Resumen de Dissection of cellular mechanics and molecular response to stress (análisis de la mecánica celular y la respuesta molecular al estrés)
The analysis of cellular responses to chemical signals has been studied in great detail, but the elements involved in the recognition of physical inputs, and the mechanisms transducing and implementing cell responses to these stimuli remain barely analyzed. Mechanical stress is a prominent stimulus sensed by cells that modulates most aspects of cellular behaviour, including growth, differentiation, migration, apoptosis and also many developmental and morphogenetic processes. The c-Jun¿N-terminal kinases (JNKs) and their related cousins, p38 kinases, become activated after exposure to inflammatory cytokines as well as to diverse stress inputs. JNKs phosphorylate the DNA binding protein Jun and increase its transcriptional activity inducing a wide range of cellular responses. These responses are modulated by negative feedback loops implemented by specific dual specificity phosphatases (DSPs).
We have used an in-house developed FRET sensor to monitor in real-time the activity of the JNK pathway in Drosophila S2R+ cells subjected to static mechanical stress. We observed that the basal level of JNK activity was extremely sensitive to the strength and type of cellular attachments and membrane elasticity/stiffness. Further, cells subjected to static mechanical stretch revealed a significant increase in dJun FRET biosensor activation, whose kinetics could be monitored live. Stretch also induced dramatic changes of cell morphology and actin and tubulin cytoskeleton dynamics. Remarkably, ¿¿integrins, but not their attachment to the actin cytoskeleton via Talin, were essential for stretch-mediated cellular activation of the JNK cascade. We note however, that in the absence of either ¿PS integrin or Talin, cytoskeleton dynamics and cell shape were still affected by stretch. The Talin-independent JNK response to the mechanical stimulation of integrins at focal adhesions is thus a major element, but not the only one, in the regulation of the cytoskeleton and cell shape remodeling associated to mechanical stretch.
We have also explored the JNK signaling regulatory machinery modulating the response to mechanical insults by performing single and double knockdowns or overexpression of individual regulators of the pathway. In this way, we discovered new functions for JNK cascade kinases and RhoGTPases and the existence of collateral interactions with other MAPK members like ERK and P38s in S2R+ cells at rest. Further, epistatic tests with combinations of RNAis revealed a change in the regulatory function of these elements from resting to stretch condition uncovering their plasticity. Mathematical simulations also allowed us to generate specific regulatory gene network models with predictive capabilities.
Finally, we aimed to study in real time the mechanics and dynamics of the activity of the JNK pathway during dorsal closure (DC) in Drosophila. Segmentation and 3D tracking of trajectories of individual nuclei highlighted that the entire process of closure was driven by pure deformation of the epithelial tissue devoid of intercalation or division. Further, the cells in the medio-lateral region of the dorsal epidermis exhibited stronger deformation indicating that as DC proceeds, this domain experiences the highest stretch and tension. To correlate the mechanics with the dynamics of the dJun-FRET biosensor, we expressed this with an En-Gal4 driver and thus only in posterior cells in each segment. Implementing spectral detection and confocal microscopy techniques in parallel, we were able to monitor FRET in vivo in Drosophila embryos during DC. Preliminary analyzed data determined a dynamic phosphorylation of dJun during the process and a rough correlation with the tensional maps traced by linear deformation analysis.
