We conclude that partitioning of endosomes and lysosomes is an ordered, yet imprecise, process, and that the organelle copy number is maintained by the daughter cells.

SummaryOrganelles in the endocytic pathway interact and communicate through the crucial mechanisms of fusion and fission. However, any specific link between fusion and fission has not yet been determined. To study the endosomal interactions with high spatial and temporal resolution, we enlarged the endosomes by two mechanistically different methods: by expression of the MHC-class-II-associated chaperone invariant chain (Ii; or CD74) or Rab5, both of which increased the fusion rate of early endosomes and resulted in enlarged endosomes. Fast homotypic fusions were studied, and immediately after the fusion a highly active and specific tubule formation and fission was observed. These explosive tubule formations following fusion seemed to be a direct effect of fusion. The tubule formations were dependent on microtubule interactions, and specifically controlled by Kif16b and dynein. Our results show that fusion of endosomes is a rapid process that destabilizes the membrane and instantly induces molecular-motor-driven tubule formation and fission.

Early endosomal antigen 1 (EEA1) is a cytosolic protein that specifically binds to early endosomal membranes where it has a crucial role in the tethering process leading to homotypic endosome fusion. Green fluorescent protein-tagged EEA1 (EEA1-GFP) was bound to the endosomal membrane throughout the cell cycle, and measurements using fluorescent recovery after photobleaching showed two fractions: one rapidly exchanging with the cytosolic pool, and the other with a long half-life. The exchange consists of a release and binding process, and we have separated these two by using GFP and photoactivable GFP. The release rate was identical to the exchange rate, showing that the dissociation characteristics determine the cycling of this molecule. During mitosis, we found that the dissociation rate was markedly accelerated and, in addition, the long-lived fraction was markedly reduced. This indicates that a fusion arrest in mitosis is not the result of EEA1 not binding to early endosomes, but rather due to the marked shift in membrane-binding characteristics. This might be a general mechanism to fine-tune and control tethering and fusion of early endosomes.

Vascular endothelial cells present luminal chemokines that arrest rolling leukocytes by activating integrins. It appears that several chemokines must form higher-order oligomers to elicit proper in vivo effects, as mutants restricted to forming dimers have lost the ability to recruit leukocytes to sites of inflammation. Here, we show for the first time that the chemokine RANTES/CCL5 binds to the surface of human endothelial cells in a regular filamentous pattern. Furthermore, the filaments bound to the surface in a heparan sulfate-dependent manner. By electron microscopy we observed labeling for RANTES on membrane projections as well as on the remaining plasma membrane. Mutant constructs of RANTES restricted either in binding to heparin, or in forming dimers or tetramers, appeared either in a granular, non-filamentous pattern or were not detectable on the cell surface. The RANTES filaments were also present after exposure to flow, suggesting that they can be present in vivo. Taken together with the lacking in vivo or in vitro effects of RANTES mutants, we suggest that the filamentous structures of RANTES may be of physiological importance in leukocyte recruitment.