Abstract

Mechanical force is known to modulate the activity of the Jun N-terminal kinase (JNK) signaling cascade. However, the effect of mechanical stresses on JNK signaling activation has previously only been analyzed by in vitro detection methods. It still remains unknown how living cells activate the JNK signaling cascade in response to mechanical stress and what its functions are in stretched cells.We assessed in real-time the activity of the JNK pathway in Drosophila cells by Fluorescence Lifetime Imaging Microscopy (FLIM), using an intramolecular phosphorylation-dependent dJun-FRET (Fluorescence Resonance Energy Transfer) biosensor. We found that quantitative FRET-FLIM analysis and confocal microscopy revealed sustained dJun-FRET biosensor activation and stable morphology changes in response to mechanical stretch for Drosophila S2R+ cells. Further, these cells plated on different substrates showed distinct levels of JNK activity that associate with differences in cell morphology, integrin expression and focal adhesion organization.These data imply that alterations in the cytoskeleton and matrix attachments may act as regulators of JNK signaling, and that JNK activity might feed back to modulate the cytoskeleton and cell adhesion. We found that this dynamic system is highly plastic; at rest, integrins at focal adhesions and talin are key factors suppressing JNK activity, while multidirectional static stretch leads to integrin-dependent, and probably talin-independent, Jun sensor activation. Further, our data suggest that JNK activity has to coordinate with other signaling elements for the regulation of the cytoskeleton and cell shape remodeling associated with stretch.

Highlights

  • Cells, whether in isolation or in tissues, invariably face and respond to a wide variety of external stimuli

  • Treatment of dJun-FRET biosensor transfected S2R+ cells with L-JNKI1, a cell-permeable inhibitor of Jun N-terminal kinase (JNK) including the minimal 20 aminoacid inhibitory sequence of IB1, which is not fluorescent. 10 mM L-JNKI1 led to a robust increase of fluorescence lifetime (FL) to 2.5460.17 ns in 2 hours (Figure 1A and Figure S1)

  • FRET/Fluorescence Lifetime Imaging Microscopy (FLIM) is a robust method for quantification of JNK signaling activity

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Summary

Introduction

Whether in isolation or in tissues, invariably face and respond to a wide variety of external stimuli. While the analysis of cellular responses to chemical signals has been studied in great detail, the elements involved in the recognition of physical inputs, e.g. hypoxia, osmotic shock, ionizing radiation or mechanical stretching, and the mechanisms transducing and implementing cell responses to these stimuli remain barely analyzed. These responses include a variety of conserved adaptive behaviors such as wound healing, cell migration, extravasation, secretion and necrotic or apoptotic death [1]. In some cases, organs and tissues adapt their morphologies and functions in response to acute or chronic mechanical stress [5], e.g. pressure overload causes cardiovascular hypertrophy, and muscle disuse results in atrophy

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