Novel zebrafish approaches to investigate leaderless protein secretion in vivo

点击翻译按钮获取中文摘要

All cells within the body traffic their proteins either to compartments within the cell, or to the cell surface. At the cell surface they can either remain attached to the cell or they are secreted so that these proteins can be "seen" outside the cell, and away from the cell. The vast majority of surface or secreted proteins have a specific signal directing them to automatically traffic there. Recently it has been found that a small number of proteins, which play a role outside the cell but were assumed to be externalised only upon cell death, are in fact secreted under certain circumstances. One group of secreted proteins are inflammatory cytokines (messengers), critical in signaling to blood cells the location of an injury or infection and so essential to maintain a healthy immune system throughout life. We now know that activation of the damage-sensing "P2X7" cell surface receptor causes release (secretion) of inflammatory mediators (specifically "interleukin-1) into the bloodstream. Interestingly, when interleukin-1 exits the cell, it is enveloped in small, protected membrane packets, that we have termed "microvesicles". These packets are likely to prevent this highly inflammatory cytokine from acting at random sites within the body, and may provide a means of targeting it to particular cells where the microvesicles can dock. The cellular machinery coordinating microvesicle formation and cytokine secretion is complex. Several pathways involved in this process have been proposed, based on studies using a single type of cell grown in culture. We have developed a new system where we can study fluorescent cytokine in a whole live organism, namely the transparent zebrafish larva. For the first time, this offers us the opportunity to view the cells that secrete interleukin-1, and to see where interleukin-1 is targeted in a live organism. In this project we will optimise computational approaches to quantify vesicle formation, tracking and targeting in a whole organism using our fluorescent zebrafish lines. We will then test which cellular components are responsible for interleukin-1 secretion by adding inhibitors to these components and measuring changes in response to an infection. We will also use these fluorescent zebrafish to screen for small molecules that are effective in blocking inflammation, so that we can identify the critical parts of the cell that are required to for a healthy immune response. In the future, beyond this project, this will help us to develop new therapies to fight disease.