Periodic Reporting for period 4 - CIL2015 (Dissecting the cellular mechanics of contact inhibition of locomotion)
Reporting period: 2021-03-01 to 2022-08-31
The goal of this project is to investigate the mechanisms and functions of a process called contact inhibition of locomotion (CIL). CIL is a process that involves a cessation of cell motion or repulsion after contact between two cells. This has been a widely studied cell biological phenomenon for decades, yet we understand little about how it is regulated. Furthermore, we are only now beginning to understand how this cell behaviour may be functioning during normal biological processes and disease states. An additional aim of this project is to gain a better understanding of how cells move; cell migration is critical for animal development and numerous biological processes (e.g. inflammation, immunity, cancer) and we are also using contact inhibition of locomotion as a paradigm to understand how cells control their motion. A better understanding of how cells migrate is essential for controlling a variety of disease processes, from cancer metastasis to autoimmunity, and the migratory mechanisms highlighted by this work will therefore have broad reaching implications.
This project is divided into 3 main Aims. Aims 1 and 2 use Drosophila (fruit fly) embryonic macrophages (white blood cells of the fly) as a model system to understand cell migration and contact inhibition. Aim 3 involves extending our understanding of CIL to other model systems. The specific Aims are:
1) Dissect the cytoskeletal dynamics and biomechanics controlling CIL
Cell migration is driven by cytoskeletal machinery inside cells and actin is a polymer network that is the critical cytoskeletal driver of cell movement. This network flows inside cells in a process called retrograde flow, and it is this treadmill of actin (analogous to a tank tread) that generates the propulsive forces driving motion. While we know that this flow of actin drives cell movement, we have little understanding of how it is regulated both from a signalling or from a biomechanical perspective. In previous work we showed that CIL is controlled in part by the mechanics of the actin retrograde flow and this aim uses CIL as a paradigm to understand how actin flow is coordinated to generate cell motion.
2) Genetically Dissect the Signaling mechanisms modulating CIL
The goal of this Aim is to exploit our ability to easily screen for genetic regulation of cell behaviours in fruit flies to understand what signalling pathways control CIL in Drosophila macrophages.
3) Extend our knowledge of CIL to other cell types and physiological processes
The goal of this Aim is to begin extending our knowledge of CIL that we have gained from studying Drosophila macrophage migration to other cell types and model systems.
This project is divided into 3 main Aims. Aims 1 and 2 use Drosophila (fruit fly) embryonic macrophages (white blood cells of the fly) as a model system to understand cell migration and contact inhibition. Aim 3 involves extending our understanding of CIL to other model systems. The specific Aims are:
1) Dissect the cytoskeletal dynamics and biomechanics controlling CIL
Cell migration is driven by cytoskeletal machinery inside cells and actin is a polymer network that is the critical cytoskeletal driver of cell movement. This network flows inside cells in a process called retrograde flow, and it is this treadmill of actin (analogous to a tank tread) that generates the propulsive forces driving motion. While we know that this flow of actin drives cell movement, we have little understanding of how it is regulated both from a signalling or from a biomechanical perspective. In previous work we showed that CIL is controlled in part by the mechanics of the actin retrograde flow and this aim uses CIL as a paradigm to understand how actin flow is coordinated to generate cell motion.
2) Genetically Dissect the Signaling mechanisms modulating CIL
The goal of this Aim is to exploit our ability to easily screen for genetic regulation of cell behaviours in fruit flies to understand what signalling pathways control CIL in Drosophila macrophages.
3) Extend our knowledge of CIL to other cell types and physiological processes
The goal of this Aim is to begin extending our knowledge of CIL that we have gained from studying Drosophila macrophage migration to other cell types and model systems.
While the goal of this project was to understand the mechanisms controlling CIL, we realized that we needed a better understanding of the processes governing normal cell migration. For example, in Aim 1 our goal was to dissect how actin flows are involved in cell repulsion. However, we have little understanding of how these flows are controlled during normal migration, nor do we have good analytical approaches to quantify and describe these flows. We therefore took a step back and developed techniques to understand the biomechanics of actin flows during normal Drosophila macrophage migration with the goal of then extending these new techniques to CIL and cell motility in other cell types. We have now developed novel approaches to visualize and quantify actin dynamics in migrating macrophages using novel image analysis tools and computational packages. This software allowed us to automatically track the speed and direction of the flowing actin network in macrophages as well as mammalian cell types. The enabled us to precisely dissect the regulation of actin dynamics in cells during migration, which resulted in several publications. We have subsequently used these tools to highlight a number of novel regulators of actin flows in Drosophila macrophages, resulting in a manuscript that is currently in preparation.
We have also unveiled an unexpected physiological function of CIL in Drosophila macrophages. We revealed that macrophages are the primary producers of extracellular matrix (ECM) in developing embryos and that these cells need to rapidly and efficiently spread throughout the animal to evenly assemble the ECM. We subsequently revealed that the embryonic ECM is surprisingly dynamic during early development in terms of its self-assembly and its rapid turnover (the ECM was once thought to be incredibly long-lived and stable).
An additional achievement that I would like to highlight is related to extrapolating to model systems and physiological processes outside of flies, which has led to one published manuscript and a second in preparation. First, we developed an approach to easily screen for CIL behaviours in cultured mammalian cells. We discovered that melanoma cells invade through epithelial monolayers upon collision rather than simply ceasing motion, suggesting that loss of CIL may indeed be related to enhanced metastatic capacity. However, we discovered that cells of a second cancer type, fibrosarcoma, showed a robust repulsion response revealing that not all cancer cells lose CIL capacity as was originally assumed. In a second project we investigated the behaviour of fibrotically active fibroblasts (keloids). It is well known that fibrotic fibroblasts produce a highly aligned ECM, which is thought to alter the mechanical properties of the tissue. In a manuscript that is currently in preparation, we revealed that keloid dermal fibroblasts (KDF), unlike normal dermal cells (NDF), develop a highly aligned supracellular network in culture, which subsequently drives the alignment of the ECM (see attached image). We subsequently revealed that KDF cells show an enhanced CIL behaviour which we hypothesise is helping to drive or maintain their alignment. Additionally, we highlighted the cellular mechanisms that allow these fibrotic cells to align, which we hypothesise may allow for clinical intervention.
We have also unveiled an unexpected physiological function of CIL in Drosophila macrophages. We revealed that macrophages are the primary producers of extracellular matrix (ECM) in developing embryos and that these cells need to rapidly and efficiently spread throughout the animal to evenly assemble the ECM. We subsequently revealed that the embryonic ECM is surprisingly dynamic during early development in terms of its self-assembly and its rapid turnover (the ECM was once thought to be incredibly long-lived and stable).
An additional achievement that I would like to highlight is related to extrapolating to model systems and physiological processes outside of flies, which has led to one published manuscript and a second in preparation. First, we developed an approach to easily screen for CIL behaviours in cultured mammalian cells. We discovered that melanoma cells invade through epithelial monolayers upon collision rather than simply ceasing motion, suggesting that loss of CIL may indeed be related to enhanced metastatic capacity. However, we discovered that cells of a second cancer type, fibrosarcoma, showed a robust repulsion response revealing that not all cancer cells lose CIL capacity as was originally assumed. In a second project we investigated the behaviour of fibrotically active fibroblasts (keloids). It is well known that fibrotic fibroblasts produce a highly aligned ECM, which is thought to alter the mechanical properties of the tissue. In a manuscript that is currently in preparation, we revealed that keloid dermal fibroblasts (KDF), unlike normal dermal cells (NDF), develop a highly aligned supracellular network in culture, which subsequently drives the alignment of the ECM (see attached image). We subsequently revealed that KDF cells show an enhanced CIL behaviour which we hypothesise is helping to drive or maintain their alignment. Additionally, we highlighted the cellular mechanisms that allow these fibrotic cells to align, which we hypothesise may allow for clinical intervention.
We believe that the role of CIL in controlling ECM distribution and organisation is paradigm-changing, providing novel insight into mechanisms controlling animal development as well as human disease. With regards to expected results, we currently have a preprint in revision and are preparing a manuscript on keloid cell and ECM alignment.
Additionally, through this project we developed a number of computational algorithms to track cells, analyse actin dynamics, and quantify fibre organization in biological images, which we have made freely available through publication or through deposition into software repositories. These algorithms are broadly applicable to a wide range of biological disciplines, beyond the work in this proposal.
Additionally, through this project we developed a number of computational algorithms to track cells, analyse actin dynamics, and quantify fibre organization in biological images, which we have made freely available through publication or through deposition into software repositories. These algorithms are broadly applicable to a wide range of biological disciplines, beyond the work in this proposal.