Even though endocytic receptor sorting was first appreciated over two decades ago (e.g., Brown et al., 1983
; Farquhar, 1983
; Steinman et al., 1983
), our understanding of the principles of this process has been limited. A major reason for this has been the lack of direct assays to visualize signaling receptor sorting in the endosome. Here we directly visualized, in living cells, endosomal sorting between two prototypic members of the largest known family of signaling receptors for which sequence-specific recycling is critical for physiological regulation of cell signaling (Pippig et al., 1995
; Lefkowitz et al., 1998
; Xiang and Kobilka, 2003
). We resolve sorting at the level of single trafficking events on individual endosomes, and define a kinetic and affinity-based model for how sequence-dependent receptors are sorted away from bulk-recycling and degrading proteins.
By analyzing individual sorting and recycling events on single endosomes, we demonstrate a remarkable diversity in recycling pathways emanating from the same organelle (Scita and Di Fiore, 2010
). The traditional view has been that recycling to the plasma membrane is mediated by a uniform set of endosomal tubules from a single endosome. In contrast to this view, we demonstrate that the recycling pathway is highly specialized, and that specific cargo can segregate into specialized subsets of tubules that are biochemically, biophysically, and functionally distinct. Receptor recycling plays a critical role in controlling the rate of cellular re-sensitization to signals (Lefkowitz et al., 1998
; Sorkin and von Zastrow, 2009
), and recent data suggest that the sequence-dependent recycling of signaling receptors is selectively controlled by signaling pathways (Yudowski et al., 2009
). The physical separation between bulk and sequence-dependent recycling that we demonstrate here allows for such selective control without affecting the recycling of constitutively cycling nutrient receptors. Further, such physical separation might also reflect the differences in molecular requirements that have been observed between bulk and sequence-dependent recycling (Hanyaloglu and von Zastrow, 2007
Endosome-associated actin likely plays a dual role in endosomal sorting, both of which contribute to sequence-dependent entry of cargo selectively into special domains. First, by stabilizing the specialized endosomal tubules relative to the much more dynamic tubules that mediate bulk recycling, the local actin cytoskeleton could allow sequence-dependent cargo to overcome a kinetic barrier that limits their entry into the bulk pathway. Supporting this, we show that most endosomal tubules are highly transient, lasting less than a few seconds (), which allows enough time for entry of the fast-diffusing bulk recycling cargo, but not the slow-diffusing sequence-dependent cargo (), into these tubules. A subset of these tubules representing the sequence-dependent recycling pathway is stabilized by the presence of an actin cytoskeleton (). This stabilization allows time for B2AR to diffuse into these tubules (Fig S3
), which eventually pinch off membranes that can directly fuse with the plasma membrane (). Interestingly, inhibition of actin caused a decrease in the total number of tubules by approximately 25% (), suggesting that the actin cytoskeleton plays a role in maintaining the B2AR-containing subset of tubules, and not just in the sorting of B2AR into these tubules.
Second, a local actin cytoskeleton could provide the machinery for active concentration of recycling proteins like the B2AR, which interact with actin-associated sorting proteins (ERM and ERM-binding proteins) through C-terminal sequences (Weinman et al., 2006
; Wheeler et al., 2007
; Lauffer et al., 2009
, Fehon et al., 2010
), in specialized recycling tubules. Consistent with this, the C-terminal sequence on B2AR was both required and sufficient for sorting to the endosome and for recycling, and a distinct actin-binding sequence was sufficient for both receptor entry into tubules and recycling ( and Fig S4
). PDZ-interacting sequences have been identified on several signaling receptors, including multiple GPCRs, with different specificities for distinct PDZ-domain proteins (Weinman et al., 2006
). Further, actin-stabilized subsets of tubules were present even in the absence of B2AR in the endosome. We propose that, using a combination of kinetic and affinity-based sorting principles, discrete Actin-Stabilized SEquence-dependent Recycling Tubule (ASSERT) domains could thus mediate efficient sorting of sequence-dependent recycling cargo away from both degradation and bulk recycling pathways that diverge from the same endosomes.
Our results, therefore, uncover an additional role for actin polymerization in endocytic sorting, separate from its role in endosome motility. It will be interesting to investigate the mechanism and signals that control the nucleation of such a spatially localized actin cytoskeleton on the endosome. The lack of obvious concentration of the canonical Arp2/3 activators, WASP and WAVE, suggests a novel mode of actin nucleation involving cortactin. Cortactin can act as a nucleation-promoting factor for Arp2/3, at least in vitro
(Ammer and Weed, 2008
), and can interact with dynamin (Schafer et al., 2002
; McNiven et al., 2000
), which makes it an attractive candidate for coordinating actin dynamics on membranes. Interestingly, inhibition of WASH, a recently described Arp regulator that is present on B2AR tubules, has been reported to result in an increase in endosomal tubules (Derivery et al., 2009
). Although its role in sequence-dependent recycling remains to be tested, this suggests the presence of multiple actin-associated proteins with distinct functions on the endosome.
The simple kinetic and affinity-based principle that we propose likely provides a physical basis for sequence-dependent sorting of internalized membrane proteins between essentially opposite fates in distinct endosomal domains. Proteins that bind sequence-dependent degrading receptors and are required for their degradation (Whistler et al., 2002
; Marley and von Zastrow, 2010
) might act as scaffolds and provide a similar kinetic barrier to prevent them from accessing the rapid bulk-recycling tubules. Entry of these receptors into the involution pathway might then be accelerated by their association with the well-characterized ESCRT-associated domains on the vacuolar portion of endosomes (Hurley, 2008
; Saksena et al., 2007
; Williams and Urbe, 2007
), complementary to the presently identified ASSERT domains on a subset of endosomal tubules.
Such diversity at the level of individual trafficking events to the same destination from the same organelle raises the possibility that there exists yet further specialization amongst the pathways that mediate exit out of the endosome, including in the degradative pathway and the retromer-based pathway to the trans-Golgi network. Importantly, the physical separation in pathways that we report here potentially allows for cargo-mediated regulation as a mode for controlling receptor recycling to the plasma membrane. Such a mechanism can provide virtually an unlimited level of selectivity in the post-endocytic system using minimal core trafficking machineries, as has been observed for endocytosis at the cell surface (Puthenveedu and von Zastrow, 2006
). As the principles of such sorting depend critically on kinetics, the high-resolution imaging used here to analyze domain kinetics and biochemistry, and to achieve single-event resolution in living cells, provides a powerful method to elucidate biologically important sorting processes in the future.