LAUSR

Significance In eukaryotic cells, membranous organelles exchange trafficking vesicles that bud from the precursor and are transported to the target membrane where they fuse. Many of the required proteins are known. Moreover, some steps such as budding and fusion have been reconstituted using artificial membranes and a minimalistic set of proteins. However, it is not known whether these vesicles are functional in the complex environment of a cell. Here, we have introduced such artificial vesicles into living cells and find that only few components are needed for function in membrane traffic, with all other factors recruited from the cytoplasm. Thus, eukaryotic cells have a remarkable capacity for self-organization that can be exploited for targeting tags to specific regions in the cell. There is still a large gap in our understanding between the functional complexity of cells and the reconstruction of partial cellular functions in vitro from purified or engineered parts. Here we have introduced artificial vesicles of defined composition into living cells to probe the capacity of the cellular cytoplasm in dealing with foreign material and to develop tools for the directed manipulation of cellular functions. Our data show that protein-free liposomes, after variable delay times, are captured by the Golgi apparatus that is reached either by random diffusion or, in the case of large unilamellar vesicles, by microtubule-dependent transport via a dynactin/dynein motor complex. However, insertion of early endosomal SNARE proteins suffices to convert liposomes into trafficking vesicles that dock and fuse with early endosomes, thus overriding the default pathway to the Golgi. Moreover, such liposomes can be directed to mitochondria expressing simple artificial affinity tags, which can also be employed to divert endogenous trafficking vesicles. In addition, fusion or subsequent acidification of liposomes can be monitored by incorporation of appropriate chemical sensors. This approach provides an opportunity for probing and manipulating cellular functions that cannot be addressed by conventional genetic approaches. We conclude that the cellular cytoplasm has a remarkable capacity for self-organization and that introduction of such macromolecular complexes may advance nanoengineering of eukaryotic cells.