Recycling from endosomes to the plasma membrane

Abstract

Summary V Chapter?Summary Many membrane proteins are, after endocytic uptake, efficiently recycled back to the plasma membrane. The aim of the studies presented in this thesis was to determine pathways and molecular mechanisms that are involved in recycling. Plasma membrane-derived clathrin-coated vesicles fuse, after uncoating, with sorting endo-somes. In this organelle recycling proteins are sorted from lysosomally directed proteins. Recycling is thought to occur either directly from sorting endosomes to the plasma membrane or via a second compartment that is formed in the perinuclear area and is composed of tubular recycling endosomes. The nature of the putative recycling vesicles that derive from these two endosome populations was unknown, but it has been suggested that endocytosed membrane constituents recycle by default. However, in a previous study we characterized a novel class of clathrin coated buds on recycling endosomes (Stoorvogel et al., 1996. J. Cell Biol. 132, 21-33), and proposed that clathrin-coated vesicles might be involved in the recycling pathway from sorting endosomes. In chapter 2 we now show that these endosome associated clathrin-coated buds contain dynamin-2, a GTPase that already had an established function in the fission of clathrin-coated vesicles at the plasma membrane. To study recycling processes, we monitored the trafficking of the transferrin receptor (TfR), a prototype recycling protein, in cells which overexpress a tem-perature- sensitive dynamin-1 mutant (dyn ts ). At the non-permissive temperature, ~30% of endocytosed transferrin (Tf) was retained in recycling endosomes by dyn ts cells. At these same conditions dynamin-labeled clathrin-coated buds accumulated on tubular TfR-containing endo-somes. In addition, recycling endosomes formed a more elaborate tubular network, suggesting that fission of TfR containing clathrin-coated vesicles from tubular endosomes requires func-tional dynamin. In contrast, exit from sorting endosomes was normal in these cells. From these results, it was concluded that TfR recycling from recycling endosomes to the plasma membrane is mediated by clathrin-coated vesicles and requires a functional dynamin, while exit from sort-ing endosomes is a dynamin-independent process. In chapter 3 we made use of the reversible phosphatidyl inositide (PI) 3-kinase inhibitor LY294002 to study the requirements for PI 3-kinase in TfR recycling. LY294002 did not inter-fere with the entry of TfR into sorting endosomes, nor with transport to, or release from recy-cling endosomes. However, egress of endocytosed Tf from sorting endosomes was significant-ly delayed. LY294002 and dyn ts had synergistic effects on Tf recycling kinetics, indicating that 98?they interfered with two distinct recycling pathways which can partly compensate for each oth-ers loss of function. The inhibitory effect of LY294002 on Tf recycling was reversed upon removal of the drug, indicating that its inhibitory effect was not due to delayed transport from sorting endosomes to recycling endosomes. In addition, the ultrastructure of recycling endo-somes was unaffected by this drug, as was the assembly of clathrin/dynamin coated buds on this compartment. In contrast, the diameter of sorting endosomes was significantly increased, indi-cating accumulation of membrane at this compartment. Together, these data indicate that endo-99 Summary SE RE plasma membrane 1 2 3 4 dynamin PI 3-kinase Ca 2+ Ca 2+ Ca 2+ ? Figure 1. Model for TfR recycling (1) TfR is endocytosed via clathrin-coated pits at the plasma membranes. 100-150 nm clathrin-coated vesicles pinch off from the plasma membrane and, after being uncoated, fuse with sorting endosomes (SE). This fusion may be Ca 2+ dependent. (2) TfR can recycle from sorting endosomes to the plasma membrane directly in a PI 3-kinase dependent manner. This transport is probably mediated by transport vesicles. Fusion of sorting endo-some- derived vesicles with the plasma membrane requires an influx of extracellular Ca 2+ . (3) Alternatively, TfR can be transported from sorting endosomes to recycling endosomes (RE). It is still unclear if this transport step is vesicle mediated, or whether tubular extensions detach from sorting endosomes and migrate to the perinuclear area where they can form the recycling compartment. (4) Transport from recycling endosomes to the plasma membrane is mediated by clathrin-coated vesicles that pinch off from recycling endosomes in a dynamin-dependent manner. The fusion of these vesicles with the plasma membrane is also dependent on an influx of extracellular Ca 2+ .?cytosed TfR recycles to the plasma membrane directly from sorting endosomes in a PI 3-kinase dependent manner and via recycling endosomes from where clathrin/dynamin coated vesicles are involved in further transport to the plasma membrane. In chapter 4 we show that Ca 2+ plays an important role in membrane fusion events along the endocytic pathway of the TfR. The membrane permeable Ca 2+ chelator BAPTA/AM interfered with the function of sorting endosomes, most likely with the fusion of plasma membrane derived endocytic vesicles with this compartment. Recycling of endocytosed Tf was severely affected, while there was an accumulation of 100-150 nm vesicles, with the morphological char-acteristics of plasma membrane derived vesicles. These vesicles were primarily found in aggre-gates or in association with sorting endosomes. Ca 2+ channel antagonists also severely inhibited Tf recycling, both from sorting endosomes and from recycling endosomes. Instead, we observed an accumulation of recycling vesicles underneath the plasma membrane, suggesting that fusion of transport vesicles with the plasmamembrane was affected. Taken together, these data suggest that local release of endosomal Ca 2+ may be required for the homotypic and het-erotypic fusion of endocytic vesicles and sorting endosomes, while an influx of extracellular Ca 2+ may be required for fusion of recycling vesicles with the plasma membrane. 100 Chapter 5