Novel zebrafish approaches to investigate leaderless protein secretion in vivo

体内的所有细胞都将其蛋白质运输到细胞内的隔室或细胞表面。在细胞表面，它们可以保持附着在细胞上，或者它们被分泌，使得这些蛋白质可以在细胞外“看到”，并且远离细胞。绝大多数表面或分泌蛋白质都有一个特定的信号，引导它们自动运输到那里。最近发现，少数蛋白质在细胞外发挥作用，但被认为只有在细胞死亡时才被外化，实际上在某些情况下被分泌。一组分泌的蛋白质是炎性细胞因子（信使），在向血细胞发出损伤或感染位置的信号中至关重要，并且对于维持整个生命的健康免疫系统至关重要。我们现在知道，损伤感应“P2 X7”细胞表面受体的激活导致炎症介质（特别是“白细胞介素-1”）释放（分泌）到血流中。有趣的是，当白细胞介素-1离开细胞时，它被包裹在小的、受保护的膜包中，我们称之为“微泡”。这些包可能会阻止这种高度炎症性细胞因子在体内的随机位点起作用，并可能提供一种将其靶向微泡可以停靠的特定细胞的方法。 协调微泡形成和细胞因子分泌的细胞机制是复杂的。基于使用在培养物中生长的单一类型细胞的研究，已经提出了参与该过程的几种途径。我们已经开发了一个新的系统，我们可以研究荧光细胞因子在整个活的有机体，即透明的斑马鱼幼虫。这是我们第一次有机会观察分泌白细胞介素-1的细胞，并观察白细胞介素-1在活生物体中的靶向位置。在这个项目中，我们将优化计算方法来量化囊泡的形成，跟踪和靶向在整个生物体使用我们的荧光斑马鱼线。然后，我们将测试哪些细胞成分负责白细胞介素-1分泌，通过添加抑制剂到这些成分和测量响应感染的变化。我们还将使用这些荧光斑马鱼来筛选有效阻断炎症的小分子，以便我们可以识别健康免疫反应所需的细胞关键部分。在未来，除了这个项目，这将有助于我们开发新的疗法来对抗疾病。

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.