Neuronal Polarity
Authors: Bettina Winckler, Ira Mellman
Journal: Neuron (1999)

Many differentiated cells elaborate distinct plasma membrane domains that, although physically continuous, can differ markedly in composition. One of the more dramatic examples is the vertebrate neuron that extends two distinctive processes, axons and dendrites. Axons and dendrites differ in the complement of membrane proteins on their plasma membrane, as well as in the composition of cytoskeletal components and other organelles (2Craig A.M Banker G Annu. Rev. Neurosci. 1994; 17: 267-310Crossref PubMed Scopus (649) Google Scholar). These biochemical differences are responsible for the functional differences that enable neurons to send and receive electrical signals in a vectorial fashion. How are membrane proteins sorted correctly to axons and dendrites, and how is the resulting differential membrane protein distribution maintained? The question of how different membrane components are transported to distinct plasma membrane domains has been far more extensively studied in epithelial cells than in neurons. Despite the obvious differences, concepts arising from molecular sorting in epithelia, particularly the MDCK canine kidney cell line, have provided a good starting point for thinking about how the analogous processes occur in neurons. MDCK cells possess two distinct plasma membrane domains, apical and basolateral, which differ in both lipid and membrane protein composition. Membrane proteins destined to either the apical or basolateral domain are sorted in the trans-Golgi network (TGN) into distinct vesicle populations that are subsequently transported to the appropriate domain where they fuse with the plasma membrane (8Keller P Simons K J. Cell Sci. 1997; 110: 3001-3009Crossref PubMed Google Scholar). Preferential inclusion into apical or basolateral exocytic vesicles is based on targeting determinants in the transported cargo. Basolateral sorting signals have been identified in the cytoplasmic tails of a large number of proteins (12Mellman I Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1336) Google Scholar). These signals are often, but not always, based on the presence of a critical tyrosine or dileucine motif. During endocytosis and transport from the TGN to lysosomes, tyrosine-based motifs are known to interact with the μ subunits of the AP-2 and AP-1 adaptor complexes, respectively. These interactions recruit them to clathrin-coated vesicles that are targeted from the plasma membrane or TGN to the endosomal compartment. The transport of tyrosine signal–containing proteins to the basolateral domain might also be accomplished by as yet unidentified adaptor complexes. Transport to the apical domain of MDCK cells is frequently controlled by sequences in the transmembrane domain or by N- or O-glycans in the extracellular domain (8Keller P Simons K J. Cell Sci. 1997; 110: 3001-3009Crossref PubMed Google Scholar). GPI-linked proteins are also sorted to the apical domain (11Lisanti M.P Caras I.W Davitz M.A Rodriguez-Boulan E J. Cell Biol. 1989; 109: 2145-2156Crossref PubMed Scopus (375) Google Scholar), primarily based on their N-glycan complement, although their lipid tails may also play some role (1Benting J.H Rietveld A.G Simons K J. Cell Biol. 1999; 146: 313-320Crossref PubMed Scopus (210) Google Scholar). The importance of extracellular carbohydrate determinants in apical sorting implies a critical role for a carbohydrate receptor, but such a receptor has yet to be identified. There is another element to apical targeting that has gained wide attention in recent years, the participation of detergent-insoluble glycolipid domains, or "rafts." Several lines of evidence gave rise to the hypothesis that rafts serve as sorting platforms for apically directed proteins (13Simons K Ikonen E Nature. 1997; 387: 569-572Crossref PubMed Scopus (8092) Google Scholar). GPI-linked and at least some apical proteins with membrane-spanning domains (e.g., influenza virus HA) partition into these microdomains, which are enriched in glycosphingolipids and cholesterol. This association with lipid rafts leads to insolubility in nonionic detergents at 4°C and low density in sucrose gradients. A complication to this parsimonious paradigm for protein sorting results from the fact that not all epithelial cells are created equally. Hepatocytes, for example, do not sort apical and basolateral proteins in the TGN, but rather send all membrane proteins to the basolateral domain. Sorting is accomplished "indirectly" in endosomes after internalization, with basolateral proteins being returned to the sinusoidal surface by recycling and apical proteins delivered to the bile canaliculus by transcytosis. Indeed, even in MDCK cells, polarized sorting in endosomes must play an important role given the continuous endocytic activity that occurs at both the apical and basolateral plasma membranes. The endosomal system has been studied extensively by characterizing the transport of recycling and transcytosing ligands and their receptors. Distinct apical and basolateral early endosomes receive cargo from either the apical or basolateral surface and then pass the cargo to pericentriolar recycling endosomes, which have exit paths to both apical and basolateral domains (12Mellman I Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1336) Google Scholar). Does sorting in polarized neurons involve similar trafficking routes and machinery? Initial work on polarized sorting in MDCK cells relied on the analysis of viruses whose glycoproteins are transported to one domain or the other and that subsequently bud from opposite poles. Influenza HA protein is targeted apically, while VSV G protein is directed to the basolateral surface. In a classic series of experiments, 3Dotti C.G Simons K Cell. 1990; 62: 63-72Abstract Full Text PDF PubMed Scopus (358) Google Scholar observed that these same viral proteins were also expressed in a polarized fashion in rat hippocampal neurons in culture: the influenza HA protein was expressed on the axon, while VSV G protein appears on the somatodendritic surface. They proposed that the apical domain corresponds to the axonal domain and the somatodendritic to the basolateral. The dendrite/basolateral and axonal/apical sorting paradigm has proven to be a powerful one, but, as we shall see, limits to the analogy are becoming increasingly apparent. 7Jareb M Banker G Neuron. 1998; 20: 855-867Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar recently used recombinant Herpes viruses to express various basolateral receptors and mutants with well characterized sorting signals (e.g., LDL receptor [LDLR]). As predicted, basolateral proteins all appeared on the somatodendritic surface. Moreover, for LDLR, the same amino acid residues found to be important for basolateral targeting in MDCK cells were likewise important for sorting to dendrites. Apical targeting was a different matter. In MDCK cells, elimination of basolateral targeting information results in the reversed transport of many proteins to the apical surface. Presumably, the cryptic carbohydrate-based apical sorting system was revealed in the absence of a dominant basolateral signal. In hippocampal neurons, however, the mutant LDLRs were expressed equally at the axonal and somatodendritic domains. Thus, neurons use the same signals for somatodendritic targeting as MDCK cells do for basolateral targeting but were unable to recognize the LDLR apical targeting information for axonal transport. Although similar results were obtained for several other receptors, the universality of basolateral targeting signals may not be complete. The basolateral protein transferrin receptor (TfR) localizes to the somatodendritic domain, but its targeting signal to dendrites may be at least partly distinct from the targeting signal to the basolateral domain (16West A.E Neve R.L Buckley K.M J. Neurosci. 1997; 17: 6038-6047Crossref PubMed Google Scholar). The cytoplasmic tail of mGluR2 is necessary for somatodendritic targeting, but the 60–amino acid segment required bears no sequences reminiscent of "conventional" basolateral targeting signals (14Stowell J.N Craig A.M Neuron. 1999; 22: 525-536Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Although these results suggest that neurons may have an additional mechanism for somatodendritic targeting, such a conclusion must be tempered by the fact that typical basolateral signals are highly degenerate, impossible to recognize by sequence alone. A given cytoplasmic tail may also contain multiple signals, regions that mask one or more signals, or binding sites for cytoskeletal elements that may override even conventional sorting signals. This is particularly a problem in the case of multispan membrane proteins (e.g., mGluR2) whose targeting mechanisms are as yet not as well understood. Even in the case of the very well studied TfR, a predominant role for any individual amino acid has not been revealed, further complicating comparative analysis between neurons and epithelia. Accordingly, it remains likely that somatodendritic targeting in neurons relies on the same signals as does basolateral targeting in MDCK cells. Additionally, mechanisms must be in place to ensure proper localization of receptors to dendritic sublocales (different dendrites, spines, etc.). How about transport to the axonal domain? Most apical proteins expressed in hippocampal neurons are expressed on both axons and dendrites (7Jareb M Banker G Neuron. 1998; 20: 855-867Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), rather than just on axons. This suggests that apical sorting signals are not recognized as axonal sorting determinants by neuronal cells. This extends even to the distribution of the GPI-anchored protein Thy1, characterized by Dotti and colleagues (9Ledesma M.D Simons K Dotti C.G Proc. Natl. Acad. Sci. USA. 1998; 95: 3966-3971Crossref PubMed Scopus (199) Google Scholar, 10Ledesma M.D Brugger B Bunning C Wieland F.T Dotti C.G EMBO J. 1999; 18: 1761-1771Crossref PubMed Scopus (115) Google Scholar) as being strictly axonal but subsequently found by others to often be randomly distributed (4Faivre-Sarrailh C Rougon G Mol. Neurobiol. 1993; 7: 49-60Crossref PubMed Scopus (26) Google Scholar, 7Jareb M Banker G Neuron. 1998; 20: 855-867Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Indeed, the ability of hippocampal neurons to target Thy1 axonally correlates with the age of the cultures: immature neurons being less capable of axonal targeting than mature neurons (10Ledesma M.D Brugger B Bunning C Wieland F.T Dotti C.G EMBO J. 1999; 18: 1761-1771Crossref PubMed Scopus (115) Google Scholar). While developmental regulation may explain some of the disparity, Thy1 can be randomly distributed in neurons that exhibit strict axonal polarity of another marker, the adhesion molecule L1/Ng-CAM (18Winckler B Forscher P Mellman I Nature. 1999; 397: 698-701Crossref PubMed Scopus (347) Google Scholar). Thus, L1/Ng-CAM, which possesses a proteinaceous membrane anchor, and Thy1 reach the axonal domain by distinct mechanisms. However, the mechanistic significance of this observation is far from clear. Thy1 and L1/Ng-CAM might take distinct vesicular routes to the axon, or they might take a single vesicle but be differentially retained after their arrival. For example, the cytoplasmic tail of L1/Ng-CAM contains an ankyrin-binding motif that might result in axonal retention. Do lipid rafts play a role in axonal targeting, as is the case for apical targeting in MDCK cells? Dotti and colleagues analyzed the distribution of Thy1, which is axonal in their hands (9Ledesma M.D Simons K Dotti C.G Proc. Natl. Acad. Sci. USA. 1998; 95: 3966-3971Crossref PubMed Scopus (199) Google Scholar, 10Ledesma M.D Brugger B Bunning C Wieland F.T Dotti C.G EMBO J. 1999; 18: 1761-1771Crossref PubMed Scopus (115) Google Scholar). Disruption of rafts by depletion of glycosphingolipids with fumonisin B1 led to increased appearance of Thy1 in dendrites. Furthermore, addition of a precursor to sphingomyelin and glucosyl ceramide to immature neuronal cultures, which had low levels of sphingomyelin and did not sort Thy1 exclusively to axons, improved axonal targeting. Although these data imply that lipid rafts can play a role for axonal delivery, the extent to which this is true remains unclear. As mentioned above, neurons that target L1/Ng-CAM axonally may still exhibit randomly distributed Thy1. Remarkably, our own preliminary results suggest that Thy1 is detergent insoluble at 4°C regardless of its localization to axons or dendrites, while axonal L1/Ng-CAM is detergent soluble. Another conundrum is presented by the targeting of the Alzheimer's disease precursor protein (β-APP). When expressed in MDCK cells, β-APP is transported to the basolateral domain due to a tyrosine-dependent basolateral targeting/endocytosis signal. Yet in neurons, β-APP first appears axonally, followed by transcytosis to the somatodendritic domain (19Yamazaki T Selkoe D.J Koo E.H J. Cell Biol. 1995; 129: 431-442Crossref PubMed Scopus (186) Google Scholar, 15Tienari P.J et al.EMBO J. 1996; 15: 5218-5229Crossref PubMed Scopus (123) Google Scholar). These data imply that the apical targeting information in β-APP is dominant to basolateral targeting signals when in the context of hippocampal neurons. As discussed above, there do appear to be differences between neuronal and epithelial cells in their use of targeting information responsible for accurate apical transport, so perhaps this otherwise unappealing possibility may in fact be the case. Unfortunately, there is little information concerning the mechanism of β-APP targeting to the axon. There is conflicting evidence as to whether β-APP partitions into lipid rafts. Moreover, the fact that β-APP is subject to proteolytic processing on the secretory pathway may contribute at least in part to the impression that it is largely transported to the axonal domain. For example, neurons may cleave a greater fraction of β-APP prior to its exit from the TGN than do MDCK cells. If so, the resulting soluble β-APP fragment, no longer attached to the β-APP cytoplasmic domain upon reaching its presumptive sorting site in the TGN, might be taken axonally due to its lumenal carbohydrate chains. In any event, the evidence clearly supports the fact that at least some intact β-APP must reach the axonal plasma membrane. That this β-APP proceeds to the somatodendritic domain suggests that the basolateral targeting information in the β-APP cytoplasmic tail can at least be recognized for somatodendritic targeting following endocytosis; this would be formally equivalent to what happens in epithelial cells when basolateral proteins are "missorted" to the apical side (12Mellman I Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1336) Google Scholar). As mentioned earlier, the endosomal system plays a crucial role as a sorting station in epithelial cells, to maintain polarity in the face of continued endocytosis and recycling in all cells and as the unique site for the separation of basolateral and apical components in others. The question emerges of whether neurons sort directly or indirectly: that is, do axonal proteins reach their final destination after first being inserted into and endocytosed from the somatodendritic plasma membrane or by being selectively packaged in axonally directed transport vesicles directly at the TGN? Since rigorous biochemical studies are not possible in cultured neurons, this has proved to be a difficult point to test. One assay to assess direct delivery has been to examine the expression pattern of a given plasma membrane marker early after viral infection and see if it changes over time. By this assay, several somatodendritic proteins seem to follow a direct route to the somatodendritic domain (pIgR, LDLR, mGluR2); while at least one protein (β-APP) seems to follow a direct delivery route to the axon followed by transcytotic delivery to the cell body. But what about resident axonal plasma membrane proteins? Indirect sorting to the axon is a possibility since transcytotic pathways do exist in neurons. pIgR, which is transcytosed from the basolateral to the apical side in epithelial cells, is similarly transcytosed from the somatodendritic to the axonal surface (6Ikonen E Parton R.G Hunziker W Simons K Dotti C.G Curr. Biol. 1993; 3: 635-644Abstract Full Text PDF PubMed Scopus (25) Google Scholar). In both neurons and MDCK cells, pIgR transcytosis is stimulated by dimeric IgA, the ligand for pIgR. A small percentage of TfR also appears in the axon by transcytosis as was demonstrated directly by chasing a photoactivated pool of fluorescent Tf from the dendrite to the axon (5Hémar A Olivo J.-C Williamson E Saffrich R Dotti C.G J. Neurosci. 1997; 17: 9026-9034Crossref PubMed Google Scholar). Once membrane proteins are successfully delivered to the axonal or somatodendritic domain, mechanisms must exist to keep them from entering the inappropriate domain by lateral diffusion. In epithelial cells, this function is provided by a molecular fence presumably corresponding to the tight junction, a region of close apposition between adjacent cells where the extrafacial leaflets of their plasma membrane lipid bilayers coalesce to form characteristic "strands." Tight junctions prevent the paracellular movement of solutes across epithelial cell layers and are also thought to restrict the diffusion of membrane proteins and lipids between the apical and basolateral domains. Neurons in culture do not appear to engage in cell–cell contacts that could result in the construction of a tight junction between axon and cell body, yet they clearly established biochemically distinct axonal and somatodendritic domains. While endocytosis and transcytosis may serve to retrieve incorrectly localized components, it would seem that such a mechanism would be insufficient to cope with the rate of randomization that would ensue were axonal and somatodendritic components free to diffuse between the two domains. Indeed, neurons have developed the means to restrict the diffusion of membrane proteins, if not membrane lipids. Selective restriction to diffusion involves either extracellular and intracellular mechanisms. Interactions between membrane proteins and extracellular matrix components may ensure proper localization of some membrane proteins. Perhaps more striking, however, are the connections between membrane proteins and cytoskeletal elements. Voltage-gated sodium channels (VGNC) accumulate on the axonal initial segment and Nodes of Ranvier where they play critical roles in propagating action potentials. Immobilization of VGNCs at these sites reflects their ability to bind a brain-specific ankyrin G splice isoform found at initial segments and Nodes of Ranvier (20Zhou D Lambert S Malen P.C Carpenter S Boland L.M Bennett V J. Cell Biol. 1998; 143: 1295-1304Crossref PubMed Scopus (461) Google Scholar). The adhesion protein neurofascin also has a cytoplasmic tail motif enabling interaction with the same ankyrin G isoform and thus is also found enriched at initial segments and Nodes of Ranvier. The initial segment not only anchors resident membrane proteins like VGNCs to its specialized cytoskeleton but also acts as a general diffusion barrier to other membrane proteins (18Winckler B Forscher P Mellman I Nature. 1999; 397: 698-701Crossref PubMed Scopus (347) Google Scholar). Although many membrane proteins are highly mobile in the plasma membrane in the distal portion of the axon, as well as in dendrites and the axon hillock, they become almost entirely immobile in the initial 60–90 μm of the axon. Since this is true even for GPI-anchored proteins such as Thy1, the restricted diffusion cannot be attributed to cytoplasmic tail–cytoskeleton interactions. Instead, it seems far more likely that the dense packing of membrane proteins that do interact with ankyrin G at the initial segment form a simple but effective physical barrier. Other membrane proteins simply cannot penetrate the molecular fence, or, if they do, they get stuck in place. One might expect that membrane lipids would not be so impeded, an expectation that is consistent with observations showing that fluorescent lipid analogs such as DiI are capable of diffusing from axon to cell body and vice versa (17Winckler B Poo M.-M Nature. 1996; 379: 213Crossref PubMed Scopus (27) Google Scholar). The ganglioside GM1 does seem to exhibit axonal polarity (10Ledesma M.D Brugger B Bunning C Wieland F.T Dotti C.G EMBO J. 1999; 18: 1761-1771Crossref PubMed Scopus (115) Google Scholar), but this may reflect the propensity of this lipid to form large rafts unable to cross the fence region. Although its biochemical features are incompletely understood, it is already clear that the initial segment fence involves an array of other cytoplasmic proteins. In addition to ankyrin G, one can also find F-actin and spectrin; disruption of F-actin with drugs such as latrunculin B results in a loss of fence activity and eventual randomization of axonal components (18Winckler B Forscher P Mellman I Nature. 1999; 397: 698-701Crossref PubMed Scopus (347) Google Scholar) (Figure 1). These cytoskeletal proteins appear to form a detergent-insoluble complex that also contains at least two proteins normally associated with endocytosis via clathrin-coated vesicles: dynamin I and amphiphysin II. Conceivably, these proteins belie endocytic activity at the initial segment whose purpose might be to internalize hapless proteins that diffuse into the fence and return them to their domains of origin. Were such a correction mechanism not to exist, the initial segment would become a black hole that might eventually accumulate all axonal and somatodendritic proteins. Although a role for the axonal initial segment in maintaining polarity seems clear, it is tempting to suggest that it may also be a critical determining factor in generating polarity. By providing a barrier to diffusion, construction of the initial segment molecular fence would provide the means by which a developing neuron can build upon an ability to sort axonal and somatodendritic components to stabilize the morphogenesis of biochemical distinct plasma membrane domains.