneous Detection ◽

Diabetic Rats ◽

High Temporal Resolution ◽

Pharmacokinetics And Pharmacodynamics ◽

Affinity Reagents ◽

Versatile Tool ◽

Multiple Analytes

AbstractReal-time biosensors that can continuously measure circulating biomolecules in vivo would provide valuable insights into a patients’ health status and their response to therapeutics even when there is considerable variability in pharmacokinetics and pharmacodynamics across patient populations. Unfortunately, current real-time biosensors are limited to a handful of analytes (e.g. glucose and blood oxygen) and are limited in sensitivity (high nanomolar). In this work, we describe a general approach for continuously and simultaneously measuring multiple analytes with picomolar sensitivity and sub-second temporal resolution. As exemplars, we report the simultaneous detection of glucose and insulin at picomolar concentrations in live diabetic rats. Using our system, we demonstrate the capacity to resolve inter-individual differences in the pharmacokinetic responses to insulin and discriminate profiles from different insulin formulations at a high temporal resolution. Critically, our approach is general and could be readily modified to continuously and simultaneously measure other circulating analytes in vivo by swapping the affinity reagents, thus making it a versatile tool for biomedical research.

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In vivo real-time dynamics of ATP and ROS production in axonal mitochondria show decoupling in mouse models of peripheral neuropathies

Acta Neuropathologica Communications ◽

10.1186/s40478-019-0740-4 ◽

2019 ◽

Vol 7 (1) ◽

Cited By ~ 10

Author(s):

Gerben van Hameren ◽

Graham Campbell ◽

Marie Deck ◽

Jade Berthelot ◽

Benoit Gautier ◽

...

Keyword(s):

Real Time ◽

Mouse Models ◽

Ros Production ◽

Peripheral Neuropathies ◽

Time Dynamics

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Extracellular signal-regulated kinase pathway is differentially involved in β-agonist-induced hypertrophy in slow and fast muscles

AJP Cell Physiology ◽

10.1152/ajpcell.00466.2006 ◽

2007 ◽

Vol 292 (5) ◽

pp. C1681-C1689 ◽

Cited By ~ 48

Author(s):

H. Shi ◽

C. Zeng ◽

A. Ricome ◽

K. M. Hannon ◽

A. L. Grant ◽

...

Keyword(s):

Fiber Type ◽

Erk Signaling ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal ◽

Slow Twitch ◽

Fast Twitch ◽

Erk Activity ◽

Fast Muscles ◽

Slow And Fast Muscles

The molecular mechanisms controlling β-adrenergic receptor agonist (BA)-induced skeletal muscle hypertrophy are not well known. We presently report that BA exerts a distinct muscle- and muscle fiber type-specific hypertrophy. Moreover, we have shown that pharmacologically or genetically attenuating extracellular signal-regulated kinase (ERK) signaling in muscle fibers resulted in decreases ( P < 0.05) in fast but not slow fiber type-specific reporter gene expressions in response to BA exposure in vitro and in vivo. Consistent with these data, forced expression of MAPK phosphatase 1, a nuclear protein that dephosphorylates ERK1/2, in fast-twitch skeletal muscle ablated ( P < 0.05) the hypertrophic effects of BA feeding (clenbuterol, 20 parts per million in water) in vivo. Further analysis has shown that BA-induced phosphorylation and activation of ERK occurred to a greater ( P < 0.05) extent in fast myofibers than in slow myofibers. Analysis of the basal level of ERK activity in slow and fast muscles revealed that ERK1/2 is activated to a greater extent in fast- than in slow-twitch muscles. These data indicate that ERK signaling is differentially involved in BA-induced hypertrophy in slow and fast skeletal muscles, suggesting that the increased abundance of phospho-ERK1/2 and ERK activity found in fast-twitch myofibers, compared with their slow-twitch counterparts, may account, at least in part, for the fiber type-specific hypertrophy induced by BA stimulation. These data suggest that fast myofibers are pivotal in the adaptation of muscle to environmental cues and that the mechanism underlying this change is partially mediated by the MAPK signaling cascade.

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Signal Pathways Which Promote Invasion and Metastasis: Critical and Distinct Contributions of Extracellular Signal-Regulated Kinase and Ral-Specific Guanine Exchange Factor Pathways

Molecular and Cellular Biology ◽

10.1128/mcb.21.17.5958-5969.2001 ◽

2001 ◽

Vol 21 (17) ◽

pp. 5958-5969 ◽

Cited By ~ 112

Author(s):

Yvona Ward ◽

Warner Wang ◽

Elisa Woodhouse ◽

Ilona Linnoila ◽

Lance Liotta ◽

...

Keyword(s):

Dominant Negative ◽

Signal Pathways ◽

Nucleotide Exchange ◽

3T3 Cells ◽

Invasion And Metastasis ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal ◽

Erk Activity

ABSTRACT Approximately 50% of metastatic tumors contain Ras mutations. Ras proteins can activate at least three downstream signaling cascades mediated by the Raf–MEK–extracellular signal-regulated kinase family, phosphatidylinositol-3 (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (RalGEFs). Here we investigated the contribution of RalGEF and ERK activation to the development of experimental metastasis in vivo and associated invasive properties in vitro. Each pathway contributes distinct properties to the metastatic phenotype. Following lateral tail vein injection, 3T3 cells transformed by constitutively active Raf or MEK produced lung metastasis that displayed circumscribed, noninfiltrating borders. In contrast, 3T3 cells transformed by Ras(12V,37G), a Ras effector mutant that activates RalGEF but not Raf or P13 kinase, formed aggressive, infiltrative metastasis. Dominant negative RalB inhibited Ras(12V,37G)-activated invasion and metastasis, demonstrating the necessity of the RalGEF pathway for a fully transformed phenotype. Moreover, 3T3 cells constitutively expressing a membrane-associated form of RalGEF (RalGDS-CAAX) formed invasive tumors as well, demonstrating that activation of a RalGEF pathway is sufficient to initiate the invasive phenotype. Despite the fact that Ras(12V,37G) expression does not elevate ERK activity, inhibition of this kinase by a conditionally expressed ERK phosphatase demonstrated that ERK activity was necessary for Ras(12V,37G)-transformed cells to express matrix-degrading activity in vitro and tissue invasiveness in vivo. Therefore, these experiments have revealed a hitherto-unknown but essential interaction of the RalGEF and ERK pathways to produce a malignant phenotype. The generality of the role of the RalGEF pathway in metastasis is supported by the finding that Ras(12V,37G) increased the invasiveness of epithelial cells as well as fibroblasts.

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Real-time dynamics of prostaglandin F2α release from uterus and corpus luteum during spontaneous luteolysis in the cow

Reproduction ◽

10.1530/rep.1.00183 ◽

2004 ◽

Vol 128 (2) ◽

pp. 189-195 ◽

Cited By ~ 24

Author(s):

Koumei Shirasuna ◽

Hitomi Asaoka ◽

Tomas J Acosta ◽

Missaka P B Wijayagunawardane ◽

Masayuki Ohtani ◽

...

Keyword(s):

Corpus Luteum ◽

Real Time ◽

Prostaglandin F2α ◽

Paracrine Factor ◽

Time Dynamics ◽

Physiological Relevance ◽

A Site ◽

Local Release ◽

Venous Plasma

Prostaglandin (PG) F2α released from the uterus in a pulsatile fashion is essential to induce regression of the corpus luteum (CL) in the cow. In addition to the uterus, the CL has also been recognized as a site of PGF2α production. Therefore, this study aimed to determine the detailed dynamics of the releasing profile of CL-derived PGF2α together with uterus-derived PGF2α during spontaneous luteolysis in the cow. Non-lactating Holstein cows (n = 6) were surgically implanted with a microdialysis system (MDS) on day 15 (oestrus = day 0) of the oestrous cycle. Simultaneously, catheters were implanted to collect ovarian venous plasma ipsilateral to the CL as well as jugular venous plasma. The concentrations of PGF2α, 13,14-dihydro-15-keto-PGF2α (PGFM) and progesterone in the MDS and plasma samples were determined by enzyme immunoassays. The intra-luteal PGF2α secretion slightly increased after the onset of luteolysis (0 h) and drastically increased from 24 h, and was maintained at high levels towards the following oestrus. Furthermore, PGF2α was released from the CL into the ovarian vein in a pulsatile manner during spontaneous luteolysis. Also, the fact that intra-luteal secretion of PGF2α and PGFM showed a positive correlation indicates the existence of a local metabolic pathway for PGF2α in the CL. In conclusion, the present study clarified the real-time dynamics of uterus-derived PGF2α and CL-derived PGF2α during spontaneous luteolysis in the cow, and gives the first in vivo evidence that the CL releases PGF2α during spontaneous luteolysis in the cow. Although the physiological relevance of CL-derived PGF2α appears to be restricted to a local role as an autocrine/paracrine factor in the CL, overall results support the concept that the local release of PGF2α within the regressing CL amplifies the luteolytic action of PGF2α from the uterus.

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Intercellular propagation of extracellular signal-regulated kinase activation revealed by in vivo imaging of mouse skin

eLife ◽

10.7554/elife.05178 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 94

Author(s):

Toru Hiratsuka ◽

Yoshihisa Fujita ◽

Honda Naoki ◽

Kazuhiro Aoki ◽

Yuji Kamioka ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Mouse Skin ◽

Egf Receptors ◽

M Cell ◽

Erk Activation ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal ◽

Erk Activity

Extracellular signal-regulated kinase (ERK) is a key effector of many growth signalling pathways. In this study, we visualise epidermal ERK activity in living mice using an ERK FRET biosensor. Under steady-state conditions, the epidermis occasionally revealed bursts of ERK activation patterns where ERK activity radially propagated from cell to cell. The frequency of this spatial propagation of radial ERK activity distribution (SPREAD) correlated with the rate of epidermal cell division. SPREADs and proliferation were stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) in a manner dependent on EGF receptors and their cognate ligands. At the wounded skin, ERK activation propagated as trigger wave in parallel to the wound edge, suggesting that ERK activation propagation can be superimposed. Furthermore, by visualising the cell cycle, we found that SPREADs were associated with G2/M cell cycle progression. Our results provide new insights into how cell proliferation and transient ERK activity are synchronised in a living tissue.

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