Signalling and regulation of autophagy-related pathways during cellular differentiation of African trypanosomes.

长期以来，溶酶体一直被视为废物管理的细胞器。然而，在过去的20年里，这种观点发生了变化。溶酶体现在被认为是维持内稳态(一种由细胞维持稳定的内部、物理和化学条件以实现正常功能的状态)、细胞生存和分化的中央细胞器。自噬(源自希腊语“自动”自体和“吞噬”吞噬)是细胞将细胞内成分、大分子、细胞器或细胞内病原体定位到溶酶体以进行降解和循环的基本过程。自噬在细胞生长、分化或对诸如营养限制等应激因素的反应中是必不可少的。此外，自噬的缺陷或失去调节可能会导致癌症或神经退行性疾病的进展。在真核生物中，蛋白激酶和激活酶复合体产生蛋白质磷酸化。这些可逆的调节修饰对于控制自噬，从而促进细胞存活和分化是必不可少的。最近的研究已经确定了一种非常规的分泌途径，称为溶酶体胞吐作用，它依赖于自噬机制。溶酶体胞吐作用是溶酶体不能降解的蛋白质释放的基本机制，从而避免了对细胞的潜在毒性作用。此外，它还被认为是破坏宿主细胞膜以允许线虫入侵的原因。事实上，蠕虫将其溶酶体内容物直接释放到细胞外环境中，以穿透宿主细胞的膜，使其能够被感染宿主内的蠕虫入侵。研究蛋白激酶如何通过磷酸化事件调节自噬和溶酶体胞吐作用，对于了解昆虫传播的寄生虫如何入侵并在人类或动物宿主中生存具有重要意义。这些生物坚持在不断变化的环境中生存，这些环境需要微调和快速的适应才能生存。探索真核信号网络多样性的一个易于处理的模型是动质体寄生虫。动体寄生虫具有很大的临床相关性，影响着数百万患有利什曼病、恰加斯病和睡眠病等疾病的人和动物，导致严重的社会经济影响。动体对环境非常敏感，需要自噬来适应宿主转变过程中的剧烈变化。我建议使用动体寄生虫布鲁氏锥虫(非洲昏睡病和牲畜‘nagana’的病原体)来表征在生命周期分化过程中调节自噬和溶酶体胞吐的信号通路。在这个项目中，我将开发一种不偏不倚的方法，将高通量实时成像系统的优化、针对基因组中编码的所有蛋白激酶的基因沉默屏幕和质谱学相结合。这些结果将揭示布鲁氏毛滴虫自噬和溶酶体胞吐的磷酸化的分子组成和调控。然后将评估这些调节成分的功能，以确定它们在寄生虫生命周期中的分化作用。这一研究结果不仅将有助于更好地了解动体寄生虫如何适应不同寄主在其复杂的生命周期中遇到的不同寄主并在体内持续存在，而且将为以布鲁氏锥虫为模型研究自噬和胞吐途径的调节和串扰奠定基础。

For a long time, lysosomes have been seen as waste management organelles. Over the last two decades, however, this view has evolved. Lysosomes are now considered as central organelles for the maintenance of homeostasis (a state of steady internal, physical and chemical conditions maintained by the cell for proper functioning), cellular survival and differentiation. Autophagy (from greek "Auto" self and "phagos" eating) is an essential process by which cells target intracellular components, macromolecules, organelles or intracellular pathogens to the lysosome for degradation and recycling. Autophagy is essential during cell growth, differentiation, or in response to stressors such as nutrient limitation. Moreover, defects or loss of regulation of autophagy can contribute to cancer or to the progression of neurodegenerative diseases. In eukaryotes, protein kinases and kinase enzyme complexes create protein phosphorylations. These reversible regulatory modifications are essential to control autophagy and therefore promote cell survival and differentiation. Recent studies have identified an unconventional secretory pathway called lysosome exocytosis that is dependent on the autophagy machinery. Lysosome exocytosis is a fundamental mechanism for the release of proteins that lysosomes could not degrade, thus avoiding potential toxic effect for the cell. Additionally, it has been implicated in the breach of host cellular membrane to allow invasion by the worm C. elegans. Indeed, the worm releases its lysosome contents directly into the extracellular environment to perforate the membrane of the host cells, allowing their invasion by the worm within the infected host.Studying how protein kinases regulate autophagy and lysosome exocytosis by phosphorylation events is of major importance to understand how insect-transmitted parasites invade and survive in human or animal hosts. These organisms persist in changing environments that demand finely-tuned and rapid adaptation for survival. A tractable model to explore the diversity of eukaryotic signalling networks are kinetoplastid parasites. Kinetoplastid parasites are of great clinical relevance affecting millions of people and animals with diseases such as Leishmaniasis, Chagas disease and Sleeping Sickness, leading to critical social-economic implications. Kinetoplastid are exquisitely sensitive to their environment and require autophagy to adapt drastic changes during host transitions.I propose to use the kinetoplastid parasite Trypanosoma brucei (causative agent of African sleeping sickness and livestock 'nagana') to characterise signalling pathways that regulate autophagy and lysosome exocytosis during lifecycle differentiation. In this project, I will develop an unbiased approach that will combine the optimisation of a high-throughput live imaging system, a gene silencing screen targetting all protein kinases encoded in the genome and mass spectrometry. Results obtained will reveal the molecular composition and regulations by phosphorylation of autophagy and lysosome exocytosis in T. brucei. The function of these regulatory components will then be evaluated for their roles in differentiation during the parasite lifecycle. The results of this study will not only provide a better understanding of how kinetoplastid parasites adapt to the different hosts encountered during their complex lifecycles and persist in vivo, but will lay the foundations for the use of T. brucei as model to study the regulation of, and crosstalk between, the autophagy and exocytosis pathways.