Regulation of TMEM55A/B-dependent positioning of lysosomes

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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.