Periodic Reporting for period 1 - PulsatERK (Intracellular ERK signalling dynamics mediated epidermal stem cell fate control)
Période du rapport: 2019-05-01 au 2021-04-30
The current understanding of cell biology suggests that cells receive cues or signals from their ‘microenvironment’ (cells and proteins in their immediate surroundings), which are transduced internally, resulting in a cellular response. This is referred to as the ‘cue-signal-response’ paradigm. Cells in different parts of an organism behave differently; for instance, cells in the eye behave differently from those in the liver. The cue-signal-response paradigm would suggest that given the cells in different organs reside is different microenvironments, they receive different signals which activate different pathways internally thereby generating different responses. However, recent data showed a curious observation in the cells that reside in the human skin. Skin cells that reside in the same microenvironment appear to activate the same signalling pathway (called ‘ERK’) – which is not too surprising. However, they can give rise to sharply divergent cellular responses. For instance, some cells maintain stemness, whereas some differentiate – and both these cell types appear to respond to the external cues via the ERK signalling pathway. This led to the question of how the cells in the skin could identify the ERK signal that was intended to guide the cells to differentiate from the one that was aimed to maintain stemness. Recent data has suggested that ERK signalling in skin cells manifests in a dynamic manner – i.e. it appears in pulses, and different skin cell subpopulations may have different signatures of these dynamics. Some pulses have high ERK levels, others have low levels. Some have a high frequency of pulses; the others have low frequencies of ERK pulses. The central hypothesis of the action ‘Intracellular ERK signalling dynamics mediated epidermal stem cell fate control’ was that skin cells were able to decipher and decode information directly from these ERK pulses to make decisions on whether they would maintain stemness or differentiate. This would allow them to utilize the same signalling pathway but make different cellular decisions.
A deeper understanding of the question mentioned above has long reaching consequences. Not only would it uncover complex aspects of cellular decision making which would dramatically improve our understanding of basic biology, but it would also allow scientists to develop strategies that enable fine tune control over cellular behaviour and improve the current technologies related to the field of regenerative medicine. These strategies could significantly improve the outlook for patients that struggle with debilitating medical conditions.
The objectives of the project were to disentangle the signalling profiles associated with the various subpopulations in the human skin and identify the profiles attributed to each subpopulation. To achieve this, the fellow needed to establish strategies to identify different cellular subtypes in living human skin cells. This required the transfer of a novel technology that the fellow had developed to micropattern adherent cells for use in human epidermal cell types. The final goal was to develop tools that would permit the testing of the central hypothesis for this action by exogenously controlling the ERK signalling dynamics in the skin cells and query if the cell states were able to be controlled by controlling the dynamic profiles of ERK signalling activity.
A deeper understanding of the question mentioned above has long reaching consequences. Not only would it uncover complex aspects of cellular decision making which would dramatically improve our understanding of basic biology, but it would also allow scientists to develop strategies that enable fine tune control over cellular behaviour and improve the current technologies related to the field of regenerative medicine. These strategies could significantly improve the outlook for patients that struggle with debilitating medical conditions.
The objectives of the project were to disentangle the signalling profiles associated with the various subpopulations in the human skin and identify the profiles attributed to each subpopulation. To achieve this, the fellow needed to establish strategies to identify different cellular subtypes in living human skin cells. This required the transfer of a novel technology that the fellow had developed to micropattern adherent cells for use in human epidermal cell types. The final goal was to develop tools that would permit the testing of the central hypothesis for this action by exogenously controlling the ERK signalling dynamics in the skin cells and query if the cell states were able to be controlled by controlling the dynamic profiles of ERK signalling activity.
The work required establishing strategies to identify different cellular subtypes in human skin cells in a manner that avoids having to kill the cells. While this can be done using fluorescent reporters, we needed the cells to have a fluorescent reporter of live ERK signalling activity and human skin cells are highly sensitive to being engineered to express multiple fluorescent reporters making the generation of a cell line that reported cell fates and ERK signalling incredibly challenging. The fellow achieved this is two different ways. First by taking advantage of the variable expression levels of cell adhesion proteins called integrins in the different cellular subpopulations. And second, by transferring a novel technology that the fellow had previously developed to permit patterning of adherent cells and employ it to generate a ‘micro-epidermis’ tissue model that reliably segregated the different cellular subpopulations along the geometry of the micropatterned colonies. As a next step, the fellow engineered skin cells to robustly report ERK signalling levels in living cells using a novel strategy called ‘Kinase Translocation Reporters’ (KTR) – which have multiple advantages than approaches that have been previously employed (like FRET). Furthermore, the fellow developed a highly robust and bespoke image analysis pipeline to be able to reliably extract the ERK signalling level readouts from individual cells from extended live imaging videos of the KTR reporter cells. Using the above two tools, the fellow was able to record live cell videos of the different cell compartments and using unsupervised strategies of clustering the different cellular subtypes in the skin cell population, the fellow was able to disentangle the ERK dynamics signatures associated with the different cellular cell sub-compartments.
During the disruption caused due to the Covid pandemic, the fellow trained in developing detailed mathematical models and programming using python and developed mathematical descriptions of complex biological behaviour which contributed to a manuscript that is currently under review.
During the disruption caused due to the Covid pandemic, the fellow trained in developing detailed mathematical models and programming using python and developed mathematical descriptions of complex biological behaviour which contributed to a manuscript that is currently under review.
The current state of the art to identify a specific cellular subtype in the human skin requires the cell to first be killed and then base the classification of the cell into one of the various categories via transcriptomic or proteomic analysis. This project was able to establish a method that permits the identification of different skin cell subtypes without the need to kill the cells by using cells that report the levels of ERK signalling, the project describes a technique to identify which cell type belongs to which cellular sub-compartment.
This project represents an important step towards a more rigorous and holistic understanding of the intracellular signalling dynamics and how they relate to the different cellular decisions that are made during tissue development and homeostasis. A deeper understanding of how cells make these decisions can allow scientists to engineer innovative solutions to resolve debilitating conditions for people all over the world.
In addition to undertaking the scientific tasks, the fellow founded and was the founding president of a group called the London Postdoc Network that developed multiple workshops to upskill postdoctoral researchers around the UK during the covid pandemic crisis. This included workshops where the attendees received tips from recent principal investigators on how to apply for an independent lab, tips from editors on how to improve the chances of having their research published, to non-academic workshops like how to spin out a start-up company based on your research. These workshops were all online, and free to attend and were very well attended. We routinely hosted about 300 attendees per event that we organized. This group not only served to provide our postdoctoral colleagues with critically important skills that would help them with their future, but also served as a way for them to congregate and network – which was incredibly valuable, especially during the pandemic.
This project represents an important step towards a more rigorous and holistic understanding of the intracellular signalling dynamics and how they relate to the different cellular decisions that are made during tissue development and homeostasis. A deeper understanding of how cells make these decisions can allow scientists to engineer innovative solutions to resolve debilitating conditions for people all over the world.
In addition to undertaking the scientific tasks, the fellow founded and was the founding president of a group called the London Postdoc Network that developed multiple workshops to upskill postdoctoral researchers around the UK during the covid pandemic crisis. This included workshops where the attendees received tips from recent principal investigators on how to apply for an independent lab, tips from editors on how to improve the chances of having their research published, to non-academic workshops like how to spin out a start-up company based on your research. These workshops were all online, and free to attend and were very well attended. We routinely hosted about 300 attendees per event that we organized. This group not only served to provide our postdoctoral colleagues with critically important skills that would help them with their future, but also served as a way for them to congregate and network – which was incredibly valuable, especially during the pandemic.