Cellular Sorting Visualized
Recycling or Degradation: a New Assay for Cellular Sorting
- Fig. 1: Trafficking of transferrin and LDL in early endosomes. Upon internalization both molecules are transported to the early endosome (EE) from where they are differentially sorted: transferrin is recycled back to the plasma membrane (PM) while LDL is targeted to the late endosome (LE) and lysosome (Lys) for degradation.
- Fig. 2: The cell-free sorting assay. Isolated endosomes double labeled with fluorescent transferrin (green) and LDL (red) are incubated in the presence of cytosol and ATP at 37°C. This leads to the sorting and separation of the two labels in vitro, which can be determined by fluorescence microscopy.
- Fig. 3: Imaging of endosomes. The amount of endosomes double labeled with transferrin (green) and LDL (red) (see arrows) decreases in the presence of ATP. Minus ATP represents the negative control and plus ATP the positive control. Size bar = 2 µm.
- Fig. 4: Requirements for endosome sorting. Docking and fusion factors, such as the PI(3)-kinase, Rab proteins, EEA1 and NSF, but not SNARE proteins, are required for cargo sorting within the early endosome
- From left to right: Dr. Silvio Rizzoli, group leader at the European Neuroscience Institute, Göttingen; Prof. Reinhard Jahn, director at the Max Planck Institute for Biophysical Chemistry Göttingen. In front: Sina-Victoria Barysch, PhD student at the Max Planck Institute for Biophysical Chemistry, Göttingen.
The early endosomes are cellular sorting stations which receive material from the plasma membrane by fusing with cargo-loaded incoming vesicles. They later sort these cargoes towards different cellular destinations. We have visualized this sorting process using a novel approach which is based on fluorescence microscopy . Surprisingly, we found that fusion and sorting, which were thought to function via completely different machineries, are connected on a molecular level.
The Role of Endosomes in Cellular Sorting
Early endosomes represent major sorting platforms in eukaryotic cells. They constitute the first compartment of the endocytic pathway to which recently internalized material is delivered. This material is then sorted and packed into budding vesicles which are targeted to different intracellular destinations. These outgoing trafficking pathways include direct recycling of receptors to the plasma membrane, delivery of vesicles to the Golgi apparatus, and maturation of early endosomes into late endosomes (which then fuse with lysosomes, the degradative compartment in the cell).
All incoming vesicular carriers join the endosomal compartment by membrane fusion. Conversely, all outgoing trafficking pathways involve sorting and the formation of carrier vesicles that bud from the early endosomes (with the exception of traffic to late endosomes, which only involves organelle maturation). Thus, the elementary steps underlying endosome function are fusion with incoming vesicles, cargo sorting and budding of new vesicles.
Early endosomal trafficking and function have been studied for decades by using two cargoes: transferrin and the low density lipoprotein (LDL). Transferrin is a protein that is responsible for transporting iron into the cells: upon binding to iron, it binds to its receptor, it is taken up by the cell and is transported to the early endosome. There, the iron is released, and transferrin, together with its receptor, is recycled back to the plasma membrane for new rounds of iron uptake. LDL, a cholesterol-containing lipoprotein complex, is transported into the endosomes by a very similar mechanism. However, from the endosome it is then transported in the maturing endosome to the late endosome/lysosome, for its degradation and release of cholesterol .
Therefore, transferrin and LDL are differentially trafficked from the early endosome and need to be sorted and separated from the latter (fig. 1).
A New Assay to Investigate Cellular Sorting
Fusion of cargo-containing incoming vesicles with the early endosome has been investigated in great detail - but how are molecules sorted and separated again, once they are mixed in the endosome? Studying this is difficult because it requires the visualization of this highly heterogeneous organelle, which is only 200-250 nm in diameter. It therefore falls below the diffraction limit of conventional microscopy, which is one of the reasons why sorting-specific assays are scarce. We have now developed a novel microscopy-based cell free assay for sorting and budding from early endosomes, by taking advantage of their ability to segregate different cargoes into different carrier vesicles. We loaded the endosomes in living cells by incubating them with fluorescent transferrin and LDL. We then disrupted the cells, and collected the endosomes. We found that isolated endosomes can efficiently separate the two markers in vitro (fig. 2). In our assay we visualize single endosomes by fluorescence microscopy and identify those which contain both fluorescent molecules. The amount of double labeled organelles decreases with in vitro incubation due to sorting and cargo segregation, and serves as a convenient readout (fig. 3). We found that the process required physiological temperature, an energy source and cytosolic proteins .
Fusion and Sorting Are Coupled on the Molecular Level
On our way of identifying the nature of these cytosolic proteins we decided to look at several of the characterized endosomal docking and fusion factors. Fusion of endocytic vesicles with early endosomes has been studied intensely in recent years. It involves the sequential recruitment of protein complexes that orchestrate the initial contact between the membranes (tethering and docking), followed by fusion. Rab5 and its effectors (such as the class III phosphatidyl-inositol-(3)-kinase or the early endosomal autoantigen 1, EEA1), as well as the SNARE fusion machinery and the SNARE cofactor NSF, have been identified as key players in these processes [3, 4].
Surprisingly, inhibition of docking and fusion molecules such as the Rab proteins, EEA1 or phosphatidyl-inositol-(3)-kinase completely blocked the ability of the endsome to separate transferrin and LDL . This may indicate that a fusion step is required for the sorting process. However, this was not the case, as blocking the function of SNARE proteins (the molecules responsible for the final fusion step) did not affect sorting. Interestingly, however, we found that NSF, a fusion cofactor which keeps the SNAREs in a primed and ready-for-fusion state, but has no direct role in fusion, is required for endosomal cargo sorting .
Altogether, these data suggest that sorting/budding and docking/fusion are tightly connected on a molecular level, at least in early endosomes - a concept which is somewhat unexpected as the two pathways have opposing functions, one aiming to separate cargoes, and one to unite them (fig. 4).
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