Neuron, Vol.

40, 277295, October 9, 2003, Copyright 2003 by Cell Press

Neuronal Polarity and Trafficking

Review

April C. Horton1 and Michael D. Ehlers1,2,3,* 1 Department of Neurobiology 2 Department of Cell Biology 3 Department of Pharmacology and Cancer Biology Duke University Medical Center Box 3209 Durham, North Carolina 27710

Among the most morphologically complex cells, neurons are masters of membrane specialization. Nowhere is this more striking than in the division of cellular labor between the axon and the dendrites. In morphology, signaling properties, cytoskeletal organization, and physiological function, axons and dendrites (or more properly, the somatodendritic compartment) are radically different. Such polarization of neurons into domains specialized for either receiving (dendrites) or transmitting (axons) cellular signals provides the underpinning for all neural circuitry. The initial specification of axonal and dendritic identity occurs early in neuronal life, persists for decades, and is manifested by the presence of very different sets of cell surface proteins. Yet, how neuronal polarity is established, how distinct axonal and somatodendritic domains are maintained, and how integral membrane proteins are directed to dendrites or accumulate in axons remain enduring and formidable questions in neuronal cell biology. Introduction Neurons reside at a pinnacle of cellular specialization. With their long extended axon and elaborate dendritic arbor, neurons establish the circuitry that detects, stores, and transmits information that is essential to the function of all complex organisms. Although neurons come in many shapes and sizes, they all polarize into discrete functional domains. The broadest level is between the axon and the somatodendritic compartment (Craig and Banker, 1994; Winckler and Mellman, 1999). Dendrites themselves can be further polarized (e.g., apical versus basolateral dendrites), and individual dendritic segments may have distinct molecular compositions and functional properties suggesting localized polarity. Neuronal polarity is established early on in development as neurons differentiate and extend processes and must be subsequently maintained over a life span comprising decades. Both the maintenance and establishment of neuronal polarity involve coordinated and widespread regulation of the cytoskeleton and membrane trafficking machinery (Foletti et al., 1999; da Silva and Dotti, 2002). Given that neurons are cells with surface areas and cytoplasmic volumes 10,000 times greater than most eukaryotic cells, these regulatory mechanisms must be writ large. How is it then that neuronal polarity is established? How is it maintained?
* Correspondence: ehlers@neuro.duke.edu

How are membrane constituents trafficked through such large volumes and long distances to ensure polarity? The establishment of subcellular domains with distinct molecular components and functional properties is a fundamental problem of cell biology. Studies in nonneuronal cells such as yeast, fibroblasts, and epithelial cells have provided substantial insight into the highly conserved mechanisms that are used to establish and maintain polarity. For example, in one of the simplest eukaryotic cells, the budding yeast Saccharomyces cerevisiae, membrane addition is directed specifically to the tip of the growing bud (Pruyne and Bretscher, 2000). Before cytokinesis, membrane addition is redirected to the bud neck, allowing fission. Genetic studies in yeast have provided important insights into the mechanisms that allow such exquisite control over the precise location of membrane addition (Chant, 1999). Studies of genetically tractable multicellular organisms including Drosophila and C. elegans have likewise provided clues as to how cells establish polarity. Screens in these organisms have revealed genes and protein complexes necessary for asymmetric cell division during embryogenesis. These complexes have subsequently been discovered to have broader roles in establishing cellular polarity (Wodarz, 2002). Finally, one of the most well-characterized mammalian models of cell polarity is that of the polarized epithelial cell. These cells, such as those lining the gut or renal tubules, have distinct apical and basolateral domains separated by tight junctions, each with their own repertoire of membrane proteins specialized to accomplish the unique functions of each domain (Mostov et al., 2003). Studies of protein targeting in epithelial cells have provided a model for how proteins might be targeted to the axonal or somatodendritic domains in neurons. Cell polarity, particularly as it has been studied in epithelial cells and neurons, has traditionally been seen as a segregation of protein and lipid repertoires. That is, there exist distinct sets of membrane proteins, including receptors, ion channels, transporters, and adhesion molecules, that are specific for a particular cellular domain. The mechanisms of cell polarization have thus been defined as those that establish these distinct protein locales. Such mechanisms include barriers in the membrane to protein diffusion (Kobayashi et al., 1992; Winckler et al., 1999; Nakada et al., 2003), stabilization of protein complexes at the membrane by scaffolding proteins (Harris and Lim, 2001), and selective targeting of proteins along the secretory or endosomal pathways (Jacob and Naim, 2001; Keller et al., 2001; Kreitzer et al., 2003) (Figure 1). These mechanisms likely all contribute to the maintenance of neuronal polarity. However, there are also important questions about cell polarity that are not answered by looking exclusively at membrane protein localization. For example, how is neuronal polarity established to begin with? What intracellular and extracellular cues determine which neurite becomes the axon and which the dendrites? What cytoskeletal rearrangements must occur to allow axonal and dendritic outgrowth and branching? How might

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Figure 1. The Secretory and Endocytic Pathways of Eukaryotic Cells (1) Newly synthesized membrane cargo leaves the endoplasmic reticulum (ER) at specialized ER subdomains termed ER exit sites. This cargo traffics to the Golgi apparatus, where it is sorted into carriers destined for the plasma membrane (2), endosomes (3), or back to the ER (not indicated). Proteins may also enter the endocytic pathway following clathrin-mediated endocytosis from the plasma membrane (4). Within the endosomal system, proteins may be recycled to the plasma membrane or targeted to lysosomes (5).

these cytoskeletal rearrangements influence the establishment of membrane domains and the trafficking of proteins? What organelles accomplish the targeting of membrane proteins to locations as specific as a node of Ranvier or a single synapse? And what then regulates the distribution of these organelles? Finally, neuronal polarity has been presented in its simplest form as a binary process, with the neuron divided into two domains: axonal and somatodendritic. However, clearly there are important specializations even within these domains, as neither has a homogeneous plasma membrane. Determining what mechanisms accomplish specialization within domains, such as those distinguishing apical from basolateral dendrites, presents an additional layer of complexity to the problem of neuronal polarity. In this review, we describe how recent studies in diverse cell types and organisms have informed studies of neuronal polarity by uncovering conserved mechanisms of polarity establishment, maintenance, and membrane protein targeting. We also discuss roles for selective trafficking, fusion, and retention in the targeting of axonal and dendritic proteins. Finally, we outline important issues that remain unresolved in neuronal polarity and trafficking. Establishment of Neuronal Polarity Neurons originate as the terminal product of successive rounds of division and differentiation by neural precursor cells (Zhao et al., 1999). Initially relatively small, nonpolarized cells, neurons proceed to develop distinct axonal and dendritic domains as these processes grow out from the neuronal soma (Bourrat and Sotelo, 1988; da Silva and Dotti, 2002). Because cultured neurons also undergo these developmental processes (Dotti et al., 1988; Craig and Banker, 1994; Bradke and Dotti, 1999),

neuronal polarization has been studied both in vivo and in vitro. In both cases, after a few nascent processes extend from the soma, one of these differentiates into the axon and enters a more rapid phase of growth. Barring experimental manipulation, such as axonal amputation (Goslin and Banker, 1989) or the application of mechanical tension (Lamoureux et al., 2002), the remaining neurites are destined to become dendrites. Thus, the initial event in establishing a polarized neuron is the determination of a single axon, a process termed axon specification (Fukata et al., 2002b). What signals specify the axon? In all polarized cells, one of the first steps in establishing polarity is the establishment of a spatial landmark that marks one section of the plasma membrane as different from the rest (Nelson, 2003). In budding yeast, that landmark is the bud scar (Chant and Herskowitz, 1991), while in epithelial cells, landmarks are the sites of adhesive contact with other cells (Yeaman et al., 1999). Subsequent growth of the yeast bud or establishment of an epithelial apicobasal axis involves numerous protein complexes, cytoskeletal rearrangements, and membrane trafficking events, but these all lie downstream of the initial distinction of one region of the plasma membrane as a polarity landmark. Might neurons utilize cellular programs similar to those used by yeast and epithelial cells to establish spatial landmarks? As discussed below, recent experiments showing conserved polarity mechanisms at work in the process of axon specification suggest that this is indeed the case. Cytoskeletal and Membrane Rearrangements During axon specification, one of several seemingly identical nascent neurites undergoes drastic changes in cytoskeletal and membrane composition, resulting in a neuritic process morphologically and functionally distinct from those that grow and develop into dendrites (Dotti and Banker, 1987; Goslin and Banker, 1989). Early distinctions between the axon and other neurites include an exclusion of polyribosomes from the axon and a higher concentration of intracellular membranes in the axonal growth cone (Deitch and Banker, 1993). The axonal growth cone develops characteristics distinct from the remaining dendritic growth cones, accumulating axon-specific proteins such as the growth-associated protein GAP-43 (Goslin et al., 1988). Cytoskeletal rearrangements include a reorganization of microtubules from the mixed polarity of the neurite to the exclusively plus-end distal microtubules of the axon (Baas et al., 1989), likely by the transport of preassembled plus-end directed microtubules from the cell body (Baas and Ahmad, 1993). This polarized microtubule orientation is maintained in the axon of the mature neuron, while dendrites contain microtubules of mixed polarity (Baas et al., 1989). Proteins associated with microtubules help maintain orientation and influence axon specification. For example, outgrowth of the newly specified axon is facilitated by the collapsin response mediator protein-2 (CRMP-2), mammalian homolog of the C. elegans protein UNC-33 (Fukata et al., 2002a). CRMP-2 is preferentially associated with axonal microtubules, and it binds tubulin dimers, promoting their assembly into microtubule polymers (Fukata et al., 2002a). Intriguingly, overexpression of CRMP-2 increases axonal length and can even cause

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aberrant axons to sprout from established dendrites (Inagaki et al., 2001; Fukata et al., 2002a), suggesting that CRMP-2 is a key regulator of the microtubule dynamics that establish and maintain an axonal cytoskeleton distinct from that of dendrites. Axonal growth cones also have a dynamic actin cytoskeleton, in contrast to the more stable actin of dendritic growth cones (Bradke and Dotti, 1999). As dynamic actin is critical for polarized membrane addition (Smith, 1988; Bretscher, 1996), the dynamic actin of axonal growth cones may account for their rapid outgrowth. Indeed, regulators of the actin cytoskeleton are intimately involved in neuronal polarizationchief among them the Rho family of small GTPases. Rho Family GTPases The family of Rho GTPases, including Rho, Rac, and Cdc42, control many critical cellular functions, most notably regulation of the actin cytoskeleton (Etienne-Manneville and Hall, 2002). Intriguingly, Rho GTPases are also important in establishing cell polarity (Nobes and Hall, 1999) and exert profound effects on neuronal morphology (Luo, 2002). In fact, Cdc42 was one of the first genes identified as critical for polarity establishment in yeast, as Cdc42 mutants fail to develop a bud and instead become large, spherical, and multinucleate (Adams et al., 1990). Cdc42 is also required for polarization of the C. elegans zygote (Gotta et al., 2001). In yeast arrested in G1, constitutively active Cdc42 accumulates at polar caps in an actin- and secretion-dependent manner (Wedlich-Soldner et al., 2003). Activated Cdc42 is thought to traffic along actin cables to sites of secretion, where it accumulates and organizes more actin cables to the same site in a feed-forward mechanism (Figure 2A). Rho GTPases are also known to be important in neuronal morphogenesis during development (Luo, 2002). Expression of mutant Cdc42 in embryonic Drosophila neurons impairs the formation of axons and dendrites (Luo et al., 1994). Transgenic mice expressing a constitutively active Rac1 in Purkinje cells have normal dendritic development but reduced axonal arbors (Luo et al., 1996), suggesting impaired cellular polarization or membrane addition. These studies provided initial important clues that actin regulatory elements such as the Rho GTPases direct cellular polarization and morphogenesis in a conserved fashion from yeast to neurons. The Par3/Par6/aPKC Complex A satisfying functional link between the Rho GTPases and axon specification has recently been found with the identification of the Par3/Par6/aPKC complex as an evolutionarily conserved polarization signal (Wodarz, 2002) that also regulates the actin cytoskeleton (Qiu et al., 2000; Etienne-Manneville and Hall, 2001, 2003). This complex illustrates how the study of fundamental processes in other cell biological systems has provided insight into neuronal polarization. Further, the role of the Par complex in directing subsequent secretory trafficking underscores the critical role of membrane trafficking in the establishment of polarity. Par3 and Par6 are conserved multidomain scaffolding proteins that were identified in a genetic screen in C. elegans for embryos defective in the partitioning required for asymmetric cell division in embryogenesis (Watts et al., 1996). Par3 and Par6 form a complex with atypical protein kinase C (aPKC) at the anterior pole of

C. elegans embryos (Etemad-Moghadam et al., 1995), with Par3 and aPKC required for the proper localization of Par6 (Hung and Kemphues, 1999). Proper localization of this complex is critical for the establishment of anterior and posterior poles in the polarized embryo (Etemad-Moghadam et al., 1995; Watts et al., 1996; Hung and Kemphues, 1999). The Par proteins are conserved from worms to mammals (Johansson et al., 2000). Par3 and Par6 are PDZ scaffold proteins that bind atypical PKC isoforms (aPKC, e.g., PKC- and PKC-) as well as activated forms of the actin-regulatory small GTPases Cdc42 and Rac1 (Joberty et al., 2000). The Par3/Par6/aPKC complex is present in cytosol and is recruited to cell-cell contacts during the formation of epithelial tight junctions (Yamanaka et al., 2001). This recruitment is likely mediated by direct binding of Par3 to junctional adhesion molecule (JAM) (Itoh et al., 2001) and nectin (Takekuni et al., 2003). Overexpression of Par3 promotes tight junction formation (Hirose et al., 2002), while expression of a dominant-negative Par6 inhibits tight junction formation (Yamanaka et al., 2001). By contributing to tight junction formation, the Par complex helps to define and separate apical and basolateral domains in polarized epithelial cells. Given the importance of the Par complex in establishing polarity in the C. elegans embryo and epithelial cells, Shi et al. recently examined the role of the Par complex in the process of axon specification, the first step in neuronal polarization (Shi et al., 2003). They found that, in cultured hippocampal neurons, endogenous mammalian Par3 and Par6 proteins are selectively located at the tips of elongating axons. Overexpression of mPar3 or mPar6 prevents the determination of a single axon. This Par-dependent axon specification requires phosphatidylinositol 3-kinase (PI3K) activity and phosphatidylinositol 3-phosphate (PIP3), the lipid product of PI3K activation, as well as recruitment by PIP3 of the serine/ threonine kinase Akt. Before the specification of a single axon, mPar3 and mPar6 are localized at the tips of all nascent processes (Shi et al., 2003). Subsequently, dendrites lose mPar3/ mPar6 while the complex is retained in the axon. Thus, a key event in the specification of a single axon from a group of seemingly identical processes appears to be selective concentration of mPar3 and mPar6 at the tip of just one. However, it is unknown exactly how this concentration may occur. Localized signals may result in the selective degradation or destabilization of mPar3 and mPar6 in the tips of nascent dendrites. Another possibility is that a signal important for the concentration of mPar3 and mPar6 is present at the tips of all processes, with stochastic fluctuations in signal concentration resulting in a tip with a higher concentration recruiting more mPar3 and mPar6. A positive feedback loop could then trigger a signaling cascade that recruits more mPar3 and mPar6 leading to selective concentration of the polarity complex in the tip of a single process. Such a scenario is consistent with earlier observations that amputation of the nascent axon results in the specification of another process to replace the axon (Dotti and Banker, 1987; Goslin and Banker, 1989). As PI3K and PIP3 are required for axon specification (Shi et al., 2003), one candidate stochastic signal for

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Figure 2. Polarization Is Associated with Rearrangements of the Secretory Apparatus in a Variety of Cell Types (A) In budding yeast, the Rho-GTPase Cdc42 accumulates at sites of membrane addition at the tip of the yeast bud, marked by the presence of the exocyst complex (yellow outline). Cdc42-GTP organizes the actin cytoskeleton such that secretory vesicles trafficking via actin motors such as Myo2 are directed toward the bud tip. (B) Cdc42-GTP acts in migrating fibroblasts and astrocytes by recruiting the Par3/Par6 complex, which acts through atypical PKC (aPKC) to phosphorylate and thereby inactivate GSK3. Inactivation of GSK3 leads to the association of APC with the plus ends of microtubules, directing microtubule-based secretory traffic to the leading edge. (C) During epithelial cell polarization, the Golgi apparatus becomes localized to the apical one-third of the cell, and microtubules become rearranged to direct basolateral secretory traffic to the apical two-thirds of the lateral membrane. Selective fusion of apical and basolateral cargo with the appropriate domain is accomplished, at least in part, by the microtubule-dependent polarized distribution of the SNARE proteins syntaxin-3 and syntaxin-4. (D) While factors such as Cdc42, the Par complex, and the exocyst are known to be important for actin rearrangements, polarity establishment, and membrane addition in neurons, little is understood about how these or other processes direct the organelles of the neuronal secretory pathway. (E) Distributed Golgi outposts in a subpopulation of dendrites may confer local polarized secretory trafficking. Neuronal Golgi, as revealed by staining for endogenous GM130 (red), is distributed in the neuronal soma (arrow), as well as in multiple discontinuous puncta throughout a subset of dendrites (arrowheads). MAP2 staining is shown in blue. Scale bar, 10 m. (Derived from Horton and Ehlers, 2003.)

recruiting mPar3 and mPar6 is PIP3. Notably, PIP3 can also activate members of the Rho family of small GTPases such as Rac and Cdc42 by recruiting pleckstrin homology (PH) domain-containing guanine nucleotide exchange factors (Quilliam et al., 1995). As Par6 can mediate the activation of bound aPKC in response to Cdc42-GTP (Qiu et al., 2000), the Rho family GTPases may be the link between PIP3 and enhanced mPar3/ mPar6/aPKC concentration at the axonal growth cone. Effectors of the Par Complex and aPKC Although the proper localization of the Par complex is clearly critical for its function in establishing polarity, it is not yet clear which mechanisms effect polarization downstream of the Par proteins and aPKC. One possibility is that the Par proteins control cytoskeletal rearrangements important for cell polarity. This is known to be the case in the C. elegans zygote, where Par regulation of microtubule organization is critical for polarized cell division (OConnell et al., 2000). In nondividing cells, such cytoskeletal rearrangements may lie up-

stream of the reorganizations of the secretory pathway that occur during cell polarization (Bacallao et al., 1989). In migrating astrocytes, mPar6 and PKC are localized to the leading edge of the cell, and they reorient the microtubule organizing center (MTOC) to the side of the nucleus facing the direction of migration (EtienneManneville and Hall, 2001, 2003) (Figure 2B). Because post-Golgi secretory carriers traffic along microtubules (Schmoranzer and Simon, 2003), proper orientation of the microtubules by the Par complex may direct membrane addition to the leading edge of the cell. In fact, the MTOC may affect the localization of the Golgi apparatus itself (Shorter and Warren, 2002), such that a rearrangment of the microtubules could orient the entire secretory apparatus in such a way as to facilitate membrane addition to the leading edge. It is currently unknown whether the Par complex affects the orientation of microtubules or the distribution of secretory organelles in developing neurons. In addition to its traditional role in regulating actin

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dynamics, Cdc42 may also act through the Par complex to regulate microtubule organization. In migrating astrocytes, for example, Cdc42 acts through the Par3/ Par6/aPKC complex to phosphorylate and thereby inhibit glycogen synthase kinase 3 (GSK-3). Inhibition of GSK-3 allows the association of the tumor suppressor protein adenomatous polyposis coli (APC) with microtubules, stabilizing microtubule plus-ends at the leading edgethe site of membrane addition (Etienne-Manneville and Hall, 2003) (Figure 2B). In this case, Cdc42 functions to direct secretory trafficking to a particular domain of the plasma membrane, in much the same way as it functions to organize actin during the establishment of polarity in yeast (Wedlich-Soldner et al., 2003). Similar pathways may be at work in other polarized cell types. For example, in polarized Madin-Darby canine kidney (MDCK) cells, APC is associated in an actindependent manner with the basal plasma membrane (Rosin-Arbesfeld et al., 2001), the site of delivery of postGolgi carriers in these cells (Kreitzer et al., 2003). It is worth noting that it is not yet known what proteins might be targeted by activated aPKC in axonal growth cones. Also, it is not yet known what role, if any, GSK-3 and APC play in neuronal polarization. Conserved Protein Complexes Mediate Cellular Polarity Besides the Par3/Par6/aPKC complex, two additional multiprotein complexes have been identified as important in establishing polarity in both invertebrate and vertebrate cells (Bilder, 2001). In Drosophila, these complexes include the Crumbs (Crb)/Stardust (Sdt)/Discs Lost (Dlt) complex (Knust et al., 1993) and the Scribble (Scrib)/Discs Large (Dlg)/Lethal Giant Larvae (Lgl) complex (Woods and Bryant, 1991; Bilder et al., 2000; Bilder and Perrimon, 2000). Mammalian homologs to these complexes include the Crumbs/Pals1/Pals1-associated tight junction (PATJ) protein and Scrib (Vartul)/mammalian Dlg (mDlg)/mammalian Lgl (mLgl) complexes. Disruption of any of these protein complexes disrupts formation of the apical adherens junction, the spatial landmark that defines the apical and basolateral membrane domains, and subsequently the apicobasal polarity axis (Muller and Wieschaus, 1996; Bilder and Perrimon, 2000; Knust and Bossinger, 2002). Of the proteins that make up these complexes, Par3 (termed Bazooka or Baz in Drosophila), Par6, Sdt, Scrib, Dlg, and their mammalian homologs are all PDZ domain-containing proteins, illustrating the importance of PDZ-based protein scaffolds in polarity establishment. Observations in both invertebrate and vertebrate epithelia suggest a sequential mode of action for these protein complexes in polarity establishment. In Drosophila, the boundary between apical and basolateral domains is marked by sites of cadherin-mediated cellcell contact called the zonula adherens (ZA) (Tepass et al., 2001). At the ZA, Baz/Par3 is recruited first and establishes the apical plasma membrane (Muller and Wieschaus, 1996). Crb is recruited by Baz/Par3, and Baz/ Par3 is in turn stabilized at the ZA through its interaction with Crb (Bilder et al., 2003). Crb is involved in the growth of the apical membrane (Wodarz et al., 1995), and overexpression of Crb leads to aberrant apical membrane expansion. This apical growth is counterbalanced by the third protein complex, the Scrib complex, which is

recruited to the basal side of the ZA and which functions in the growth of the lateral membrane (Bilder et al., 2000). These findings suggest a dynamic feedback loop at the apical junctional complex between the Par3/Par6/aPKC, Scrib/Dlg/Lgl, and Crumbs/Pals1/PATJ complexes that organizes and defines the boundaries of polarized domains (Nelson, 2003). While the Par3/Par6/aPKC complex contributes to axon specification, it is not known whether the Crumbs/ Pals1/PATJ or Scrib/Dlg/Lgl complexes regulate polarity establishment in neurons. In nonneuronal cells, Crumbs and Scrib play opposing and balancing roles in the establishment of apical and lateral membrane identity, respectively (Bilder et al., 2000). It it thus tempting to speculate that these complexes also operate to specify axons and dendrites in neurons. In this regard, it is worthwhile to note that there are three mammalian homologs to Crb: CRB1, 2, and 3 (Lemmers et al., 2002). Of these, CRB1 is expressed primarily in the brain and retina (den Hollander et al., 2002), where its precise role is uncertain. In epithelial cells, the Crb and Scrib complexes are located in close apposition to but on opposite sides of the tight junction, the boundary between apical and basolateral domains (Lee et al., 2002; Medina et al., 2002). In neurons, the spatial distribution of Crb and Scrib complexes is much less clear. Although neurons, like classic epithelial cells, possess adherens junctions (including synapses themselves) (Fannon and Colman, 1996; Shapiro and Colman, 1999), they lack tight junctions, raising the question of where tight junction proteins such as Crb and Scrib function in neurons. Perhaps these complexes occupy analogous positions at the axon initial segment, the boundary between axonal and somatodendritic domains. Or, perhaps Crb and Scrib exhibit functional but not spatial conservation, operating at great distances from one another to create distinct axonal and dendritic identities. Much like the ability of Par3/Par6/aPKC to specifiy axons (and to promote apical identity in epithelial cells), it will be of interest to determine whether somatodendritic identity is controlled by the lateralizing activity of Scrib/Dlg/Lgl. Extrinsic Signals for Neuronal Polarity during Morphogenesis While data acquired in cultured neurons over the past two decades clearly show an intrinsic program for polarization in vitro, extracellular cues also undoubtedly play a role in guiding neuronal polarity in vivo. Candidate proteins that have been shown to influence axonal and dendritic development include the neurotrophins, bone morphogenetic proteins (BMPs), and the semaphorins. Neurotrophins have complex influences on neuronal development. For example, BDNF and NT-3 regulate dendritic growth in opposing directions in a lamina-specific, activity-dependent manner (McAllister et al., 1997). Likewise, the various BMPs have differential effects on developing neurons. Sympathetic neurons in culture form an axon spontaneously but require exposure to BMP-2 or BMP-7 in order to sprout dendrites (Lein et al., 1995). The physiological source of BMPs may be the Schwann cell, as Schwann cells produce BMPs 6 and 7 (Schluesener et al., 1995) and also stimulate dendritic outgrowth of cultured sympathetic neurons (Lein et al., 1995). The mechanisms of neurotrophin and BMP signaling are varied and likely involve activation of transcriptional pro-

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grams. As a third example, semaphorin 3A (Sema3A), in addition to its role as a chemorepellant for cortical axons (Campbell et al., 2001; Huber et al., 2003), also serves as a chemoattractant for apical cortical dendrites (Polleux et al., 2000). Sema3A signaling depends on a concentration of soluble guanylate cyclase within the apical dendrite, illustrating the polarity between apical and basolateral dendrites that exists with the somatodendritic domain. In vivo, it is likely that a combination of extrinsic cues and intrinsic cellular programs leads to axon specificationthe first step in the establishment of the polarized neuron with distinct axonal and dendritic subdomains. Once these domains are established, however, further mechanisms must exist to maintain the highly specialized polar neuron in the face of continued membrane flux, protein turnover, and activity-dependent changes in synaptic composition. Maintenance of Neuronal Polarity Once the neuron has developed morphologically distinct axonal and dendritic domains, preservation of polarity requires maintenance of their proper protein, lipid, and cytoskeletal composition. The divergent composition of the axonal and dendritic membranes is in turn maintained by targeting delivery of proteins and lipids to subdomains of the plasma membrane as well as by restricting unwanted mobility or mixing of membrane constituents. Targeted delivery of membrane protein-laden vesicles to the plasma membrane begins with polarized trafficking through the secretory and endocytic pathways (Figure 1). As discussed above, the organelles of the secretory pathway (particularly the Golgi apparatus and post-Golgi carriers) are themselves reorganized by the cytoskeletal changes that occur during polarity establishment. This creates a polarized secretory pathway oriented in such a way as to facilitate directional membrane trafficking (Bacallao et al., 1989; Kreitzer et al., 2003). For example, during epithelial cell polarization, the Golgi is relocated to the apical region of the cytoplasm (Bacallao et al., 1989) (Figure 2C). Concomitant with this relocation of the Golgi, protein delivery ceases to occur over the entire membrane, as it does in the nonpolarized cell, and proceeds to occur only in the apical two-thirds of the lateral plasma membrane (Kreitzer et al., 2003). In neurons, the Golgi is primarily somatic before polarization, whereas it expands and extends into one or more of the larger dendrites during periods of rapid dendritic outgrowth and branching (Horton and Ehlers, 2003) (Figure 2D and 2E), suggesting directed flow of secretory traffic. Microtubules and Polarized Traffic Microtubules act as a superstructure for vesicle and organelle transport, and their intrinsic polarity (plus ends and minus ends) makes them well-suited for instituting cellular polarity (Baas, 1999). In most interphase nonneuronal cells, microtubules are oriented radially with the plus ends extending out from a central MTOC (Piehl and Cassimeris, 2003). In neurons, there is no central MTOC. Instead, microtubules extend throughout the axon and dendrites with different polarities in the two domains. In axons, the microtubules are all oriented with the plus

ends outward, while in dendrites microtubules may have either the plus or minus ends outwardly directed (Baas et al., 1988, 1989). These differences in microtubule arrangement produce distinct patterns and pathways for membrane trafficking. In nonneuronal cells, carriers leave the perinuclear Golgi and traffic outward to the plasma membrane along microtubules powered by plus-end directed microtubule-based motors such as the kinesins (Kreitzer et al., 2000) (see the review by Goldstein [2003], this issue of Neuron). Directed transport of post-Golgi carriers to cell subdomains occurs, in part, by adaptor complexes that link sorting signals within cargo to specific molecular motors associated with microtubules (Nakagawa et al., 2000; Setou et al., 2000). During cell polarization, microtubules reorganize (Bacallao et al., 1989). That reorganization of microtubules leads to polarity is suggested by experiments showing that disruption of the microtubule cytoskeleton can cause a mistargeting of apical proteins to the basolateral membrane (Gilbert et al., 1991; Kreitzer et al., 2003) and basolateral proteins to the apical domain (De Almeida and Stow, 1991). This mistargeting involves, in part, a cytoplasmic redistribution of apical post-Golgi transport intermediates (Kreitzer et al., 2003). In neurons, microtubule remodeling is likewise essential for polarity and post-Golgi exocytosis (Baas, 1999). This reorganization involves the appearance of minusend distal microtubules in newly polarized dendrites (Baas et al., 1988, 1989), which arrive via transport from the soma (Sharp et al., 1995; Baas, 1999), as well as the localization of microtubule-associated proteins such as tau isoforms and MAP2 to axons and dendrites, respectively (Matus et al., 1981; Binder et al., 1985; Baas, 1999). Because post-Golgi carriers traffic along microtubules (Schmoranzer and Simon, 2003), it is likely that the unique distribution of the microtubules in neurons underlies significant differences in protein trafficking between neurons and nonneuronal cells and may explain differences in the trafficking of axonal and dendritic proteins. One model proposes that post-Golgi carriers of dendritic proteins traffic mainly via minus-end directed motors, explaining why carriers of dendritic proteins such as the transferrin receptor are restricted from entering axons (Burack et al., 2000). In contrast, carriers of axonal proteins may be transported via plus-end directed motors. As plus-end directed motors are present in both axons and dendrites, these carriers could enter both domains. Hence, axonal proteins would require additional mechanisms, such as selective fusion or retention (see below), to ensure their surface polarization on the axonal membrane. Further, the microtubule cytoskeleton is important in determining Golgi distribution (Shorter and Warren, 2002). In this regard, it is interesting to note that transport-competent neuronal Golgi is distributed as discrete outposts in a subpopulation of dendrites (Gardiol et al., 1999; Horton and Ehlers, 2003) (Figures 2D and 2E), perhaps due to the bidirectionally oriented microtubule network. The Exocyst and Directed Membrane Addition Once polarized intracellular delivery of membrane proteins and lipids occurs via post-Golgi or endocytic carriers, addition to the plasma membrane takes place at sites specialized to allow membrane fusion. In axons,

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membrane addition occurs largely at growth cones (Craig et al., 1995). In dendrites, the situation is less clear and more complex, but multiple locations of membrane fusion likely exist (Maletic-Savatic and Malinow, 1998; Hartmann et al., 2001). Sites of exocytosis are characterized by membrane-associated protein complexes that direct and facilitate fusion events (Chen and Scheller, 2001; Jahn et al., 2003; Li and Chin, 2003). In other cell types, and likely also in neurons, one protein complex that defines such sites of directed membrane addition is the exocyst complex. The exocyst is made up of eight tightly associated proteinsSec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84that are highly conserved from yeast to mammals (TerBush and Novick, 1995; Hsu et al., 1996; TerBush et al., 1996). In yeast, the exocyst acts as the effector for the Rab family GTPase Sec4 in the fusion of carriers with the plasma membrane (Guo et al., 1999). Through its presence on the plasma membrane, the exocyst directs membrane addition to specific locations. Early in budding, membrane is targeted by the exocyst to the bud tip. When the bud matures, the exocyst becomes relocated to the bud neck, where it directs membrane addition to allow cytokinesis (Finger et al., 1998; Wang et al., 2002). The exocyst is expressed in both polarized and nonpolarized mammalian cells, albeit with different subcellular localizations. In nonpolarized epithelial cells, the exocyst is cytoplasmic and post-Golgi carrier fusion occurs nonselectively over the entire membrane (Kreitzer et al., 2003). With polarization, the exocyst becomes localized to the plasma membrane in the region near tight junctions (Grindstaff et al., 1998), and as discussed above, post-Golgi carrier fusion becomes limited to the apical two-thirds of the basolateral membrane (Kreitzer et al., 2003). Overexpression of Sec10 causes polarized MDCK cells to become taller and to form more tubules and cysts (Lipschutz et al., 2000), suggesting that not only does the exocyst participate in targeted membrane fusion but also that the exocyst may be the rate-limiting factor in plasma membrane addition. Although it remains unclear precisely how the exocyst becomes localized to plasma membrane domains, cytoskeletal changes associated with polarization almost certainly contribute. Consistent with this notion, the actin-regulatory GTPase Cdc42 interacts with the exocyst subunit Sec3 in yeast, and Cdc42 mutants exhibit disrupted localization of exocyst proteins and randomized protein secretion patterns at their cell surface (Zhang et al., 2001). Additionally, in mammalian cells, Sec5 binds activated RalAa Rasrelated small GTPase involved in actin remodeling and vesicular transport (Sugihara et al., 2002). In mammals, the exocyst is expressed in both neuronal and nonneuronal tissue (Hsu et al., 1996). In cultured neurons, the exocyst is located in the cell body, in a punctate distribution along dendrites, and at the tips of growing neurites (Hazuka et al., 1999; Murthy et al., 2003). It is unknown whether this localization is predictive of the actual sites of membrane addition in neurons, as it is in other cell types. However, membrane addition in growing axons is thought to occur at the growth cones (Craig et al., 1995), consistent with the presence of the exocyst at the tips of extending neurites (Hazuka et al., 1999; Murthy et al., 2003). Furthermore,

expression of dominant-negative Sec8 or Sec10 impairs neurite outgrowth in PC12 cells (Vega and Hsu, 2001), and Drosophila Sec5 mutants have impaired axon elaboration at the neuromuscular junction (Murthy et al., 2003). Thus, the exocyst appears to play a role in directing the membrane addition that accompanies axon outgrowth and possibly dendritic outgrowth as well. Interestingly, neurotransmitter release is not impaired in neurons cultured from Drosophila Sec5 mutants (Murthy et al., 2003), suggesting that there exist both exocystdependent and -independent membrane fusion pathways in neurons (Sans et al., 2003). It is possible that the exocyst is more important for the constitutive exocytosis events underlying process outgrowth and maintenance of membrane protein complements, while exocytosis at the presynaptic terminal is specialized to allow rapid, exocyst-independent neuronal signaling. In addition to its role in directing sites of membrane addition, the exocyst is also involved mechanistically in the targeting of membrane proteins. In some cell types, the exocyst selectively directs the targeting or fusion of basolateral proteins at the plasma membrane. Specifically, the injection of antibodies against Sec6 or Sec8 partially blocks delivery of basolateral proteins but does not affect delivery of apical proteins in MDCK cells (Grindstaff et al., 1998). Also, overexpression of Sec10 enhances the surface delivery of basolateral proteins in MDCK cells (Lipschutz et al., 2000). The role of the exocyst in targeting membrane proteins in neurons is clearly different, as in neurons the exocyst is necessary for constitutive membrane fusion in both dendrites and axons and not just in one domain (Murthy et al., 2003). Therefore, while the exocyst may be involved in directing the specific site of protein insertion into the neuronal plasma membrane, mechanisms for segregation between axons and dendrites must exist upstream of the exocyst. SNAREs and Localized Exocytosis Soluble NSF attachment protein receptor (SNARE) proteins are core components of the vesicle fusion apparatus that have been been extensively studied due to their importance in mediating docking and calcium-dependent fusion of synaptic vesicles at presynaptic terminals (Augustine et al., 1999; Jahn et al., 2003) (also see the review by Augustine et al., [2003], this issue of Neuron). Composed of trimolecular helical bundles of syntaxin, synaptobrevin/VAMPs, and SNAP-25 (each of which has multiple isoforms), SNAREs mediate membrane fusion between various intracellular vesicles and organelles as well as constitutive exocytotic fusion events at the plasma membrane (McNew et al., 2000; Parlati et al., 2002; Hu et al., 2003). The importance of SNARE proteins in constitutive membrane addition in neurons was first suggested by experiments using reagents that inhibit SNARE protein function. In these experiments, disrupting SNARE function impaired the growth of both axons (Osen-Sand et al., 1993; Igarashi et al., 1996) and dendrites (Grosse et al., 1999; Martinez-Arca et al., 2001). Interestingly, toxins that selectively cleave certain SNARE proteins have differential effects on the outgrowth of axons and dendrites (Osen-Sand et al., 1996). For example, botulinum neurotoxin C (BoNT-C), which cleaves syntaxin and SNAP-25, inhibits neurite outgrowth in general, while botulinum neurotoxin-A (BoNT-A), which

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cleaves only SNAP-25, reduces axonal outgrowth without affecting dendritic outgrowth (Osen-Sand et al., 1996). These experiments suggest that specific SNARE proteins are either differentially distributed or functionally different in the axonal versus the dendritic domains. In addition, SNARE proteins themselves are differentially distributed on the plasma membrane of polarized cells, suggesting that certain carrier populations will be selectively incorporated into different areas of the plasma membrane. In polarized MDCK cells, the t-SNAREs syntaxin-3 and syntaxin-4 are targeted to the apical and basolateral plasma membrane domains, respectively (Low et al., 1996) (Figure 2C). Syntaxin-3 along with the v-SNARE VAMP7/TI-VAMP function together in the selective fusion of apical carriers with the apical plasma membrane (Galli et al., 1998; Low et al., 1998). This restricted distribution of SNAREs may be even more microscopic, as SNARE proteins concentrate in microclusters of 200 nm to define docking and fusion sites for secretory vesicles (Lang et al., 2001). Recently, it has been shown that the delivery of syntaxin-3 to the apical domain via microtubules is necessary for the restricted delivery of apical carriers to the apical membrane (Kreitzer et al., 2003). This finding raises the possibility that cytoskeletal organization and microtubulebased transport is important not only for the directed transport of cargo to specific plasma membrane domains but also for localizing the protein complexes that organize fusion-competent plasma membrane domains in the first place. Polarized delivery of lipids and proteins to axons and dendrites is ensured locally by the presence of specialized membrane subdomains, characterized by the exocyst and SNARE proteins, that coordinate targeted vesicle fusion with the plasma membrane. Once membrane is delivered, however, additional mechanisms must ensure that proteins and lipids do not simply diffuse within the plasma membrane to nonspecific locations. Barriers to this sort of diffusion, then, are critical to maintaining neuronal polarity. Diffusion Barriers Within the lipid bilayer of the plasma membrane, lipids and proteins may diffuse freely unless anchored to scaffolding complexes or the cytoskeletal matrix (Borgdorff and Choquet, 2002; Fujiwara et al., 2002; Serge et al., 2002; Tovar and Westbrook, 2002). This diffusion would rapidly lead to a mixing of components of polarized membranes (Balda et al., 1996), were barriers not in place to prevent it. In polarized epithelial cells, such a barrier is the tight junction, or zonula occludens, the site of intercellular contacts at the most apical part of the lateral membrane (Farquhar and Palade, 1963). These tight junctions function as an intracellular fence, preventing the mixing of apical and basolateral membrane components (van Meer et al., 1986). Might such a fence be at work in neurons, preventing the mixing of axonal and dendritic membranes? Experiments monitoring the diffusion of fluorescently labeled lipids (Kobayashi et al., 1992) and proteins (Winckler et al., 1999) support the presence of a diffusion barrier at the axon initial segment. Recent elegant experiments tracking the movement of single molecules of the fluorophore- or gold-labeled phospholipid L--dioleoylophosphatidylethanolamine (DOPE) have shown restricted diffusion

of the lipid in the axon initial segment (Nakada et al., 2003), confirming that this region indeed functions as a diffusion barrier in neurons. Further, this barrier is present only in mature neurons and correlates with the formation of clusters of the protein ankyrin, which links the membrane to the actin cytoskeleton at axon initial segments (Berghs et al., 2000; Jenkins and Bennett, 2001). Developmentally, the neuronal diffusion barrier becomes fully effective well after the initial establishment of polarity and may be thought of as a principal means of maintaining distinct axonal and dendritic membrane identities. Targeting Membrane Proteins to Polarized Domains With the establishment of cellular subdomains and polarized membrane delivery mechanisms, individual proteins destined for these domains must be recognized, sorted, and targeted. In fact, nearly all features of neuronal specialization depend on the proper localization of membrane proteins, whether they be neurotransmitter receptors, ion channels, transporters, or adhesion molecules. One of the first steps in membrane protein localization is the segregation of somatodendritic versus axonal proteins. Although this is obviously a fundamental requirement for polarization, exactly how sorting of axonal and dendritic proteins occurs has been the subject of extensive and continuing study. One complicating problem is that there is not one predominant mechanism of protein segregation that functions across all cell types. Hence, in the study of protein sortingas opposed to the establishment of polarity or maintenance of polarized membrane additionexperimental findings in other cell types have not always proven directly applicable to neurons. Protein Sorting Occurs in Both Secretory and Endocytic Pathways In general, membrane protein sorting is accomplished along the secretory pathway, the endocytic pathway, or using a combination of both pathways (Figure 3). Along the secretory pathway, sorting occurs within the Golgi and the trans-Golgi network (TGN). From the Golgi, proteins may be trafficked to the plasma membrane (Liljedahl et al., 2001), to endosomes and lysosomes (Mullins and Bonifacino, 2001), or back to the endoplasmic reticulum (ER) (Girod et al., 1999; White et al., 1999). Of course, resident Golgi proteins must be retained in the face of all of this protein sorting and constant membrane flux. In most polarized epithelial cells, segregation of apical and basolateral proteins occurs at the level of the TGN (Keller et al., 2001). In the prototypical polarized MDCK cell, apical and basolateral proteins are sorted in the TGN into separate classes of post-Golgi carriers, which are transported and fuse preferentially with the apical or basolateral plasma membrane domains (Keller et al., 2001; Kreitzer et al., 2003) (Figure 3A). However, in a number of cell types, trafficking along the endocytic pathway is critical for the proper localization of membrane proteins. In these cases, membrane proteins are usually inserted into the plasma membrane in relatively nonspecific locations, rapidly internalized in a clathrin-dependent manner, and trafficked via transcytosis to the proper membrane domain (Tuma and Hub-

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Figure 3. Polarized Protein Trafficking Occurs along Both Secretory and Endocytic Pathways (A) The polarized MDCK cell is the prototype for studying polarized protein sorting along the secretory pathway. Sorting of apical and basolateral cargo into distinct sets of post-Golgi carriers is the primary mechanism for maintaining polarized repertoires of membrane proteins. These carriers traffic along microtubules to the appropriate plasma membrane domain. (B) In other cell types, such as the hepatocyte schematized here, transcytosis is the primary mechanism for polarized protein trafficking. In this example, cargo is delivered nonselectively to the basolateral membrane. Here, apical proteins (red arrows) are selectively endocytosed via a clathrin-dependent mechanism and transcytosed via endosomes to the apical domain.

bard, 2003). In hepatocytes, for example, transcytosis, rather than sorting at the level of the TGN, is the major mechanism for targeting apical membrane proteins (Bastaki et al., 2002). Even MDCK cells, which rely heavily upon the secretory pathway for proper localization of membrane proteins, constitutively endocytose 50% of their membrane surface in 1 hour (Bomsel et al., 1989). With membrane flux through the endocytic pathway being ten times greater than that through the secretory pathway, membrane targeting through the endocytic pathway must be important for maintaining the proper localization of membrane proteins, even if targeting of post-Golgi carriers is critical for establishing a polarized protein distribution (Bomsel et al., 1989; Mostov et al., 2003). Extensive sorting can take place along the endosomal system because not all endosomes are created equal. An illustrative example of endosomal sorting is that of the immune protein IgR. In gut epithelium, IgR is constitutively internalized from the basolateral membrane. In the absence of ligand, IgR is recycled back to the basolateral surface. However, in the presence of IgA, it is sorted away from basolateral proteins and transcytosed to the apical surface. During transcytosis, IgR traverses basolateral early endosomes, recycling endosomes, and apical endosomes before being inserted into the apical plasma membrane (Rojas and Apodaca, 2002). Hence, a complex series of sorting events can take place within the endosomal system. The dependence of a particular cell type upon either the secretory or endosomal pathways for proper local-

ization of plasma membrane proteins can even vary depending upon the developmental stage of the cell itself. For example, early in polarization of the Fischer rat thyroid cell line, protein sorting occurs mainly via transcytosis. Later in polarizationpresumably as the secretory pathway itself becomes polarizedsorting at the TGN becomes more important (Zurzolo et al., 1992). Notably, early and recycling endosomes are abundant and highly mobile in dendrites (Prekeris et al., 1999) but are largely excluded from axons (West et al., 1997; Wilson et al., 2000). Moreover, distributed Golgi outposts capable of transporting secretory cargo are present in a subpopulation of dendrites (Gardiol et al., 1999; Horton and Ehlers, 2003). Thus, the organelles for neuronal protein sorting may themselves be subject to polarization. From these observations, it is safe to assume that mechanisms segregating axonal from dendritic plasma membrane proteins in neurons operate either along the secretory pathway, at the level of the Golgi/TGN, or via an endocytic pathway (Craig and Banker, 1994; Winckler and Mellman, 1999). However, whether one pathway is more important for protein localization in neurons, whether axonal and dendritic proteins may traverse different pathways, and what signals regulate protein trafficking through the secretory and endocytic pathways remain subjects of intense investigation. Based upon the sorting of viral glycoproteins, Dotti and Simons proposed over a decade ago that neurons utilize the same protein sorting machinery as polarized epithelial cells (Dotti and Simons, 1990). They found that the viral protein influenza hemagglutinin (HA) is localized to the apical surface in epithelial cells and is concentrated on the axonal membrane in neurons. Conversely, the vesicular stomatitis virus (VSV) glycoprotein has a basolateral localization in epithelial cells and is trafficked to the somatodendritic membrane in neurons. Hence, they proposed a model in which the neuronal axonal membrane was analogous to the epithelial apical domain and the neuronal somatodendritic domain was analogous to the epithelial basolateral membrane. Now, some 13 years later, it is clear that important exceptions to the axonal/ apical and dendritic/basolateral model exist (Jareb and Banker, 1998; Poyatos et al., 2000) and the epithelial metaphor for neuronal polarity has important limitations (Colman, 1999). However, this model has provided a useful guide for subsequent studies looking for intrinsic sorting signals that direct proteins to specific membrane domains in epithelial cells and neurons. Sorting Signals Recruit Cargo to Coat Protein Complexes Intrinsic sorting signals, amino acid motifs within a given protein, can direct that protein to specific intracellular organelles or plasma membrane domains (Hunziker et al., 1991). Several general motifs have emerged that function in numerous proteins by interacting with coat protein complexes at the Golgi, on endosomes, or at the plasma membrane. Through these interactions, proteins become concentrated into budding secretory or endocytic vesicles. These signals are mainly cytoplasmic and include tyrosine-based motifs (e.g, NPXY, YXX), monoand dileucine-based motifs (e.g., DXXLL, [DE]XXXL[LI]), and clusters of acidic amino acids (Ikonen and Simons, 1998). The mechanism through which these motifs direct

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trafficking is principally through binding to coat protein components at the cytoplasmic face of membranes (Folsch et al., 1999; Bonifacino and Traub, 2003). Most notable among such coat protein components are the adaptor protein (AP) complexes AP-1, AP-2, and AP-3 (and the less well-characterized AP-4)multisubunit protein complexes that concentrate cargo into clathrin coats prior to budding from the TGN, the plasma membrane, or endosomes, respectively (Bonifacino and Traub, 2003). Both tyrosine-based and dileucine-based sorting signals are known to directly bind to subunits of AP-1, AP-2, AP-3, and AP-4 (Ohno et al., 1995; Owen and Evans, 1998; Schaefer et al., 2002). In some polarized cells, expression of the epithelial cell-specific AP1B isoform is necessary for targeting proteins to the basolateral membrane (Folsch et al., 1999). Neurons also express a version of AP3 with neuron-specific isoforms of the and subunits (Pevsner et al., 1994; Newman et al., 1995; Simpson et al., 1996). These cell type-specific adaptor proteins could account for some of the variability that is observed in protein sorting in different cell types. Another class of coat proteins that are important for recognizing cytoplasmic sorting signals are the Golgilocalized, -ear-containing, ARF binding proteins (GGAs). The GGAs recognize the acidic-cluster-dileucine signals of mannose-6-phosphate receptors (MPRs) in the TGN, an interaction necessary for sorting enzymes to lysosomes from the Golgi (Puertollano et al., 2001; Shiba et al., 2002). GGAs function, at least in part, by interacting with AP-1 and incorporating MPRs into vesicles budding from the TGN via a clathrin-dependent mechanism (Doray et al., 2002). The distribution and function of GGAs in neurons has yet to be examined. As discussed below, a number of the dendritic sorting motifs discovered thus far in neurons are similar to the tyrosine- and dileucine-based motifs described here. Thus, it is probable that at least a subset of dendritic proteins are sorted by incorporation into carriers through coat protein interactions. Dendritic Targeting The ability of dendrites to receive and integrate synaptic inputs requires that a cadre of proteins, including neurotransmitter receptors, adhesion molecules, ion channels, and certain transporters be properly localized with high spatial precision. Many of these proteins, such as glutamate receptors (Stowell and Craig, 1999; Ruberti and Dotti, 2000), are excluded from axons, which have their own unique protein repertoire specialized for propagating action potentials some distance from the cell body and enabling rapid Ca2-dependent neurotransmitter release. The signals which mediate such precise protein targeting in neurons are only beginning to be dissected. One of the first dendritic proteins to yield insight into the molecular mechanisms of dendritic targeting was not a neurotransmitter receptor or an ion channel. Rather, it was the recycling transmembrane transferrin receptor (TfR). This type II membrane protein is a useful model protein for studying dendritic targeting because it is strictly excluded from the axon in cultured neurons (Cameron et al., 1991). Expression of TfR with various N-terminal deletions uncovered a dendritic targeting motif (West et al., 1997) that overlaps the tyrosine-based

rapid internalization motif (Fuller and Simons, 1986). Later, live imaging experiments using a GFP fusion of the TfR revealed that post-Golgi carriers containing TfRGFP were excluded from entering axons (Burack et al., 2000). Hence, it is likely that the tyrosine-based signal sorts the TfR into post-Golgi carriers that selectively deliver their cargo to dendrites. Tyrosine-based motifs have also been shown to be important for the selective dendritic delivery of the polymeric Ig receptor (pIgR) (de Hoop et al., 1995) and the low-density lipoprotein receptor (Brown et al., 1997). A dileucine-based sorting signal has recently been shown to be responsible for the dendritic targeting of the Shal K channel (Rivera et al., 2003). The proper spatial distribution of these multisubunit ion channels is critical for fine-tuning the electrophysiological properties of dendrites and axons, and different K channel families have distinct distributions. For example, certain Shaker K channels are exclusively axonal in CNS neurons (Monaghan et al., 2001), while Shal K channels tend to be somatodendritic (Sheng et al., 1992; Veh et al., 1995). Interestingly, the dileucine motif that directs the dendritic polarization of Shal K channels acts in a dominant fashion, as it redirects the normally axonal Kv1.3 and Kv1.4 to dendrites (Rivera et al., 2003). Novel dendritic targeting motifs have been identified in the C termini of the metabotropic family of glutamate receptors (mGluRs), although the required amino acids have not been precisely mapped. Although normally excluded from axons, mGluR2 can be redirected to axons by attaching the 60 amino acid C terminus of the axonally targeted mGluR7 (Stowell and Craig, 1999). Group I mGluRs, including mGluR1 and mGluR5, are interesting in that their subcellular localization depends upon alternative splicing (Francesconi and Duvoisin, 2002). A tripeptide motif in the C terminus of mGluR1 excludes the splice variant mGluR1b from dendrites. However, this motif is masked by the longer C-terminal tail of mGluR1a, which is polarized to dendrites (Francesconi and Duvoisin, 2002). Despite their importance in excitatory neurotransmission and the overwhelming recent interest in their trafficking related to activity-dependent insertion or endocytosis at synapses (Carroll and Zukin, 2002; Malinow and Malenka, 2002), little is known about what mediates the dendritic polarization of ionotropic glutamate receptors. One clue to the dendritic localization of AMPARs is the observation that the proximal C-terminal tail of the AMPAR subunit GluR1 contains dominant dendritic sorting information. While the cytoplasmic tail of GluR1 is not necessary for its proper localization to dendrites, the proximal C terminus does contain information that is sufficient for rerouting the normally axonal hemagglutinin to dendrites (Ruberti and Dotti, 2000). Further, phosphorylation of a specific residue within the C terminus of GluR1 regulates sorting of the receptor within endosomes toward recycling or degradative pathways (Ehlers, 2000), perhaps contributing to dendritic polarization. It is likely that, for glutamate receptors, sorting signals such as these may function alongside selective retention at the plasma membrane by scaffolding complexes in ensuring the precise spatial localization required for neurotransmission (Sheng and Kim, 2002).

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Axonal Targeting As discussed above, axons contain a repertoire of proteins that are quite distinct from dendrites. Neurotransmitter transporters such as the GABA transporter GAT1 (Minelli et al., 1995), transporters for the biogenic amines (Sur et al., 1996; Nirenberg et al., 1997), and the glycine transporter GLYT2 (Poyatos et al., 2000) are concentrated in presynaptic terminals, along with the proteins required for Ca2-activated neurotransmitter release. Along the axonal shaft, Na and K channels are localized to allow action potential propagation, while axonal adhesion molecules guide axonal outgrowth and facilitate the formation of intercellular synaptic contacts. The search for axonal targeting motifs has revealed disparate signals working along multiple trafficking pathways rather than the stereotypical tyrosine- and dileucinebased motifs uncovered thus far for dendritic targeting. Recently, progress has been made on uncovering the basis for the axonal targeting of the Kv1 (Shaker) family of voltage-gated K channels. Unlike the exclusively dendritic Shal K channels discussed above, certain Shaker K channels are axonal in CNS neurons (Sheng et al., 1992; Veh et al., 1995; Monaghan et al., 2001). The axonal localization of the Kv1.2 member of this family depends upon the N-terminal T1 tetramerization domain and not upon a cytoplasmic tyrosine-based internalization motif or the C-terminal PDZ binding domain (Gu et al., 2003). The Kv1.2 T1 domain also confers axonal polarity when fused to the normally nonpolarized CD4 and the normally dendritic TfR, indicating that this domain may be a general axonal targeting motif that functions in a number of proteins, not only multimeric ion channels (Gu et al., 2003). Another class of axonal targeting motifs has been revealed in NgCAM/L1, a member of the large group of Ig superfamily adhesion molecules that plays an important role in axon guidance and fasciculation (Kamiguchi and Lemmon, 1998). Intracellularly, NgCAM resides in transport carriers and vesicles throughout the axon and dendrites (Burack et al., 2000). However, it appears to reach the plasma membrane only in axons (Sampo et al., 2003) (but see Wisco et al., 2003). The cytoplasmic and transmembrane domains of NgCAM are dispensable for axonal localization in hippocampal neurons. Instead, the five extracellular fibronectin type III-like repeats (FnIII) of NgCAM are both necessary and sufficient to confer axonal targeting (Sampo et al., 2003). However, additional axonal targeting information may reside in the NgCAM cytoplasmic tail, as replacing the C-terminal tail of the LDLR with the NgCAM C terminus redirects the normally nonpolarized LDLR to the axonal membrane, perhaps via a distinct transcytotic mechanism (Wisco et al., 2003). While direct delivery is the primary method for polarizing dendritic proteins (Burack et al., 2000), some axonal proteins are delivered to both neuronal domains and achieve their polarization through selective endocytosis from the dendritic plasma membrane. For example, the sodium channel Nav1.2 is localized to the axon following selective depletion from the somatodendritic membrane (Garrido et al., 2001). This has recently also been shown to be the case for VAMP2 (Sampo et al., 2003), a synaptic vesicle v-SNARE required for exocytic neurotransmitter release at presynaptic terminals. Using live cell antibody

feeding experiments to track the transient appearance of membrane proteins at the somatodendritic surface and subsequent transport into intracellular endosomes, Sampo et al. found that VAMP2 reaches the surface of both axons and dendrites but is selectively endocytosed from the latter (Sampo et al., 2003). Although the precise molecular events remain unclear, one important clue is that the cytoplasmic domain of VAMP2, and in particular a known endocytosis signal (Grote and Kelly, 1996), is required for polarized targeting to the axonal surface (Sampo et al., 2003). This makes sense given the robust endocytosis of VAMP2 from dendrites, and it suggests that this endocytic signal is inoperative over most of the axonal surface, perhaps by restricted distribution of specific endocytic adaptors to dendrites or by selective incorporation of dendritic VAMP2 into endocytic zones (Blanpied et al., 2002). Alternatively, the endocytic signal may be masked in axons via posttranslational modification or protein interactions. In this regard, it is interesting to note that atypical PKC isoforms (e.g., PKC-) that play a central role in axon specification and polarity (Shi et al., 2003) have been reported to phosphorylate VAMP2 and regulate membrane protein localization in nonneuronal cells (Braiman et al., 2001). What is the molecular basis for these distinct targeting mechanisms (Figure 4)? As post-Golgi carriers laden with axon-bound cargo can traffic into both axons and dendrites (Burack et al., 2000), it is possible that downstream mechanisms, such as selective fusion only with the axonal plasma membrane, mediate axonal polarization. There is a precedent for fusion selectivity in MDCK cells, where certain SNAREs are selectively localized to the apical or basolateral domains (Low et al., 1996) (Figure 2C). Intriguingly, the mutations in the T1 domain that affect the interface with the accessory subunit Kv also reduce axonal targeting (Gu et al., 2003). Kv, in turn, interacts directly with syntaxin1A (Fili et al., 2001). It is thus tempting to speculate that the Kv-dependent SNARE interaction allows selective fusion with regions of the axonal plasma membrane decorated with this or other SNAREs. On the other hand, how the FnIII repeats within the extracellular domain of NgCAM confer axonal targeting remains mysterious but could similarly involve a recruitment of NgCAM into a subpopulation of postGolgi transport carriers destined for axonal exocytosis. However, there is not complete agreement on these results, as NgCAM may also undergo rapid endocytosis from the dendritic cell surface (Wisco et al., 2003) by virtue of a well-characterized tyrosine-based endocytosis motif (Kamiguchi and Lemmon, 1998). Although further mechanistic clarifications will be necessary, these results raise the intriguing possibility that both selective delivery and selective retention mechanisms operate (Figure 4). These data raise a number of questions. First, a major point of contention is whether NgCAM actually resides transiently on the somatodendritic domain before reaching the axonal surface. This issue must be definitively resolved before one can say whether selective fusion or transcytosis is the most appropriate model for axonal protein targeting. In any case, the finding of an extracellularly situated axonal targeting motif is quite interesting in light of recent studies demonstrating crucial roles for extracellular domains in the targeting of apical mem-

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the distribution of the organelles and adaptors of the endocytic machinery, such that they function selectively within a given domain. Further, it is not known what factors may determine the fusion competence of carriers of axonal versus dendritic proteins, although plasma membrane subdomains rich in axon- or dendrite-specific t-SNARES is one possibility. Third, proteins that are delivered to both axonal and somatodendritic domains must seemingly reside in distinct transport carriers from proteins selectively delivered to the axonal plasma membrane. But what is the nature of these carriers and where are they derived from? Are they defined by distinct sets of SNARE proteins, motor molecules, and Rab GTPases? Further, where along the secretory pathway do proteins destined for the soma and dendrites diverge from those destined for the axonal hinterland? Ultimately, answering these questions will require expanding our knowledge of the entire life cycle of membrane proteins destined for axons and dendritesfrom their initial synthesis at the endoplasmic reticulum to their incorporation at correct plasma membrane sites. Paramount will be defining the cellular distribution, molecular composition, cargo utilization, and dynamic behavior of relevant organelles and transport carriers. Beyond a Binary View of Neuronal Polarity The establishment and maintenance of axonal and somatodendritic domains is the most apparent and most fundamental level of neuronal polarity. However, this two-compartment model is a gross oversimplification of neuronal specialization. Neither axons nor dendrites are homogeneous membranes. Each has specialized subdomains whose function requires that proteins be targeted much more specifically than simple axonal or dendritic segregation (for further discussion, see the reviews by Bredt and Nicoll [2003]; Goda and Davis [2003]; and Salzer [2003] in this issue of Neuron). For example, somatodendritically targeted K channels can be restricted to just the most proximal segments by specific targeting motifs (Lim et al., 2000). On the other hand, electrophysiological experiments have demonstrated a progressive increase in the number of AMPAtype glutamate receptors (and an associated increase in synaptic conductance) at distal dendrites of CA1 pyramidal neurons in the hippocampus (Stricker et al., 1996; Magee and Cook, 2000; Andrasfalvy and Magee, 2001). Such studies suggest dendritic polarization in the proximal-distal dimension. Even more clear is the morphological differences in specific dendritic arbors (see Jan and Jan [2003], this issue of Neuron), with the classic apical versus basolateral dendritic arborizations of pyramidal neurons being a prime example of polarity within the somatodendritic domain. Such dendritic polarization may be accomplished by extrinsic signals such as secreted semaphorins (Polleux et al., 2000) or neurotrophins (Huang and Reichardt, 2003) and the intrinsic action of cytoskeletal regulatory proteins and morphologycontrolling genes (Gao et al., 2000; Brenman et al., 2001). How such signals impinge on the cytoskeletal architecture and associated organization of membrane trafficking systems in dendrites remain crucial questions for future inquiry.

Figure 4. Multiple Mechanisms Function in Neurons for the Targeting of Dendritic and Axonal Proteins (A) In this schematized model, axonal and dendritic proteins are sorted at the level of the Golgi into carriers that preferentially deliver their cargo to axons or dendrites, respectively. (B) In some cases, carriers of axonal proteins can traffic within dendrites but are not competent to fuse with the dendritic membrane, ensuring selective surface delivery only in the axon. (C) In other cases, axonal proteins are delivered to the dendritic plasma membrane alongside dendritic proteins, either in distinct or perhaps in common post-Golgi carriers. After a transient period on the cell surface, axonal proteins are selectively endocytosed and trafficked via transcytosis to the axonal membrane.

brane proteins in epithelial cells (Nelson and Yeaman, 2001). It will be important to identify the relevant protein binding partners of the NgCAM FnIII ectodomain and to determine the generality of this mechanism for targeting other axonal proteins. In this regard, it is worth noting that the third FnIII domain of NgCAM/L1 spontaneously homomultimerizes, binds to integrins, and is subject to proteolytic cleavage by plasmin (Silletti et al., 2000), raising the possibility that axonal targeting may be regulated by protein assembly or proteolytic events. Second, why are some proteins, like VAMP2, retained on the axonal membrane and selectively endocytosed from the somatodendritic membrane? One possibility is that there are dendrite-specific endocytosis factors required for the polarization of membrane proteins via transcytosis. Or perhaps endocytic signals are selectively masked in axons (e.g., by protein interactions or posttranslational modifications). Alternately, endocytic adaptors themselves may be polarized to axons or dendrites. It will be important to determine what regulates

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Axons are likewise distinguished by specialized subdomains created by sophisticated trafficking mechanisms and cytoskeletal rearrangements. Notable among these subdomains are the axon initial segment, where action potentials are generated, and the nodes of Ranvier that enable saltatory conduction along myelinated axons (Salzer, 2002). Each of these regions requires a high density of Na channels for proper function (Catterall, 1981). Na channels at the initial segment are selectively clustered in a complex consisting of ankyrin G, neurofascin, and the cytoskeletal protein spectrin IV (Zhou et al., 1998; Jenkins and Bennett, 2001), which is itself targeted specifically to nodes and the initial segment (Berghs et al., 2000). Intriguingly, in the retinal ganglion cell axon, Na channels are distributed in an isoform-specific manner, with Nav1.2 distributed diffusely along the unmyelinated axonal region, while Nav1.6 is selectively delivered to the initial segment and the nodes of the myelinated region in a developmentally regulated manner (Boiko et al., 2001, 2003; Kaplan et al., 2001). Na channels are then retained at the nodes via a local diffusion barrier provided by interactions between the paranodal glial membrane and the axon (Rios et al., 2003). In this way, trafficking and retention of Na channels provides a functional specialization of different axonal regions (see the review by Salzer, in this issue). Might the selective delivery of two Na channel isoforms to different axonal regions indicate a level of post-Golgi sorting beyond simply axonal versus somatodendritic? Live cell imaging experiments of axonally targeted proteins indicate that different proteins can in fact be trafficked via distinct carrier types (Kaether et al., 2000). Another axonal function that requires selective delivery to the plasma membrane is the process of outgrowth. During axonal outgrowth, while the primary growth cone extends, the growth of collateral axonal branches is suppressed (Kalil et al., 2000). This process requires the microtubule-based motor KIF2A, suggesting a role for directed vesicular transport (Homma et al., 2003). There is also the concept of planar cell polarity, the multicellular polarity whereby epithelial cells and other groups of cells acquire polarity orthogonal to their apical-basal axes (i.e., within the plane of the epithelia) (Mlodzik, 2002). Examples include the aligned orientation of stereocilial bundles in hair cells of the inner ear (Montcouquiol et al., 2003), actin spikes in Drosophila wing cells (Eaton, 1997), and ommatidia within the fly eye (Mlodzik, 1999). Due to the lack of obvious permanent cues along different body axes and the requirement for tissue-level organization, planar cell polarity is necessarily more complex than apical-basolateral polarity. Genetic studies in Drosophila have revealed a prominent role for the frizzled (Fz)/disheveled (Dsh) pathway, as well as other genes such as Rho family GTPases and flamingo which, intriguingly, play important roles in cell polarity (Etienne-Manneville and Hall, 2001; Habas et al., 2003; Wittmann et al., 2003) and dendrite morphogenesis (Luo, 2002; see Jan and Jan, this issue). In the inner ear, the transmembrane PDZ protein Vangl2, the mammalian homolog of the Drosophila Van Gogh/strabismus tissue polarity gene, controls planar organization such that in the loop-tail mouse, which has a mutation in Vangl2, the stereociliary bundles of cochlear hair cells

are not uniformly aligned (Montcouquiol et al., 2003). It remains to be seen whether planar cell polarity processes control higher-order organization of neural tissues, but some structures, such as cortical layers or retinal laminae, may be particularly well-suited. Conclusions More study is clearly needed. The identification of the Par3/Par6/aPKC complex as an evolutionarily conserved polarity signal that specifies axon identity raises many questions about how this complex is localized and regulated in developing and mature neurons. Determining the signals that operate upstream and downstream of Par3/Par6/aPKC, including potential extrinsic signals directing polarization, will be a critical next step. Furthermore, our understanding of organelle organization and membrane trafficking systems in axons and dendrites remains in its infancy. How organelles and transport machinery become partitioned and polarized is a crucial topic for future investigation. In particular, very little knowledge exists regarding the regulation of secretory organelles and endocytic sorting compartments during neuronal polarization. In addition, identification of additional intrinsic sorting signals and the protein (or lipid) machinery that recognizes them will assist in a molecular dissection of neuronal polarity and protein targeting. Perhaps even more important will be monitoring the life cycle of individual proteins in living neurons from the point of initial synthesis to their arrival at their ultimate cellular destination. Also important will be deciphering the relationship between protein sorting for purposes of polarity and sorting utilized to maintain more localized membrane microdomains along axonal and dendritic segments. In this regard, uncovering links between the general protein trafficking machinery and neuron-specific mechanisms that specify axons and dendrites will help illuminate the process of making processes.
Acknowledgments We thank Vann Bennett, Marc Caron, Juliet Hernandez, Chris Nicchitta, Isabel Perez-Otano, Derek Scott, and Ryan Terry-Lorenzo for critical comments and suggestions. In addition, we apologize to those whose work we did not cite due to space limitations. Work from the laboratory of M.D.E. is supported by the National Institutes of Health (NS39402 and MH64748) and a grant from the Ruth K. Broad Biomedical Research Foundation. A.C.H. has received support from the Medical Scientist Training Program of the NIH and a Broad Foundation Fellowship. References Adams, A.E., Johnson, D.I., Longnecker, R.M., Sloat, B.F., and Pringle, J.R. (1990). CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae. J. Cell Biol. 111, 131142. Andrasfalvy, B.K., and Magee, J.C. (2001). Distance-dependent increase in AMPA receptor number in the dendrites of adult hippocampal CA1 pyramidal neurons. J. Neurosci. 21, 91519159. Augustine, G.J., Santamaria, F., and Tanaka, K. (2003). Local Calcium Signaling in Neurons. Neuron 40, this issue, 331346. Augustine, G.J., Burns, M.E., DeBello, W.M., Hilfiker, S., Morgan, J.R., Schweizer, F.E., Tokumaru, H., and Umayahara, K. (1999). Proteins involved in synaptic vesicle trafficking. J. Physiol. 520, 3341.

Neuron 290

Baas, P.W. (1999). Microtubules and neuronal polarity: lessons from mitosis. Neuron 22, 2331. Baas, P.W., and Ahmad, F.J. (1993). The transport properties of axonal microtubules establish their polarity orientation. J. Cell Biol. 120, 14271437. Baas, P.W., Deitch, J.S., Black, M.M., and Banker, G.A. (1988). Polarity orientation of microtubules in hippocampal neurons: uniformity in the axon and nonuniformity in the dendrite. Proc. Natl. Acad. Sci. USA 85, 83358339. Baas, P.W., Black, M.M., and Banker, G.A. (1989). Changes in microtubule polarity orientation during the development of hippocampal neurons in culture. J. Cell Biol. 109, 30853094. Bacallao, R., Antony, C., Dotti, C., Karsenti, E., Stelzer, E.H., and Simons, K. (1989). The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium. J. Cell Biol. 109, 28172832. Balda, M.S., Whitney, J.A., Flores, C., Gonzalez, S., Cereijido, M., and Matter, K. (1996). Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J. Cell Biol. 134, 1031 1049. Bastaki, M., Braiterman, L.T., Johns, D.C., Chen, Y.H., and Hubbard, A.L. (2002). Absence of direct delivery for single transmembrane apical proteins or their Secretory forms in polarized hepatic cells. Mol. Biol. Cell 13, 225237. Berghs, S., Aggujaro, D., Dirkx, R., Jr., Maksimova, E., Stabach, P., Hermel, J.M., Zhang, J.P., Philbrick, W., Slepnev, V., Ort, T., and Solimena, M. (2000). betaIV spectrin, a new spectrin localized at axon initial segments and nodes of ranvier in the central and peripheral nervous system. J. Cell Biol. 151, 9851002. Bilder, D. (2001). PDZ proteins and polarity: functions from the fly. Trends Genet. 17, 511519. Bilder, D., and Perrimon, N. (2000). Localization of apical epithelial determinants by the basolateral PDZ protein Scribble. Nature 403, 676680. Bilder, D., Li, M., and Perrimon, N. (2000). Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289, 113116. Bilder, D., Schober, M., and Perrimon, N. (2003). Integrated activity of PDZ protein complexes regulates epithelial polarity. Nat. Cell Biol. 5, 5358. Binder, L.I., Frankfurter, A., and Rebhun, L.I. (1985). The distribution of tau in the mammalian central nervous system. J. Cell Biol. 101, 13711378. Blanpied, T.A., Scott, D.B., and Ehlers, M.D. (2002). Dynamics and regulation of clathrin coats at specialized endocytic zones of dendrites and spines. Neuron 36, 435449. Boiko, T., Rasband, M.N., Levinson, S.R., Caldwell, J.H., Mandel, G., Trimmer, J.S., and Matthews, G. (2001). Compact myelin dictates the differential targeting of two sodium channel isoforms in the same axon. Neuron 30, 91104. Boiko, T., Van Wart, A., Caldwell, J.H., Levinson, S.R., Trimmer, J.S., and Matthews, G. (2003). Functional specialization of the axon initial segment by isoform-specific sodium channel targeting. J. Neurosci. 23, 23062313. Bomsel, M., Prydz, K., Parton, R.G., Gruenberg, J., and Simons, K. (1989). Endocytosis in filter-grown Madin-Darby canine kidney cells. J. Cell Biol. 109, 32433258. Bonifacino, J.S., and Traub, L.M. (2003). Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72, 395447. Borgdorff, A.J., and Choquet, D. (2002). Regulation of AMPA receptor lateral movements. Nature 417, 649653. Bourrat, F., and Sotelo, C. (1988). Migratory pathways and neuritic differentiation of inferior olivary neurons in the rat embryo. Axonal tracing study using the in vitro slab technique. Brain Res. 467, 1937. Bradke, F., and Dotti, C.G. (1999). The role of local actin instability in axon formation. Science 283, 19311934.

Braiman, L., Alt, A., Kuroki, T., Ohba, M., Bak, A., Tennenbaum, T., and Sampson, S.R. (2001). Activation of protein kinase C zeta induces serine phosphorylation of VAMP2 in the GLUT4 compartment and increases glucose transport in skeletal muscle. Mol. Cell. Biol. 21, 78527861. Bredt, D.S., and Nicoll, R.A. (2003). AMPA receptor trafficking at excitatory synapses. Neuron 40, this issue, 361379. Brenman, J.E., Gao, F.B., Jan, L.Y., and Jan, Y.N. (2001). Sequoia, a tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila. Dev. Cell 1, 667677. Bretscher, M.S. (1996). Getting membrane flow and the cytoskeleton to cooperate in moving cells. Cell 87, 601606. Brown, M.D., Banker, G.A., Hussaini, I.M., Gonias, S.L., and VandenBerg, S.R. (1997). Low density lipoprotein receptor-related protein is expressed early and becomes restricted to a somatodendritic domain during neuronal differentiation in culture. Brain Res. 747, 313317. Burack, M.A., Silverman, M.A., and Banker, G. (2000). The role of selective transport in neuronal protein sorting. Neuron 26, 465472. Cameron, P.L., Sudhof, T.C., Jahn, R., and De Camilli, P. (1991). Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis. J. Cell Biol. 115, 151164. Campbell, D.S., Regan, A.G., Lopez, J.S., Tannahill, D., Harris, W.A., and Holt, C.E. (2001). Semaphorin 3A elicits stage-dependent collapse, turning, and branching in Xenopus retinal growth cones. J. Neurosci. 21, 85388547. Carroll, R.C., and Zukin, R.S. (2002). NMDA-receptor trafficking and targeting: implications for synaptic transmission and plasticity. Trends Neurosci. 25, 571577. Catterall, W.A. (1981). Localization of sodium channels in cultured neural cells. J. Neurosci. 1, 777783. Chant, J. (1999). Cell polarity in yeast. Annu. Rev. Cell Dev. Biol. 15, 365391. Chant, J., and Herskowitz, I. (1991). Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway. Cell 65, 12031212. Chen, Y.A., and Scheller, R.H. (2001). SNARE-mediated membrane fusion. Nat. Rev. Mol. Cell Biol. 2, 98106. Colman, D.R. (1999). Neuronal polarity and the epithelial metaphor. Neuron 23, 649651. Craig, A.M., and Banker, G. (1994). Neuronal polarity. Annu. Rev. Neurosci. 17, 267310. Craig, A.M., Wyborski, R.J., and Banker, G. (1995). Preferential addition of newly synthesized membrane protein at axonal growth cones. Nature 375, 592594. da Silva, J.S., and Dotti, C.G. (2002). Breaking the neuronal sphere: regulation of the actin cytoskeleton in neuritogenesis. Nat. Rev. Neurosci. 3, 694704. De Almeida, J.B., and Stow, J.L. (1991). Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells. Am. J. Physiol. 261, C691C700. de Hoop, M., von Poser, C., Lange, C., Ikonen, E., Hunziker, W., and Dotti, C.G. (1995). Intracellular routing of wild-type and mutated polymeric immunoglobulin receptor in hippocampal neurons in culture. J. Cell Biol. 130, 14471459. Deitch, J.S., and Banker, G.A. (1993). An electron microscopic analysis of hippocampal neurons developing in culture: early stages in the emergence of polarity. J. Neurosci. 13, 43014315. den Hollander, A.I., Ghiani, M., de Kok, Y.J., Wijnholds, J., Ballabio, A., Cremers, F.P., and Broccoli, V. (2002). Isolation of Crb1, a mouse homologue of Drosophila crumbs, and analysis of its expression pattern in eye and brain. Mech. Dev. 110, 203207. Doray, B., Ghosh, P., Griffith, J., Geuze, H.J., and Kornfeld, S. (2002). Cooperation of GGAs and AP-1 in packaging MPRs at the transGolgi network. Science 297, 17001703. Dotti, C.G., and Banker, G.A. (1987). Experimentally induced alteration in the polarity of developing neurons. Nature 330, 254256.

Review 291

Dotti, C.G., and Simons, K. (1990). Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture. Cell 62, 6372. Dotti, C.G., Sullivan, C.A., and Banker, G.A. (1988). The establishment of polarity by hippocampal neurons in culture. J. Neurosci. 8, 14541468. Eaton, S. (1997). Planar polarization of Drosophila and vertebrate epithelia. Curr. Opin. Cell Biol. 9, 860866. Ehlers, M.D. (2000). Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting. Neuron 28, 511525. Etemad-Moghadam, B., Guo, S., and Kemphues, K.J. (1995). Asymmetrically distributed PAR-3 protein contributes to cell polarity and spindle alignment in early C. elegans embryos. Cell 83, 743752. Etienne-Manneville, S., and Hall, A. (2001). Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta. Cell 106, 489498. Etienne-Manneville, S., and Hall, A. (2002). Rho GTPases in cell biology. Nature 420, 629635. Etienne-Manneville, S., and Hall, A. (2003). Cdc42 regulates GSK3beta and adenomatous polyposis coli to control cell polarity. Nature 421, 753756. Fannon, A.M., and Colman, D.R. (1996). A model for central synaptic junctional complex formation based on the differential adhesive specificities of the cadherins. Neuron 17, 423434. Farquhar, M.G., and Palade, G.E. (1963). Junctional complexes in various epithelia. J. Cell Biol. 17, 375412. Fili, O., Michaelevski, I., Bledi, Y., Chikvashvili, D., Singer-Lahat, D., Boshwitz, H., Linial, M., and Lotan, I. (2001). Direct interaction of a brain voltage-gated K channel with syntaxin 1A: functional impact on channel gating. J. Neurosci. 21, 19641974. Finger, F.P., Hughes, T.E., and Novick, P. (1998). Sec3p is a spatial landmark for polarized secretion in budding yeast. Cell 92, 559571. Foletti, D.L., Prekeris, R., and Scheller, R.H. (1999). Generation and maintenance of neuronal polarity: mechanisms of transport and targeting. Neuron 23, 641644. Folsch, H., Ohno, H., Bonifacino, J.S., and Mellman, I. (1999). A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells. Cell 99, 189198. Francesconi, A., and Duvoisin, R.M. (2002). Alternative splicing unmasks dendritic and axonal targeting signals in metabotropic glutamate receptor 1. J. Neurosci. 22, 21962205. Fujiwara, T., Ritchie, K., Murakoshi, H., Jacobson, K., and Kusumi, A. (2002). Phospholipids undergo hop diffusion in compartmentalized cell membrane. J. Cell Biol. 157, 10711081. Fukata, Y., Itoh, T.J., Kimura, T., Menager, C., Nishimura, T., Shiromizu, T., Watanabe, H., Inagaki, N., Iwamatsu, A., Hotani, H., and Kaibuchi, K. (2002a). CRMP-2 binds to tubulin heterodimers to promote microtubule assembly. Nat. Cell Biol. 4, 583591. Fukata, Y., Kimura, T., and Kaibuchi, K. (2002b). Axon specification in hippocampal neurons. Neurosci. Res. 43, 305315. Fuller, S.D., and Simons, K. (1986). Transferrin receptor polarity and recycling accuracy in tight and leaky strains of Madin-Darby canine kidney cells. J. Cell Biol. 103, 17671779. Galli, T., Zahraoui, A., Vaidyanathan, V.V., Raposo, G., Tian, J.M., Karin, M., Niemann, H., and Louvard, D. (1998). A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells. Mol. Biol. Cell 9, 14371448. Gao, F.B., Kohwi, M., Brenman, J.E., Jan, L.Y., and Jan, Y.N. (2000). Control of dendritic field formation in Drosophila: the roles of flamingo and competition between homologous neurons. Neuron 28, 91101. Gardiol, A., Racca, C., and Triller, A. (1999). Dendritic and postsynaptic protein synthetic machinery. J. Neurosci. 19, 168179. Garrido, J.J., Fernandes, F., Giraud, P., Mouret, I., Pasqualini, E., Fache, M.P., Jullien, F., and Dargent, B. (2001). Identification of an

axonal determinant in the C-terminus of the sodium channel Na(v)1.2. EMBO J. 20, 59505961. Gilbert, T., Le Bivic, A., Quaroni, A., and Rodriguez-Boulan, E. (1991). Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells. J. Cell Biol. 113, 275288. Girod, A., Storrie, B., Simpson, J.C., Johannes, L., Goud, B., Roberts, L.M., Lord, J.M., Nilsson, T., and Pepperkok, R. (1999). Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum. Nat. Cell Biol. 1, 423430. Goda, Y., and Davis, G.W. (2003). Mechanisms of synapse assembly and disassembly. Neuron 40, this issue, 243264. Goldstein, L.S.B. (2003). Do disorders of movement cause movement disorders and dementia? Neuron 40, this issue, 415425. Goslin, K., and Banker, G. (1989). Experimental observations on the development of polarity by hippocampal neurons in culture. J. Cell Biol. 108, 15071516. Goslin, K., Schreyer, D.J., Skene, J.H., and Banker, G. (1988). Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones. Nature 336, 672674. Gotta, M., Abraham, M.C., and Ahringer, J. (2001). CDC-42 controls early cell polarity and spindle orientation in C. elegans. Curr. Biol. 11, 482488. Grindstaff, K.K., Yeaman, C., Anandasabapathy, N., Hsu, S.C., Rodriguez-Boulan, E., Scheller, R.H., and Nelson, W.J. (1998). Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells. Cell 93, 731740. Grosse, G., Grosse, J., Tapp, R., Kuchinke, J., Gorsleben, M., Fetter, I., Hohne-Zell, B., Gratzl, M., and Bergmann, M. (1999). SNAP-25 requirement for dendritic growth of hippocampal neurons. J. Neurosci. Res. 56, 539546. Grote, E., and Kelly, R.B. (1996). Endocytosis of VAMP is facilitated by a synaptic vesicle targeting signal. J. Cell Biol. 132, 537547. Gu, C., Jan, Y.N., and Jan, L.Y. (2003). A conserved domain in axonal targeting of Kv1 (Shaker) voltage-gated potassium channels. Science 301, 646649. Guo, W., Roth, D., Walch-Solimena, C., and Novick, P. (1999). The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. EMBO J. 18, 10711080. Habas, R., Dawid, I.B., and He, X. (2003). Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrulation. Genes Dev. 17, 295309. Harris, B.Z., and Lim, W.A. (2001). Mechanism and role of PDZ domains in signaling complex assembly. J. Cell Sci. 114, 32193231. Hartmann, M., Heumann, R., and Lessmann, V. (2001). Synaptic secretion of BDNF after high-frequency stimulation of glutamatergic synapses. EMBO J. 20, 58875897. Hazuka, C.D., Foletti, D.L., Hsu, S.C., Kee, Y., Hopf, F.W., and Scheller, R.H. (1999). The sec6/8 complex is located at neurite outgrowth and axonal synapse-assembly domains. J. Neurosci. 19, 13241334. Hirose, T., Izumi, Y., Nagashima, Y., Tamai-Nagai, Y., Kurihara, H., Sakai, T., Suzuki, Y., Yamanaka, T., Suzuki, A., Mizuno, K., and Ohno, S. (2002). Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation. J. Cell Sci. 115, 24852495. Homma, N., Takei, Y., Tanaka, Y., Nakata, T., Terada, S., Kikkawa, M., Noda, Y., and Hirokawa, N. (2003). Kinesin superfamily protein 2A (KIF2A) functions in suppression of collateral branch extension. Cell 114, 229239. Horton, A.C., and Ehlers, M.D. (2003). Dual modes of endoplasmic reticulum-to-Golgi transport in dendrites revealed by live-cell imaging. J. Neurosci. 23, 61886199. Hsu, S.C., Ting, A.E., Hazuka, C.D., Davanger, S., Kenny, J.W., Kee, Y., and Scheller, R.H. (1996). The mammalian brain rsec6/8 complex. Neuron 17, 12091219. Hu, C., Ahmed, M., Melia, T.J., Sollner, T.H., Mayer, T., and Rothman, J.E. (2003). Fusion of cells by flipped SNAREs. Science 300, 1745 1749.

Neuron 292

Huang, E.J., and Reichardt, L.F. (2003). TRK receptors: Roles in neuronal signal transduction. Annu. Rev. Biochem. 72, 609642. Huber, A.B., Kolodkin, A.L., Ginty, D.D., and Cloutier, J.F. (2003). Signaling at the growth cone: Ligand-receptor complexes and the control of axon growth and guidance. Annu. Rev. Neurosci. 26, 509563. Hung, T.J., and Kemphues, K.J. (1999). PAR-6 is a conserved PDZ domain-containing protein that colocalizes with PAR-3 in Caenorhabditis elegans embryos. Development 126, 127135. Hunziker, W., Harter, C., Matter, K., and Mellman, I. (1991). Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant. Cell 66, 907920. Igarashi, M., Kozaki, S., Terakawa, S., Kawano, S., Ide, C., and Komiya, Y. (1996). Growth cone collapse and inhibition of neurite growth by Botulinum neurotoxin C1: a t-SNARE is involved in axonal growth. J. Cell Biol. 134, 205215. Ikonen, E., and Simons, K. (1998). Protein and lipid sorting from the trans-Golgi network to the plasma membrane in polarized cells. Semin. Cell Dev. Biol. 9, 503509. Inagaki, N., Chihara, K., Arimura, N., Menager, C., Kawano, Y., Matsuo, N., Nishimura, T., Amano, M., and Kaibuchi, K. (2001). CRMP-2 induces axons in cultured hippocampal neurons. Nat. Neurosci. 4, 781782. Itoh, M., Sasaki, H., Furuse, M., Ozaki, H., Kita, T., and Tsukita, S. (2001). Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions. J. Cell Biol. 154, 491497. Jacob, R., and Naim, H.Y. (2001). Apical membrane proteins are transported in distinct vesicular carriers. Curr. Biol. 11, 14441450. Jahn, R., Lang, T., and Sudhof, T.C. (2003). Membrane fusion. Cell 112, 519533. Jan, Y.-N., and Jan, L.Y. (2003). The control of dendrite development. Neuron 40, this issue, 229242. Jareb, M., and Banker, G. (1998). The polarized sorting of membrane proteins expressed in cultured hippocampal neurons using viral vectors. Neuron 20, 855867. Jenkins, S.M., and Bennett, V. (2001). Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments. J. Cell Biol. 155, 739746. Joberty, G., Petersen, C., Gao, L., and Macara, I.G. (2000). The cellpolarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat. Cell Biol. 2, 531539. Johansson, A., Driessens, M., and Aspenstrom, P. (2000). The mammalian homologue of the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for the Rho GTPases Cdc42 and Rac1. J. Cell Sci. 113, 32673275. Kaether, C., Skehel, P., and Dotti, C.G. (2000). Axonal membrane proteins are transported in distinct carriers: a two-color video microscopy study in cultured hippocampal neurons. Mol. Biol. Cell 11, 12131224. Kalil, K., Szebenyi, G., and Dent, E.W. (2000). Common mechanisms underlying growth cone guidance and axon branching. J. Neurobiol. 44, 145158. Kamiguchi, H., and Lemmon, V. (1998). A neuronal form of the cell adhesion molecule L1 contains a tyrosine-based signal required for sorting to the axonal growth cone. J. Neurosci. 18, 37493756. Kaplan, M.R., Cho, M.H., Ullian, E.M., Isom, L.L., Levinson, S.R., and Barres, B.A. (2001). Differential control of clustering of the sodium channels Na(v)1.2 and Na(v)1.6 at developing CNS nodes of Ranvier. Neuron 30, 105119. Keller, P., Toomre, D., Diaz, E., White, J., and Simons, K. (2001). Multicolour imaging of post-Golgi sorting and trafficking in live cells. Nat. Cell Biol. 3, 140149. Knust, E., and Bossinger, O. (2002). Composition and formation of intercellular junctions in epithelial cells. Science 298, 19551959. Knust, E., Tepass, U., and Wodarz, A. (1993). crumbs and stardust, two genes of Drosophila required for the development of epithelial cell polarity. Dev. Suppl., 261268.

Kobayashi, T., Storrie, B., Simons, K., and Dotti, C.G. (1992). A functional barrier to movement of lipids in polarized neurons. Nature 359, 647650. Kreitzer, G., Marmorstein, A., Okamoto, P., Vallee, R., and Rodriguez-Boulan, E. (2000). Kinesin and dynamin are required for postGolgi transport of a plasma-membrane protein. Nat. Cell Biol. 2, 125127. Kreitzer, G., Schmoranzer, J., Low, S.H., Li, X., Gan, Y., Weimbs, T., Simon, S.M., and Rodriguez-Boulan, E. (2003). Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells. Nat. Cell Biol. 5, 126136. Lamoureux, P., Ruthel, G., Buxbaum, R.E., and Heidemann, S.R. (2002). Mechanical tension can specify axonal fate in hippocampal neurons. J. Cell Biol. 159, 499508. Lang, T., Bruns, D., Wenzel, D., Riedel, D., Holroyd, P., Thiele, C., and Jahn, R. (2001). SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis. EMBO J. 20, 22022213. Lee, S., Fan, S., Makarova, O., Straight, S., and Margolis, B. (2002). A novel and conserved protein-protein interaction domain of mammalian Lin-2/CASK binds and recruits SAP97 to the lateral surface of epithelia. Mol. Cell. Biol. 22, 17781791. Lein, P., Johnson, M., Guo, X., Rueger, D., and Higgins, D. (1995). Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons. Neuron 15, 597605. Lemmers, C., Medina, E., Delgrossi, M.H., Michel, D., Arsanto, J.P., and Le Bivic, A. (2002). hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells. J. Biol. Chem. 277, 2540825415. Li, L., and Chin, L.S. (2003). The molecular machinery of synaptic vesicle exocytosis. Cell. Mol. Life Sci. 60, 942960. Liljedahl, M., Maeda, Y., Colanzi, A., Ayala, I., Van Lint, J., and Malhotra, V. (2001). Protein kinase D regulates the fission of cell surface destined transport carriers from the trans-Golgi network. Cell 104, 409420. Lim, S.T., Antonucci, D.E., Scannevin, R.H., and Trimmer, J.S. (2000). A novel targeting signal for proximal clustering of the Kv2.1 K channel in hippocampal neurons. Neuron 25, 385397. Lipschutz, J.H., Guo, W., OBrien, L.E., Nguyen, Y.H., Novick, P., and Mostov, K.E. (2000). Exocyst is involved in cystogenesis and tubulogenesis and acts by modulating synthesis and delivery of basolateral plasma membrane and secretory proteins. Mol. Biol. Cell 11, 42594275. Low, S.H., Chapin, S.J., Weimbs, T., Komuves, L.G., Bennett, M.K., and Mostov, K.E. (1996). Differential localization of syntaxin isoforms in polarized Madin-Darby canine kidney cells. Mol. Biol. Cell 7, 2007 2018. Low, S.H., Chapin, S.J., Wimmer, C., Whiteheart, S.W., Komuves, L.G., Mostov, K.E., and Weimbs, T. (1998). The SNARE machinery is involved in apical plasma membrane trafficking in MDCK cells. J. Cell Biol. 141, 15031513. Luo, L. (2002). Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity. Annu. Rev. Cell Dev. Biol. 18, 601635. Luo, L., Liao, Y.J., Jan, L.Y., and Jan, Y.N. (1994). Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev. 8, 17871802. Luo, L., Hensch, T.K., Ackerman, L., Barbel, S., Jan, L.Y., and Jan, Y.N. (1996). Differential effects of the Rac GTPase on Purkinje cell axons and dendritic trunks and spines. Nature 379, 837840. Magee, J.C., and Cook, E.P. (2000). Somatic EPSP amplitude is independent of synapse location in hippocampal pyramidal neurons. Nat. Neurosci. 3, 895903. Maletic-Savatic, M., and Malinow, R. (1998). Calcium-evoked dendritic exocytosis in cultured hippocampal neurons. Part I: transGolgi network-derived organelles undergo regulated exocytosis. J. Neurosci. 18, 68036813. Malinow, R., and Malenka, R.C. (2002). AMPA receptor trafficking and synaptic plasticity. Annu. Rev. Neurosci. 25, 103126.

Review 293

Martinez-Arca, S., Coco, S., Mainguy, G., Schenk, U., Alberts, P., Bouille, P., Mezzina, M., Prochiantz, A., Matteoli, M., Louvard, D., and Galli, T. (2001). A common exocytotic mechanism mediates axonal and dendritic outgrowth. J. Neurosci. 21, 38303838. Matus, A., Bernhardt, R., and Hugh-Jones, T. (1981). High molecular weight microtubule-associated proteins are preferentially associated with dendritic microtubules in brain. Proc. Natl. Acad. Sci. USA 78, 30103014. McAllister, A.K., Katz, L.C., and Lo, D.C. (1997). Opposing roles for endogenous BDNF and NT-3 in regulating cortical dendritic growth. Neuron 18, 767778. McNew, J.A., Parlati, F., Fukuda, R., Johnston, R.J., Paz, K., Paumet, F., Sollner, T.H., and Rothman, J.E. (2000). Compartmental specificity of cellular membrane fusion encoded in SNARE proteins. Nature 407, 153159. Medina, E., Lemmers, C., Lane-Guermonprez, L., and Le Bivic, A. (2002). Role of the Crumbs complex in the regulation of junction formation in Drosophila and mammalian epithelial cells. Biol. Cell. 94, 305313. Minelli, A., Brecha, N.C., Karschin, C., DeBiasi, S., and Conti, F. (1995). GAT-1, a high-affinity GABA plasma membrane transporter, is localized to neurons and astroglia in the cerebral cortex. J. Neurosci. 15, 77347746. Mlodzik, M. (1999). Planar polarity in the Drosophila eye: a multifaceted view of signaling specificity and cross-talk. EMBO J. 18, 6873 6879. Mlodzik, M. (2002). Planar cell polarization: do the same mechanisms regulate Drosophila tissue polarity and vertebrate gastrulation? Trends Genet. 18, 564571. Monaghan, M.M., Trimmer, J.S., and Rhodes, K.J. (2001). Experimental localization of Kv1 family voltage-gated K channel alpha and beta subunits in rat hippocampal formation. J. Neurosci. 21, 59735983. Montcouquiol, M., Rachel, R.A., Lanford, P.J., Copeland, N.G., Jenkins, N.A., and Kelley, M.W. (2003). Identification of Vangl2 and Scrb1 as planar polarity genes in mammals. Nature 423, 173177. Mostov, K., Su, T., and ter Beest, M. (2003). Polarized epithelial membrane traffic: conservation and plasticity. Nat. Cell Biol. 5, 287293. Muller, H.A., and Wieschaus, E. (1996). armadillo, bazooka, and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila. J. Cell Biol. 134, 149163. Mullins, C., and Bonifacino, J.S. (2001). Structural requirements for function of yeast GGAs in vacuolar protein sorting, alpha-factor maturation, and interactions with clathrin. Mol. Cell. Biol. 21, 7981 7994. Murthy, M., Garza, D., Scheller, R.H., and Schwarz, T.L. (2003). Mutations in the exocyst component sec5 disrupt neuronal membrane traffic, but neurotransmitter release persists. Neuron 37, 433447. Nakada, C., Ritchie, K., Oba, Y., Nakamura, M., Hotta, Y., Iino, R., Kasai, R.S., Yamaguchi, K., Fujiwara, T., and Kusumi, A. (2003). Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization. Nat. Cell Biol. 5, 626632. Nakagawa, T., Setou, M., Seog, D., Ogasawara, K., Dohmae, N., Takio, K., and Hirokawa, N. (2000). A novel motor, KIF13A, transports mannose-6-phosphate receptor to plasma membrane through direct interaction with AP-1 complex. Cell 103, 569581. Nelson, W.J. (2003). Adaptation of core mechanisms to generate cell polarity. Nature 422, 766774. Nelson, W.J., and Yeaman, C. (2001). Protein trafficking in the exocytic pathway of polarized epithelial cells. Trends Cell Biol. 11, 483486. Newman, L.S., McKeever, M.O., Okano, H.J., and Darnell, R.B. (1995). Beta-NAP, a cerebellar degeneration antigen, is a neuronspecific vesicle coat protein. Cell 82, 773783. Nirenberg, M.J., Chan, J., Vaughan, R.A., Uhl, G.R., Kuhar, M.J., and Pickel, V.M. (1997). Immunogold localization of the dopamine

transporter: an ultrastructural study of the rat ventral tegmental area. J. Neurosci. 17, 52555262. Nobes, C.D., and Hall, A. (1999). Rho GTPases control polarity, protrusion, and adhesion during cell movement. J. Cell Biol. 144, 1235 1244. OConnell, K.F., Maxwell, K.N., and White, J.G. (2000). The spd-2 gene is required for polarization of the anteroposterior axis and formation of the sperm asters in the Caenorhabditis elegans zygote. Dev. Biol. 222, 5570. Ohno, H., Stewart, J., Fournier, M.C., Bosshart, H., Rhee, I., Miyatake, S., Saito, T., Gallusser, A., Kirchhausen, T., and Bonifacino, J.S. (1995). Interaction of tyrosine-based sorting signals with clathrinassociated proteins. Science 269, 18721875. Osen-Sand, A., Catsicas, M., Staple, J.K., Jones, K.A., Ayala, G., Knowles, J., Grenningloh, G., and Catsicas, S. (1993). Inhibition of axonal growth by SNAP-25 antisense oligonucleotides in vitro and in vivo. Nature 364, 445448. Osen-Sand, A., Staple, J.K., Naldi, E., Schiavo, G., Rossetto, O., Petitpierre, S., Malgaroli, A., Montecucco, C., and Catsicas, S. (1996). Common and distinct fusion proteins in axonal growth and transmitter release. J. Comp. Neurol. 367, 222234. Owen, D.J., and Evans, P.R. (1998). A structural explanation for the recognition of tyrosine-based endocytotic signals. Science 282, 13271332. Parlati, F., Varlamov, O., Paz, K., McNew, J.A., Hurtado, D., Sollner, T.H., and Rothman, J.E. (2002). Distinct SNARE complexes mediating membrane fusion in Golgi transport based on combinatorial specificity. Proc. Natl. Acad. Sci. USA 99, 54245429. Pevsner, J., Volknandt, W., Wong, B.R., and Scheller, R.H. (1994). Two rat homologs of clathrin-associated adaptor proteins. Gene 146, 279283. Piehl, M., and Cassimeris, L. (2003). Organization and dynamics of growing microtubule plus ends during early mitosis. Mol. Biol. Cell 14, 916925. Polleux, F., Morrow, T., and Ghosh, A. (2000). Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 404, 567573. Poyatos, I., Ruberti, F., Martinez-Maza, R., Gimenez, C., Dotti, C.G., and Zafra, F. (2000). Polarized distribution of glycine transporter isoforms in epithelial and neuronal cells. Mol. Cell. Neurosci. 15, 99111. Prekeris, R., Foletti, D.L., and Scheller, R.H. (1999). Dynamics of tubulovesicular recycling endosomes in hippocampal neurons. J. Neurosci. 19, 1032410337. Pruyne, D., and Bretscher, A. (2000). Polarization of cell growth in yeast. I. Establishment and maintenance of polarity states. J. Cell Sci. 113, 365375. Puertollano, R., Aguilar, R.C., Gorshkova, I., Crouch, R.J., and Bonifacino, J.S. (2001). Sorting of mannose 6-phosphate receptors mediated by the GGAs. Science 292, 17121716. Qiu, R.G., Abo, A., and Steven Martin, G. (2000). A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCzeta signaling and cell transformation. Curr. Biol. 10, 697707. Quilliam, L.A., Khosravi-Far, R., Huff, S.Y., and Der, C.J. (1995). Guanine nucleotide exchange factors: activators of the Ras superfamily of proteins. Bioessays 17, 395404. Rios, J.C., Rubin, M., St Martin, M., Downey, R.T., Einheber, S., Rosenbluth, J., Levinson, S.R., Bhat, M., and Salzer, J.L. (2003). Paranodal interactions regulate expression of sodium channel subtypes and provide a diffusion barrier for the node of Ranvier. J. Neurosci. 23, 70017011. Rivera, J.F., Ahmad, S., Quick, M.W., Liman, E.R., and Arnold, D.B. (2003). An evolutionarily conserved dileucine motif in Shal K channels mediates dendritic targeting. Nat. Neurosci. 6, 243250. Rojas, R., and Apodaca, G. (2002). Immunoglobulin transport across polarized epithelial cells. Nat. Rev. Mol. Cell Biol. 3, 944955. Rosin-Arbesfeld, R., Ihrke, G., and Bienz, M. (2001). Actin-dependent membrane association of the APC tumour suppressor in polarized mammalian epithelial cells. EMBO J. 20, 59295939. Ruberti, F., and Dotti, C.G. (2000). Involvement of the proximal C

Neuron 294

terminus of the AMPA receptor subunit GluR1 in dendritic sorting. J. Neurosci. 20, RC78. Salzer, J.L. (2002). Nodes of Ranvier come of age. Trends Neurosci. 25, 25. Salzer, J.L. (2003). Polarized domains of myelinated axons. Neuron 40, this issue, 297318. Sampo, B., Kaech, S., Kunz, S., and Banker, G. (2003). Two distinct mechanisms target membrane proteins to the axonal surface. Neuron 37, 611624. Sans, N., Prybylowski, K., Petralia, R.S., Chang, K., Wang, Y.X., Racca, C., Vicini, S., and Wenthold, R.J. (2003). NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex. Nat. Cell Biol. 5, 520530. Schaefer, A.W., Kamei, Y., Kamiguchi, H., Wong, E.V., Rapoport, I., Kirchhausen, T., Beach, C.M., Landreth, G., Lemmon, S.K., and Lemmon, V. (2002). L1 endocytosis is controlled by a phosphorylation-dephosphorylation cycle stimulated by outside-in signaling by L1. J. Cell Biol. 157, 12231232. Schluesener, H.J., Meyermann, R., and Jung, S. (1995). Immunolocalization of vgr (BMP-6, DVR-6), a TGF-beta related cytokine, to Schwann cells of the rat peripheral nervous system: expression patterns are not modulated by autoimmune disease. Glia 13, 7578. Schmoranzer, J., and Simon, S.M. (2003). Role of microtubules in fusion of post-Golgi vesicles to the plasma membrane. Mol. Biol. Cell 14, 15581569. Serge, A., Fourgeaud, L., Hemar, A., and Choquet, D. (2002). Receptor activation and homer differentially control the lateral mobility of metabotropic glutamate receptor 5 in the neuronal membrane. J. Neurosci. 22, 39103920. Setou, M., Nakagawa, T., Seog, D.H., and Hirokawa, N. (2000). Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport. Science 288, 17961802. Shapiro, L., and Colman, D.R. (1999). The diversity of cadherins and implications for a synaptic adhesive code in the CNS. Neuron 23, 427430. Sharp, D.J., Yu, W., and Baas, P.W. (1995). Transport of dendritic microtubules establishes their nonuniform polarity orientation. J. Cell Biol. 130, 93103. Sheng, M., and Kim, M.J. (2002). Postsynaptic signaling and plasticity mechanisms. Science 298, 776780. Sheng, M., Tsaur, M.L., Jan, Y.N., and Jan, L.Y. (1992). Subcellular segregation of two A-type K channel proteins in rat central neurons. Neuron 9, 271284. Shi, S.H., Jan, L.Y., and Jan, Y.N. (2003). Hippocampal neuronal polarity specified by spatially localized mPar3/mPar6 and PI 3-kinase activity. Cell 112, 6375. Shiba, T., Takatsu, H., Nogi, T., Matsugaki, N., Kawasaki, M., Igarashi, N., Suzuki, M., Kato, R., Earnest, T., Nakayama, K., and Wakatsuki, S. (2002). Structural basis for recognition of acidic-cluster dileucine sequence by GGA1. Nature 415, 937941. Shorter, J., and Warren, G. (2002). Golgi architecture and inheritance. Annu. Rev. Cell Dev. Biol. 18, 379420. Silletti, S., Mei, F., Sheppard, D., and Montgomery, A.M. (2000). Plasmin-sensitive dibasic sequences in the third fibronectin-like domain of L1-cell adhesion molecule (CAM) facilitate homomultimerization and concomitant integrin recruitment. J. Cell Biol. 149, 1485 1502. Simpson, F., Bright, N.A., West, M.A., Newman, L.S., Darnell, R.B., and Robinson, M.S. (1996). A novel adaptor-related protein complex. J. Cell Biol. 133, 749760. Smith, S.J. (1988). Neuronal cytomechanics: the actin-based motility of growth cones. Science 242, 708715. Stowell, J.N., and Craig, A.M. (1999). Axon/dendrite targeting of metabotropic glutamate receptors by their cytoplasmic carboxyterminal domains. Neuron 22, 525536. Stricker, C., Field, A.C., and Redman, S.J. (1996). Statistical analysis of amplitude fluctuations in EPSCs evoked in rat CA1 pyramidal neurones in vitro. J. Physiol. 490, 419441.

Sugihara, K., Asano, S., Tanaka, K., Iwamatsu, A., Okawa, K., and Ohta, Y. (2002). The exocyst complex binds the small GTPase RalA to mediate filopodia formation. Nat. Cell Biol. 4, 7378. Sur, C., Betz, H., and Schloss, P. (1996). Localization of the serotonin transporter in rat spinal cord. Eur. J. Neurosci. 8, 27532757. Takekuni, K., Ikeda, W., Fujito, T., Morimoto, K., Takeuchi, M., Monden, M., and Takai, Y. (2003). Direct binding of cell polarity protein PAR-3 to cell-cell adhesion molecule nectin at neuroepithelial cells of developing mouse. J. Biol. Chem. 278, 54975500. Tepass, U., Tanentzapf, G., Ward, R., and Fehon, R. (2001). Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet. 35, 747784. TerBush, D.R., and Novick, P. (1995). Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J. Cell Biol. 130, 299312. TerBush, D.R., Maurice, T., Roth, D., and Novick, P. (1996). The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J. 15, 64836494. Tovar, K.R., and Westbrook, G.L. (2002). Mobile NMDA receptors at hippocampal synapses. Neuron 34, 255264. Tuma, P.L., and Hubbard, A.L. (2003). Transcytosis: crossing cellular barriers. Physiol. Rev. 83, 871932. van Meer, G., Gumbiner, B., and Simons, K. (1986). The tight junction does not allow lipid molecules to diffuse from one epithelial cell to the next. Nature 322, 639641. Vega, I.E., and Hsu, S.C. (2001). The exocyst complex associates with microtubules to mediate vesicle targeting and neurite outgrowth. J. Neurosci. 21, 38393848. Veh, R.W., Lichtinghagen, R., Sewing, S., Wunder, F., Grumbach, I.M., and Pongs, O. (1995). Immunohistochemical localization of five members of the Kv1 channel subunits: contrasting subcellular locations and neuron-specific co-localizations in rat brain. Eur. J. Neurosci. 7, 21892205. Wang, H., Tang, X., Liu, J., Trautmann, S., Balasundaram, D., McCollum, D., and Balasubramanian, M.K. (2002). The multiprotein exocyst complex is essential for cell separation in Schizosaccharomyces pombe. Mol. Biol. Cell 13, 515529. Watts, J.L., Etemad-Moghadam, B., Guo, S., Boyd, L., Draper, B.W., Mello, C.C., Priess, J.R., and Kemphues, K.J. (1996). par-6, a gene involved in the establishment of asymmetry in early C. elegans embryos, mediates the asymmetric localization of PAR-3. Development 122, 31333140. Wedlich-Soldner, R., Altschuler, S., Wu, L., and Li, R. (2003). Spontaneous cell polarization through actomyosin-based delivery of the Cdc42 GTPase. Science 299, 12311235. West, A.E., Neve, R.L., and Buckley, K.M. (1997). Identification of a somatodendritic targeting signal in the cytoplasmic domain of the transferrin receptor. J. Neurosci. 17, 60386047. White, J., Johannes, L., Mallard, F., Girod, A., Grill, S., Reinsch, S., Keller, P., Tzschaschel, B., Echard, A., Goud, B., and Stelzer, E.H. (1999). Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells. J. Cell Biol. 147, 743760. Wilson, J.M., de Hoop, M., Zorzi, N., Toh, B.H., Dotti, C.G., and Parton, R.G. (2000). EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts. Mol. Biol. Cell 11, 26572671. Winckler, B., and Mellman, I. (1999). Neuronal polarity: controlling the sorting and diffusion of membrane components. Neuron 23, 637640. Winckler, B., Forscher, P., and Mellman, I. (1999). A diffusion barrier maintains distribution of membrane proteins in polarized neurons. Nature 397, 698701. Wisco, D., Chang, M.C., Norden, C., and Folsch, H., Anderson, E.D., A., and Winckler, B. (2003). Two biosynthetic pathways to the axon: Pathway selection by axonal targeting signals in NgCAM is contextdependent. J. Cell Biol., in press. Wittmann, T., Bokoch, G.M., and Waterman-Storer, C.M. (2003). Regulation of leading edge microtubule and actin dynamics downstream of Rac1. J. Cell Biol. 161, 845851.

Review 295

Wodarz, A. (2002). Establishing cell polarity in development. Nat. Cell Biol. 4, E39E44. Wodarz, A., Hinz, U., Engelbert, M., and Knust, E. (1995). Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82, 6776. Woods, D.F., and Bryant, P.J. (1991). The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions. Cell 66, 451464. Yamanaka, T., Horikoshi, Y., Suzuki, A., Sugiyama, Y., Kitamura, K., Maniwa, R., Nagai, Y., Yamashita, A., Hirose, T., Ishikawa, H., and Ohno, S. (2001). PAR-6 regulates aPKC activity in a novel way and mediates cell-cell contact-induced formation of the epithelial junctional complex. Genes Cells 6, 721731. Yeaman, C., Grindstaff, K.K., and Nelson, W.J. (1999). New perspectives on mechanisms involved in generating epithelial cell polarity. Physiol. Rev. 79, 7398. Zhang, X., Bi, E., Novick, P., Du, L., Kozminski, K.G., Lipschutz, J.H., and Guo, W. (2001). Cdc42 interacts with the exocyst and regulates polarized secretion. J. Biol. Chem. 276, 4674546750. Zhao, Y., Sheng, H.Z., Amini, R., Grinberg, A., Lee, E., Huang, S., Taira, M., and Westphal, H. (1999). Control of hippocampal morphogenesis and neuronal differentiation by the LIM homeobox gene Lhx5. Science 284, 11551158. Zhou, D., Lambert, S., Malen, P.L., Carpenter, S., Boland, L.M., and Bennett, V. (1998). AnkyrinG is required for clustering of voltagegated Na channels at axon initial segments and for normal action potential firing. J. Cell Biol. 143, 12951304. Zurzolo, C., Le Bivic, A., Quaroni, A., Nitsch, L., and RodriguezBoulan, E. (1992). Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line. EMBO J. 11, 23372344.