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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - CIL2015 (Dissecting the cellular mechanics of contact inhibition of locomotion)

Teaser

Summary

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 the collision 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 even broader 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 cell 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 flies) 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 the cytoskeletal machinery inside cells. 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.

Work performed

While the goal of this project is 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 is 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 have taken 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 repulsion. We have now developed novel approaches to visualize and quantify actin dynamics in migrating macrophages using a number of novel image analysis tools and computational packages. For example, we have developed an image analysis software package that allows us to automatically track the speed and direction of the flowing actin network (attached image, panel A). We can then use this flow data to quantify various biomechanical aspects of the flow field. Quantification of the divergence within the flow field highlights sinks within the flowing network that are highly stable toward the cell body (attached image, panel B). This suggests that there are regions of the actin network that are under severe compression. Analyzing the streamlines of the flow highlight the overall direction of the flowing actin network which reveals a large confluence of streamlines and a streamline sink that is asymmetrically localized within the cell (attached image, panel C, D). These data have revealed that the flowing actin network is highly organized across the entire moving cell. Furthermore, there is a hidden asymmetry to the flowing actin network that was previously unappreciated, which is a critical driver of cell migration. For example, the orientation of the flow field is strongly correlated with the cellâ€™s instantaneous direction of motion. We are now trying to understand what controls this flow organization by analysing a variety of mutations in genes that have been hypothesized to regulate actin dynamics. We are also extrapolating these techniques to other cell types, which is revealing that this actin flow organization is a common feature of most if not all migrating cells. This work was recently presented in an oral presentation at a prestigious scientific conference and is currently in revision in a scientific journal.

An additional achievement that I would like to highlight is related to Aim 3. We have begun to examine contact inhibition behaviours in other cell types and extrapolate our knowledge gained from understanding the regulation of cell repulsion in Drosophila macrophages to other systems. We have developed an approach to easily screen for CIL behaviours in cultured mammalian cells. We can place two different cell types in culture on either side of a removable barrier, and upon removal of the barrier the cells will begin moving and collide. Using this system, we screened the behaviours of a variety of cancer cells (melanoma and fibrosarcoma cells) during collisions with an epithelial cell population. In previous work, it was shown that cancer cells would often lose their contact inhibition behaviour, which was hypothesized to help their invasive capacity. Indeed, we discovered that a melanoma cell line would invade the epithelial monolayer upon collision rather than simply cease motion suggesting that a loss of CIL may indeed be related to their ability to invade. In contrast, we discovered that fibrosarcoma cells showed a robust repulsion response showing that not all cancer cells lose their capacity for CIL. We suspect that the repulsion of fibrosarcoma cells from an epithelium may force their migration into regions of tissues that are more beneficial for their growth, and therefore cell repulsion as a result of CIL may also be a hal

Final results

The computational tools that we have developed to visualize and quantify actin flow dynamics are novel analytical techniques to understand this critical biological phenomenon. The image tracking tools and mathematical approaches that we are using were developed in collaboration with a materials scientist at the University College London. Quantitative measures like divergence and streamlines are normally used by engineers to understand fluid dynamics. We believe that this interdisciplinary interaction has led to a cross fertilization of techniques that is allowing us to better understand actin dynamics and cell migration. All software packages developed in this project will be deposited in a software repository so that other cell biologists can access these tools for their models of interest.

Our immediate goal is to publish the work highlighted in the previous section. We are also now analysing a variety of genetic mutations in order to screen for regulators of actin motion. In the longer term we hope to extend these novel computational tools to the process of contact inhibition of locomotion, which will allow us to better characterize the changes in actin flow during cell collision and repulsion.