Regulation of TMEM55A/B-dependent positioning of lysosomes

溶酶体是高度动态的细胞器。它们在细胞内的时空分布依赖于主要由运动蛋白和动力蛋白等微管马达驱动的主动转运，而马达蛋白与货物的相互作用是由存在于货物小泡上的复杂的膜蛋白系统介导的。TMEM55B是一种溶酶体双跨膜蛋白，具有较大的胞浆结构域，是一种介导运动-蛋白相互作用从而调节溶酶体定位的蛋白质。我们通过分析依赖于支架蛋白JIP4的Tme55b基因敲除小鼠，找出了TMEM55B在神经元中正确定位溶酶体的关键作用。Tmem55b基因敲除小鼠在轴突起始节段积聚溶酶体。虽然TMEM55B在动力蛋白驱动的逆行转运中发挥了作用，但我们认为它在神经元的顺行转运中发挥了重要作用。我们计划在基础条件下和在迫使顺行和逆行运输的情况下，以及新开发的线虫基因敲除线虫的神经元中，研究TMEM55B在神经元中的定向运输，以清楚地破译TMEM55B在溶酶体定位中的功能。TMEM55B有一个平行序列(TMEM55A)，两者可能部分互补。我们计划验证以前通过免疫共沉淀和质谱学鉴定的TMEM55B和TMEM55A相互作用蛋白，并确定它们对TMEM55A和TMEM55B的细胞内分选和溶酶体定位的贡献。最后，我们计划研究我们先前确定的TMEM55B的不同翻译后修饰相互之间的依赖性，以及对TMEM55B的细胞转运和溶酶体定位的依赖性。综上所述，我们的实验将提供TMEM55B对溶酶体定位的详细了解，重点是神经元运输和可能的其他不依赖于定位的体外和体内功能。

Lysosomes are highly dynamic organelles. Their spatio-temporal distribution within a cell depends on active transport mainly driven by microtubule motors such as kinesins and dynein and the interaction of motor proteins with cargoes is mediated by a sophisticated system of membrane-proteins present on cargo vesicles. TMEM55B, a lysosomal double-spanning transmembrane protein with a large cytosolic domain, is such a protein that mediates motor-protein interaction and thereby regulates lysosomal positioning. We figured out an essential role of TMEM55B for proper positioning of lysosomes in neurons by analyzing Tmem55b knockout mice that depends on the scaffold protein JIP4. Tmem55b knockout mice accumulate lysosomes in the axon initial segment. While a functional role of TMEM55B in the dynein-driven retrograde transport was suggested previously, we propose an important role in the anterograde transport in neurons. We plan to examine the directional transport of lysosomes in neurons from Tmem55b knockout mice under basal conditions and upon forcing anterograde- and retrograde transport, as well as in neurons in a newly developed Caenorhabditis elegans knockout worm to clearly decipher TMEM55B`s function in lysosomal positioning. TMEM55B has a paralogue (TMEM55A) and both might partially compensate for each other. We plan to validate TMEM55B and TMEM55A-interacting proteins, previously identified by co-immunoprecipitation and mass spectroscopy and determine their contribution on intracellular sorting of TMEM55A and TMEM55B and lysosomal positioning. Finally we plan to investigate the dependence of different posttranslational modifications of TMEM55B we determined previously on each other and on cellular trafficking of TMEM55B and lysosomal positioning. In summary, our experiments will provide a detailed understanding of TMEM55B on lysosomal positioning with an emphasis on neuronal transport and possible other, positioning independent, functions in vitro and in vivo.