Periodic Reporting for period 3 - MOVE_ME (Mechanical and Electrical Guidance of Collective Cell Migration in vivo)
Período documentado: 2023-10-01 hasta 2025-03-31
The directional and synchronised movement of large groups of cells or cell clusters - also known as directed collective cell migration (dCCM) - is key during our embryonic development and when our wounds heal, but also in unfortunate scenarios such as when tumour cells undergo metastasis. Cell clusters migrate across complex environments composed of biochemical and biophysical cues. Despite this, the field has mostly focused on studying chemical guidance (chemotaxis) of dCCM - and the role of biophysical cues such as mechanical or electrical stimuli remains comparatively less understood. In this context, the mechanisms that guide dCCM in living tissues (in vivo) remain unclear. Thus, our overall goal is to complement the classic chemocentric view by addressing whether and how biophysical cues contribute to dCCM in vivo. To tackle this challenging aim, we study durotaxis (mechanical guidance) and electrotaxis (electrical guidance) at two levels: i) Tissue level, where we map mechanical and electrical properties in vivo and we test their relative contribution to dCCM; ii) Cellular level, here we explore the mechanisms by which cells sense, respond and integrate these biophysical cues. To address this, we take advantage of the innovative toolbox we developed to study mechanical and electrical cues in living tissues. As dCCM occurs in different biological contexts, we aim to generalise our results by studying dCCM of Xenopus neural crest cells, an embryonic cell population (WP1, WP2; Figure 01a); and the migration of the recently discovered Regeneration Organizing Cells (ROCs) during animal regeneration (WP3; Figure 01b). Demonstrating durotaxis and electrotaxis in vivo has proven to be a challenging goal. Thus, we expect our research to be a breakthrough across fields, bringing new perspectives and tools to study the biophysics of dCCM in vivo. This action is opening new research avenues for my lab and for others in the field, in which the interplay of biophysical and biochemical cues from the environment could be studied, paving the way to the formulation of a new and more integrative view of dCCM. Finally, revealing this information is important as we can shed lights on the mechanisms underlying embryonic defects, improve wound healing therapies and eventually understand more about how cancer cells move. In the long term this information can contribute to the design of predictive medical approaches and therapies.
During the reported period we spent time in developing tools and performing experiments for the proposed work packages (WPs).
WP1. To define the contribution of mechanical cues to neural crest dCCM in vivo. The work is advancing smoothly, we have published a work on the use of the mechanical tools required to measure and modify tissue mechanics in vivo and ex vivo (Moreira et al 2022, Meth Mol Biol). We have also written a book chapter in which we discuss roles of viscoelasticity in collective cell migration (Saraiva and Barriga, 2021; Espina and Barriga 2021) and a review article describing the mechanical control of directed cell migration or durotaxis (Espina et al 2021, FEBS). Indeed the work has advanced well and an article describing cell mechanical responses to substrate stiffening in terms of the Piezo1 control of microtubule stability has been published (Marchant et al 2022, Nature Materials highlighted in the cover). The idea is now start using the knowledge to address whether and how Piezo1 and microtubule stability control directed motion in response to anisotropic substrates via durotaxis.
WP2. To study the influence of electric fields during neural crest dCCM in vivo. We have successfully implemented vibrating probe and more recently microelectrode systems to measure the magnitude and vector of endogenous electric fields. We validated the function of these electric fields to guide collective cell migration by using microfabricated devices for cell and embryo electrotaxis assays. We also profiled the neural crest with RNA-seq experiments and identified that Vsp1 is a new electrosensor that allow neural crest cells to sense and respond to endogenous electric fields, while not being required for cell motility. All these results were delivered to the public as Ferreira et al 2021, BioRxiv and are now under revision at Nature.
WP3. To identify the mechanical and electrical nature of ROCs’ dCCM in vivo. We have developed tools for imaging ROCs in vivo and to culture them ex vivo. The mechanics of regeneration are being characterised this year and RNA-seq experiments from regenerating tissues in the presence and absence of mechanosensing are being analysed. This is a challenging goal and we expect to use these tools and screenings to design experiments allowing to elucidate the impact of cell mechanics in ROCs migration.
WP1. To define the contribution of mechanical cues to neural crest dCCM in vivo. The work is advancing smoothly, we have published a work on the use of the mechanical tools required to measure and modify tissue mechanics in vivo and ex vivo (Moreira et al 2022, Meth Mol Biol). We have also written a book chapter in which we discuss roles of viscoelasticity in collective cell migration (Saraiva and Barriga, 2021; Espina and Barriga 2021) and a review article describing the mechanical control of directed cell migration or durotaxis (Espina et al 2021, FEBS). Indeed the work has advanced well and an article describing cell mechanical responses to substrate stiffening in terms of the Piezo1 control of microtubule stability has been published (Marchant et al 2022, Nature Materials highlighted in the cover). The idea is now start using the knowledge to address whether and how Piezo1 and microtubule stability control directed motion in response to anisotropic substrates via durotaxis.
WP2. To study the influence of electric fields during neural crest dCCM in vivo. We have successfully implemented vibrating probe and more recently microelectrode systems to measure the magnitude and vector of endogenous electric fields. We validated the function of these electric fields to guide collective cell migration by using microfabricated devices for cell and embryo electrotaxis assays. We also profiled the neural crest with RNA-seq experiments and identified that Vsp1 is a new electrosensor that allow neural crest cells to sense and respond to endogenous electric fields, while not being required for cell motility. All these results were delivered to the public as Ferreira et al 2021, BioRxiv and are now under revision at Nature.
WP3. To identify the mechanical and electrical nature of ROCs’ dCCM in vivo. We have developed tools for imaging ROCs in vivo and to culture them ex vivo. The mechanics of regeneration are being characterised this year and RNA-seq experiments from regenerating tissues in the presence and absence of mechanosensing are being analysed. This is a challenging goal and we expect to use these tools and screenings to design experiments allowing to elucidate the impact of cell mechanics in ROCs migration.
Our work has definitely advanced the filed and brought it to a new level of understanding about how biophysical stimuli contribute to dCCM. What we have published in relation to WP1 lead the field to depart from the idea that cells require a given stiffness value to migrate, but instead they require balancing their forces with the substrate; this makes sense as embryos develop in variable environments, so relying in a ratio is more convenient for robust morphogenesis than a given value of stiffness. Indeed we found that as long as their forces are balanced, clusters can migrate in soft substrates which are normally conceived as non permissive for migration. We also determined that the in vivo substrate is not only permissive, but it is rather a source of mechanical information which instruct cells to modify their own mechanics to balance their forces, deform and move. This new information reshape the way we look at morphogenesis and provide hints on why some cells (such as cancer cells) can migrate in soft environments. Hence the breakthrough nature of these results. In relation to WP2, we have provided the first quantitative evidence of an electric field forming in the migratory path of a cell collective and validated its requirement for directed motion. This is relevant as this was only proposed in vitro so far. We also provided the first sensor that is not required for cell motility but only for directional response to electric fields in living tissues. To date there is no record of such a sensor. Finally we found an explanation for the formation of electric currents; this relies in the mechanical establishment of polarised ion channel activities in the ectoderm in a PCP-dependent manner. The relevance of this mechanism is related to the idea that biophysical stimuli emerge from tissue interplay to feedback into other tissues to instruct their development, in this case by modulating directed cell migration via electrotaxis.
WP3 is advancing at a good pace. ROCs line is working, we have identified few candidates via RNA seq and we are now testing them. We are also performing a mechanical profiling of the migratory path of these cells. Our atomic force device and vibrating probes suffered a flooding but this year they should be purchased back and we can go back to this WP and the others (please see challenges encountered). In the next period we expect to reveal the mechanomolecular nature of tail regeneration in terms of mechanical charaterisation of regenerating tissues, mechanosensitive and responsive pathways involved, etc.
The idea now is to explore downstream mechanisms of Vsp1, and to explore the role of other sensors found in the screening. The impact of electric fields in other surrounding tissues and their mutual impact with cell mechanics (how mechanics impacts the electric fields and viceversa) is also in the plans. This would generate data that we could use to start preparing new grants applications to explore the integration of mechanical and electrical control of dCCM.
WP3 is advancing at a good pace. ROCs line is working, we have identified few candidates via RNA seq and we are now testing them. We are also performing a mechanical profiling of the migratory path of these cells. Our atomic force device and vibrating probes suffered a flooding but this year they should be purchased back and we can go back to this WP and the others (please see challenges encountered). In the next period we expect to reveal the mechanomolecular nature of tail regeneration in terms of mechanical charaterisation of regenerating tissues, mechanosensitive and responsive pathways involved, etc.
The idea now is to explore downstream mechanisms of Vsp1, and to explore the role of other sensors found in the screening. The impact of electric fields in other surrounding tissues and their mutual impact with cell mechanics (how mechanics impacts the electric fields and viceversa) is also in the plans. This would generate data that we could use to start preparing new grants applications to explore the integration of mechanical and electrical control of dCCM.