Pathway Selection to the Axon Depends on Multiple Targeting Signals in Ngcam

1514 Research Article Pathway selection to the axon depends on multiple targeting signals in NgCAM

Chan Choo Yap1, Rita L. Nokes2,*, Dolora Wisco1,*, Eric Anderson‡, Heike Fölsch2 and Bettina Winckler1,§ 1University of Virginia Medical School, Department of Neuroscience, 409 Lane Road, Charlottesville, VA 22908, USA 2Northwestern University, Department of Biochemistry, Molecular Biology and Cell Biology, 2205 Tech Drive, Evanston, IL 60208, USA *These authors contributed equally to this work ‡Former address: Department of Cell Biology, Yale University Medical School, 333 Cedar Street, New Haven, CT 06520, USA §Author for correspondence (e-mail: [email protected])

Accepted 12 February 2008 Journal of Cell Science 121, 1514-1525 Published by The Company of Biologists 2008 doi:10.1242/jcs.022442

Summary Similar to most differentiated cells, both neurons and epithelial targeting signal in the cytoplasmic tail of NgCAM. The axonal cells elaborate distinct plasma membrane domains that contain signal is glycine and serine rich, but only the glycine residues different membrane proteins. We have previously shown that are required for activity. The somatodendritic signal is cis- the axonal cell-adhesion molecule L1/NgCAM accumulates on dominant and needs to be inactivated in order for the axonal the axonal surface by an indirect transcytotic pathway via signal to be executed. Additionally, we show that the axonal somatodendritic endosomes. MDCK epithelial cells similarly cytoplasmic signal promotes apical targeting in MDCK cells. traffic NgCAM to the apical surface by transcytosis. In this Transcytosis of NgCAM to the axon thus requires the sequential study, we map the signals in NgCAM required for routing via regulated execution of multiple targeting signals. the multi-step transcytotic pathway. We identify both a previously mapped tyrosine-based signal as a sufficient Key words: Transcytosis, L1 cell-adhesion molecule, Axonal somatodendritic targeting signal, as well as a novel axonal targeting, Apical targeting, MDCK cells, LDLR

Introduction from the TGN to the basolateral membrane during biosynthetic Epithelial cells and neurons differentiate and maintain distinct delivery (Fields et al., 2007; Simmen et al., 2002). plasma membrane domains, such as an apical and a basolateral Sorting to the apical and axonal domains is less well understood. surface, or dendrites and axons. The distinct membrane composition Apical and axonal sorting signals have been mapped to the

Journal of Cell Science is maintained by diffusion barriers found either at the tight junctions extracellular or transmembrane domains of proteins. Additionally, in epithelial cells (Shin et al., 2006) or in the axon initial segment the ability to partition into lipid raft domains correlates with correct in neurons (Winckler et al., 1999). Additionally, transmembrane sorting for some, but not all, apical or axonal proteins (Chang et proteins are secreted in a polarized manner from the trans-Golgi al., 2006; Galvan et al., 2005; Jacob and Naim, 2001; Ledesma et network (TGN) during biosynthetic delivery, or from recycling al., 1998; Paladino et al., 2004). In 1998, a cytosolic signal for apical endosomes after internalization. The molecular bases for polarized targeting was identified in rhodopsin (Chuang and Sung, 1998), membrane trafficking are still being uncovered. It is clear that at and several more have been identified since (e.g. Hodson et al., least some of the signals and the machinery involved in polarized 2006; Takeda et al., 2003). Surprisingly, some of these apical signals trafficking are conserved between epithelial cells and neurons (Dotti share characteristics with basolateral consensus sequences, such as and Simons, 1990), but cell-type specific mechanisms have also tyrosine-based motifs or motifs taking on a beta turn secondary been described. structure. The machinery recognizing these cytosolic apical targeting Most of our current understanding about polarized sorting has signals is largely unknown. come from studying the kidney epithelial cell line MDCK and much Several axonal signals map to cytoplasmic domains as well (for less is known about sorting in neurons. For example, both basolateral reviews, see Arnold, 2007; Lai and Jan, 2006), but no consensus and somatodendritic sorting signals frequently rely on tyrosine- sequence has emerged. Rather, axonal targeting signals are diverse, based sorting signals encoded in the cytoplasmic tail of and the mechanisms of their action are not yet well understood. transmembrane proteins (West et al., 1997; Folsch, 2005; Jareb and These diverse signals might operate at distinct steps on the pathway Banker, 1998; Silverman et al., 2005). Tyrosine-based signals are to the axon. For example, an ‘axonal targeting’ signal could recognized by cytosolic adaptor protein complexes (AP-1 through promote lateral enrichment into axonally destined vesicles in the AP-4) (Folsch, 2005; Nakatsu and Ohno, 2003; Rodriguez-Boulan TGN, which may then associate with axonal motors. Subsequently, et al., 2005). Frequently, tyrosine-based signals bind more than one axonal delivery might be regulated by competence for fusion with adaptor complex and are therefore active as basolateral and as axonal secretion sites. Additionally, signals may regulate anchoring endocytosis signals (Folsch, 2005). Columnar epithelial cells, but and restricted diffusion in axons, or retrieval by endocytosis from not neurons or hepatocytes, express a subclass of AP-1 complexes, inappropriate sites (Winckler, 2004). There are a number of AP-1B that is important for correct sorting of basolateral cargo examples of axonal targeting where some mechanistic insights have proteins from recycling endosomes (Folsch et al., 2003; Gan et al., been gleamed. For example, targeting of the K+ channels Kv1.x to 2002; Ohno et al., 1999). In addition, AP-4 may sort proteins directly the axonal compartment is achieved via the tetramerization motif Somatodendritic and axonal signals in L1/NgCAM 1515

(Gu et al., 2003; Rivera et al., 2005). For several other axonal and on the epithelial cell-specific adaptor complex AP-1B proteins, axonal targeting is endocytosis dependent and the axonal (Anderson et al., 2005). In this work, we undertook a fine-mapping targeting motifs map to endocytosis signals. Therefore, some analysis of the cytoplasmic tail signals in NgCAM. axonal proteins may become enriched in the axon primarily owing Trafficking along the transcytotic pathway requires that multiple to the preferential endocytosis of ‘misplaced’ somatodendritic targeting signals are read and executed sequentially, presumably receptor pools coupled to specific retention/anchoring in the correct in different endomembrane compartments. Transcytosis therefore axonal domain, rather than from polarized secretion from the TGN necessitates a system of multiple hierarchically executed signals to axons (Garrido et al., 2001; Sampo et al., 2003; Xu et al., 2006). in which initially a somatodendritic/basolateral signal is active and Previously, we have described a different endocytosis-dependent cis-dominant over an axonal/apical signal. After somatodendritic/ pathway to the axon, namely transcytosis (Wisco et al., 2003): during basolateral delivery and endocytosis, the axonal/apical signal biosynthetic delivery, the axonal cell-adhesion molecule becomes active in endosomes, whereas the somatodendritic/ L1/NgCAM is first inserted into the somatodendritic domain, then basolateral signal is turned off. Furthermore, the transcytotic internalized into somatodendritic endosomes, and finally sorted to model predicts that the ‘recessive’ axonal signals may be executed the axon from the endosomal system. Similarly, NgCAM travels throughout the biosynthetic pathway if the somatodendritic/ to the apical domain of epithelial MDCK cells via transcytosis basolateral signal is deleted or mutated. In this work, we determined (Anderson et al., 2005). In both cell types, a sufficient axonal/apical that the previously identified basolateral signal of NgCAM also targeting signal is present in the extracellular domain (Anderson et acts as a sufficient somatodendritic targeting signal. In addition, al., 2005; Sampo et al., 2003; Wisco et al., 2003). Therefore, the we identified a second axonal targeting signal located in the cytoplasmic tail is dispensable for axonal/apical accumulation per cytoplasmic tail. This cytoplasmic axonal targeting signal also se. However, signals in the cytoplasmic tail of NgCAM are required promotes apical trafficking of NgCAM in MDCK cells. for trafficking via the transcytotic route (Wisco et al., 2003), Transcytotic routing is therefore dependent on the presence and including a signal encompassing tyrosine 33. Likewise, in MDCK hierarchical regulation of multiple sorting signals in the cytoplasmic cells, the initial basolateral targeting is dependent on tyrosine 33 tail of NgCAM.

Results The basolateral targeting signal of NgCAM is also sufficient for somatodendritic targeting We have previously shown that NgCAM reaches the axonal plasma membrane by an indirect transcytotic route via the somatodendritic surface (Wisco et al., 2003; Yap et al., 2008). Therefore, NgCAM should contain a bona fide somatodendritic

Journal of Cell Science targeting signal for initial delivery to the somatodendritic surface. In MDCK cells, a tyrosine at position 33 of the cytoplasmic tail serves as a basolateral signal. Indirect evidence using a point mutation of tyrosine

Fig. 1. NgCAM contains a sufficient somatodendritic sorting signal in its cytoplasmic tail. (A,B) Cultured hippocampal neurons were transfected with DsRed (red) and either the LDLR- based chimera LexNct1-42 (A) or LexNct1- 42(Y33A) (B). The cell-surface population of the chimeras was stained with an antibody against an extracellular epitope of LDLR (anti-Lex; green). In A,B, the top row shows the soma region containing the dendrites (arrowheads), whereas the bottom row shows the distant distal portions of the axons (arrows). (C-E) The extent of surface polarity for the three constructs LΔCT, LexNct1-42 and LexNct1-42(Y33A) (shown diagrammatically in C) was determined by measuring the average fluorescence intensity along distal axons and dendrites. The axon/dendrite polarity index (PI) is the ratio of axon/dendrite intensities. The polarity index was determined for 20-25 cells and the percentage of cells displaying preferential axonal accumulation (PI>2), preferential somatodendritic accumulation (PI<0.5) or uniform distribution is plotted in D. The average PI for 20-25 cells from three independent experiments is shown in E. **P<0.001, *P<0.01, Student’s t-test. 1516 Journal of Cell Science 121 (9)

33 suggested that somatodendritic targeting was mediated by the axon intensity by the average dendrite intensity [A/D PI; as in Wisco same signal (Wisco et al., 2003). To test more directly whether the et al. (Wisco et al., 2003)]. LΔCT was uniformly expressed on axons same tyrosine or other tail-signals are important in trafficking of and dendrites and had an A/D PI of 0.9 (±0.11 s.e.m.; n=25 cells), NgCAM in neurons, we generated chimeric proteins containing the indicating close to equal intensities on axons and dendrites (Fig. extracellular and transmembrane domains of the low-density 1D,E). When the proximal half of the NgCAM cytoplasmic tail lipoprotein receptor (LDLR) plus either the proximal half (CT1- (CT1-42) was added to LΔCT to generate LexNct1-42 (Fig. 1C), 42) or distal half (CT45-114) of the NgCAM tail (Fig. 1C, Fig. 2C). the expressed protein was highly enriched on soma and dendrites This was necessary because the ectodomain of NgCAM contains (soma panel; arrowheads, Fig. 1A; D) and was largely undetectable a sufficient axonal sorting signal, which makes it almost impossible on the axon (Fig. 1A, axon panel; arrows). LexNct1-42 had an to identify cytoplasmic signals when the extracellular domain is average A/D polarity index of 0.3 (±0.06 s.e.m.; n=10 cells), present. As a control, we used LDLR lacking its cytoplasmic tail indicating over threefold higher signal intensity on dendrites than (LΔCT). The constructs were expressed transiently in cultured on axons (Fig. 1E). hippocampal neurons using Lipofectamine 2000. DsRed was co- We then tested whether tyrosine 33 was necessary for transfected to delineate the entire arborization of the neuron. In order somatodendritic targeting of LexNct1-42 by introducing a Y to A to quantify the extent of polarized expression, we determined the substitution at position 33 of the chimera (Fig. 1C). Expression of average pixel intensity along the distal part of axons and along the this construct (LexNct1-42Y33A) showed uniform distribution (A/D dendrites, and calculated a polarity index by dividing the average PI=0.9±0.1 s.e.m.; n=27 cells; Fig. 1B,D,E) similar to LΔCT, Journal of Cell Science

Fig. 2. The cytoplasmic tail of NgCAM contains an axonal sorting signal. (A,B) Cultured hippocampal neurons were transfected with DsRed (red) and either the LDLR-based chimera LexNct45-114 (A) or LexNct1-114 (B). The cell-surface population of the chimeras was stained with an antibody against an extracellular epitope of LDLR (green). In order to appreciate the difference in staining intensity on distal axons and on dendrites, single green channel insets are shown at the bottom. (C) Diagram of LDLR- or CD4-based chimeras carrying either the entire NgCAM cytoplasmic tail (Nct1-114) or the C-terminal half of the NgCAM cytoplasmic tail (Nct45-114). (D) Axon/dendrite polarity index (A/D PI) and the percentage of cells showing uniform, axonal or somatodendritic surface accumulation are shown for the constructs depicted in C (20-25 cells were analyzed). ***P<0.0001; statistically significant differences from the ΔCT backbones. Somatodendritic and axonal signals in L1/NgCAM 1517

demonstrating directly that the NgCAM cytoplasmic tail contains targets to the basolateral domain in MDCK cells dependent on AP- a sufficient somatodendritic targeting signal that requires tyrosine33 1B (Anderson et al., 2005). By contrast, NgCAMCT43 was still for activity. The demonstration of a bone fide somatodendritic signal found axonally enriched. When the localization of full-length argues that the initial somatodendritic delivery of NgCAM observed NgCAM and NgCAMCT43 were compared on parallel coverslips, in our previous kinetic experiments is in fact signal mediated. the A/D PI of NgCAM was 6.6±0.45 s.e.m. (n=23 cells), whereas the A/D PI of NgCAMCT43 was 4.7±0.4 s.e.m. (n=32 cells). The cytoplasmic tail of NgCAM contains an axonal targeting Student’s t-test showed a significant difference between these values signal at P=0.003. The surface expression of the two constructs was not Next, we tested whether the distal half of the NgCAM tail (Nct45- significantly different (data not shown) and kinetic differences of 114) contained any additional targeting information by generating surface expression were not noted. The cytoplasmic axonal signal a chimera with LΔCT (LexNct45-114) (Fig. 2C). LexNct45-114 therefore improves axonal polarity of NgCAM. was found at low levels on soma and dendrites (arrowheads) and at higher levels along the axonal surface (arrows; Fig. 2A green). Cytoplasmic tail signals are sufficient for transcytotic routing The distal region of the axon was particularly brightly stained. For proper transcytotic routing, multiple targeting signals must be Determination of A/D PI showed two- to threefold enrichment on read and executed sequentially, probably in different intracellular the axonal surface (2.6±0.2 s.e.m.; n=21 cells; Fig. 2D). To confirm compartments. Based on work in MDCK cells, we have previously the axonal localization of LexNct45-114, we double-stained cultures proposed that the basolateral signal in the cytoplasmic tail is expressing LexNct45-114 with either a somatodendritic marker, executed first in the biosynthetic pathway, then inactivated by MAP2, or a marker for the initial segment of the axon, ankyrin G phosphorylation, thus allowing execution of the lumenal apical (Boiko et al., 2007). LexNct45-114 was enriched in the MAP2- targeting signal (Anderson et al., 2005). As the cytoplasmic tail negative process that contained ankyrin G (data not shown), contained both a second axonal signal and a somatodendritic signal identifying it as the axon. The cytoplasmic tail of NgCAM therefore (Figs 1, 2), we asked whether the NgCAM cytoplasmic tail was contains sufficient targeting information to enrich a non-polarized sufficient to route a reporter protein to the axon by the multi-step membrane protein on the axon. transcytotic route or whether the extracellular axonal signal of This observation was unexpected as a chimera of CD8α NgCAM was required for transcytotic trafficking. We therefore containing the entire cytoplasmic tail of NgCAM showed no axonal determined the pathway taken by LexNct1-114 to the axon by enrichment (Sampo et al., 2003). One possible explanation for the performing kinetic analysis, as carried out previously (Wisco et al., discrepancy is that the axonal targeting signal might be masked 2003). For this assay, we infected neuronal cultures with a (and therefore inactive) in the context of the entire cytoplasmic tail. recombinant adenovirus encoding LexNct1-114 for 4 hours, then Therefore, we created a chimera of the entire NgCAM cytoplasmic blocked transit through the Golgi by treatment with Brefeldin A tail (CT1-114) and LΔCT (LexNct1-114) (Fig. 2C). When LexNct1- (BFA) to accumulate LexNct1-114 intracellularly, and then reversed 114 was expressed in cultured hippocampal neurons, significant the transport block by washing out BFA. This assay thus allowed axonal enrichment was again observed (arrows; Fig. 2B,D; A/D us to follow a pulse of LexNct1-114 from the Golgi to the surface PI=3.8±0.6 s.e.m.; n=23 cells). Given the differences between the (see Materials and Methods for details). Initially, only cells with

Journal of Cell Science capacity of the NgCAM cytoplasmic tail to redirect LDLR or CD8α intracellular staining against LexNct1-114 without surface staining ectodomain chimeras to the axon, we created an additional chimera were observed (Fig. 3A; diamonds) demonstrating the efficiency using the frequently used CD4 as a backbone (Garrido et al., 2001; of the BFA block. The first population of cells with detectable Garrido et al., 2003b; Gu et al., 2003; Xu et al., 2006). CD4 without surface staining showed somatodendritically enriched localization its cytoplasmic tail (CD4ΔCT) is found uniformly along axons and of LexNct1-114 (Fig. 3A; circles). This somatodendritically dendrites (Fig. 2C,D; A/D PI=1.1±0.1 s.e.m.; n=14 cells). We added expressing population was replaced over time with a population NgCAM CT1-114 or CT45-114 to the CD4 extracellular and expressing LexNct1-114 on the axonal surface (Fig. 3A; squares). transmembrane domain to create CD4exNct1-114 and CD4exNct45- The observed progression of surface expression from initially 114 (Fig. 2C). Similar to the LDLR-based chimeras, the CD4- somatodendritically enriched and later axonally enriched was NgCAM chimeras were enriched on the axon (A/D PI=2.7±0.17 similar to that observed for full-length NgCAM itself (Wisco et al., s.e.m., n=18 cells; or 2.9±0.3 s.e.m., n=18 cells; Fig. 2D), albeit 2003). Interestingly, the kinetics of surface appearance was not as strongly as full-length NgCAM [A/D PI of over 5 (Wisco et significantly slowed compared with wild-type NgCAM where first al., 2003)]. In summary, we showed that the cytoplasmic tail of surface appearance could be seen as early as 2 hours after BFA NgCAM indeed contains both sufficient somatodendritic and axonal washout. Nevertheless, LexNct1-114 was capable of transcytosis targeting information. in the absence of the NgCAM extracellular domain. As the extracellular domain of NgCAM is sufficient for axonal Next, we asked whether there is something unique about the sorting without the cytoplasmic tail, we wondered whether we could basolateral/somatodendritic signal in NgCAM that allows for determine any additive benefit of having a second axonal signal in transcytosis or whether another basolateral/somatodendritic signal the cytoplasmic tail. We previously found that NgCAM without the could also support transcytotic routing of a protein. We used the cytoplasmic tail [NgCAMCT3 (Chang et al., 2006)] reaches the well-characterized basolateral signals from LDLR to answer this axonal surface with a kinetic delay of several hours compared with question. LDLR possesses two basolateral signals in its cytoplasmic full-length NgCAM. In order to determine more precisely what the tail (Matter et al., 1992). The proximal signal (between residues 5 role of the second axonal signal might be, we used the previously and 27) is tyrosine based and supports both rapid endocytosis and described NgCAMCT43 (Anderson et al., 2005), which is truncated AP-1B-dependent basolateral targeting, whereas the distal signal after amino acid 43 of the cytoplasmic tail, thereby removing the supports only basolateral targeting but not rapid endocytosis (Matter cytoplasmic axonal signal, but retaining the extracellular axonal et al., 1992; Fields et al., 2007). Simultaneous inactivation of both signal and the cytoplasmic somatodendritic signal. This construct signals abrogated somatodendritic targeting of LDLR in 1518 Journal of Cell Science 121 (9)

hippocampal neurons (Jareb and Banker, 1998). We used a truncated (Fig. 4B). Lct27 by itself did not efficiently polarize to the LDLR, which lacked the distal signal but contained the proximal somatodendritic domain at steady state (Fig. 4B,C). When Lct27 signal (Lct27) as a backbone for another set of NgCAM tail chimeras was expressed from a recombinant adenovirus, somatodendritic accumulation was similarly observed in only 20% of cells, with 72% of cells showing uniform distribution. In MDCK cells, the proximal signal was found to be entirely dependent on the epithelial- specific adaptor complex AP-1B for targeting to the basolateral surface (Fields et al., 2007). As AP-1B is expressed at very low levels in neurons (Ohno et al., 1999) when compared with epithelial cells, the poor somatodendritic restriction of Lct27 might be due to saturation of the sorting machinery at longer times of expression (see also below). We then added residues 45-114 of the NgCAM cytoplasmic tail to Lct27 to generate Lct27Nct45-114. Interestingly, this construct was axonally enriched (Fig. 4A, arrows; A/D PI=3.2±0.6 s.e.m.; n=14 cells; Fig. 4C). In order to determine whether Lct27Nct45- 114 traveled to the axon by transcytosis, we performed kinetic analysis using BFA block and release, as above, using adenoviral expression of Lct27Nct45-114. Similar to NgCAM and LexNct1- 114, Lct27Nct45-114 first appeared enriched on the somatodendritic surface after BFA release (Fig. 3B), before appearing enriched on the axonal surface, suggestive of transcytosis.

Active somatodendritic signals are dominant for sorting in the TGN One possibility for the delayed execution of the axonal signal during transcytosis is that the cytoplasmic machinery necessary for its execution might be spatially restricted to later compartments of the transcytotic route, such as endosomes, but missing from the biosynthetic pathway (presumably the TGN). We showed previously that a mutant NgCAM with a Y33A substitution did not transcytose but rather traveled directly from the TGN to the axonal surface (Wisco et al., 2003). We therefore wondered whether LexNct45- 114 (which lacked the somatodendritic and endocytic motifs

Journal of Cell Science surrounding tyrosine 33) reached the axon by transcytosis or via a direct pathway. Again, we used the BFA block/release assay after adenoviral expression to study the kinetics of surface delivery. Unlike LexNct1-114, the first detectable surface expression of LexNct45-114 was on the axon (Fig. 3C, squares) and somatodendritically enriched surface expression was not observed (Fig. 3C, circles). This observation indicates direct rather than transcytotic delivery, and suggests that the axonal signal on its own could be read and executed in the biosynthetic pathway. However, when both a somatodendritic and an endocytosis signal (such as the YRSLE motif of NgCAM) were present in the cytoplasmic tail in addition to the axonal signal, the transcytotic route predominated. Fig. 3. The presence of multiple cytoplasmic tail signals is required for Therefore, it appeared that the somatodendritic signal was dominant transcytotic routing of NgCAM chimeras. The site of initial surface delivery of over the axonal signal in the biosynthetic pathway, and was LexNct1-114 (A), Lct27Nct45-114 (B) and LexNct45-114 (C) was determined preferentially read and executed in the TGN. using the Brefeldin A (BFA) block release assay. The percentage of neurons To further investigate this signal dominance, we combined the displaying the chimera on the surface uniformly (triangles), axonally enriched (squares) or somatodendritically enriched (circles) was determined for distal basolateral signal of LDLR (which lacks rapid endocytosis multiple time points after release from BFA. Additionally, the percentage of information) with the axonal NgCAM tail signal. Given the rather cells was scored in which no surface staining was detectable, but intracellular poor somatodendritic capacity of the proximal basolateral signal (see staining was present (broken line). The chimera LexNct45-114, which contains above), we first tested how well the distal signal worked as a the cytoplasmic axonal signal of NgCAM but lacks the somatodendritic and endocytosis motifs, is delivered directly to the axonal plasma membrane (C). If somatodendritic signal in neurons. We deleted the proximal signal the somatodendritic/endocytic signal surrounding Tyr33 is present [as in located between residues 5 and 27 of the LDLR cytoplasmic tail to LexNct1-114 (A)], the first surface appearance was frequently create LΔct5-27 (Fig. 4B). LΔct5-27 showed robust somatodendritic somatodendritically enriched, suggestive of transcytosis. Inclusion of the enrichment (A/D PI=0.3±0.06 s.e.m.; n=20; Fig. 4C). Therefore, the LDLR-derived somatodendritic/endocytic signal in Lct27Nct45-114 (B) also distal signal of LDLR was active as a somatodendritic signal in led to initial somatodendritic surface appearance, suggesting transcytotic routing. One representative experiment (of a minimum of three) is shown for neurons. We then added the axonal signal residues 45-114 of the each construct. NgCAM tail (LΔct5-27Nct45-114) (Fig. 4B). This construct therefore Somatodendritic and axonal signals in L1/NgCAM 1519

Fig. 4. Axonal targeting by the cytoplasmic axonal targeting signal in NgCAM requires the inactivation of the somatodendritic signal. (A) Lct27Nct45-114 (green) localizes preferentially to the axonal surface (arrows) of a transfected hippocampal neuron. DsRed was co-transfected to delineate all neuronal processes (red). Arrowheads indicate dendrites. (B) Diagrammatic depiction of LDLR-based NgCAM cytoplasmic tail chimeras. Chimeras contain either the proximal (amino acid residues 5-27) basolateral sorting signal of LDLR, which is co-linear with the endocytosis motif, or the distal (amino acid residues 28-50) basolateral signal of LDLR, which does not contain an endocytosis motif. Point mutations at Tyr35 and 37 inactivate the distal basolateral sorting signal. (C) The axon/dendrite polarity index was determined for the chimeras depicted in B. **P<0.001, ***P<0.0001; statistically significant differences between bracketed constructs.

Journal of Cell Science contains both a somatodendritic and an axonal signal, but lacks a (A/D PI=1.4±0.13 s.e.m.; n=15 cells; Fig. 5A,C) and was easily rapid endocytosis signal. Interestingly, LΔct5-27Nct45-114 showed detected throughout the dendrites (arrows; Fig. 5C) and the axon somatodendritic enrichment (A/D PI=0.5±0.1 s.e.m.; n=15 cells; Fig. (arrowheads). To further confirm the axonal targeting capacity of 4C), similar to the backbone LΔct5-27 alone. In order to test whether the mapped domain, residues 43-78 were added onto the silencing of the somatodendritic signal would allow the execution somatodendritic LexNct1-42 to generate LexNct1-78. As expected, of the axonal signal, we inactivated the distal basolateral signal by this construct again showed axonal enrichment (A/D PI=3.3±0.5 introducing two point mutations in the crucial tyrosine residues at s.e.m., n=17; Fig. 5A). Therefore, a glycine-rich 15 amino acid positions 35 and 37 (Matter et al., 1994) to create LΔct5-27YAYA residue stretch (SASGSGAGSGVGSPG) in the NgCAM Nct45-114 (Fig. 4B). We find that LΔct5-27YAYA Nct45-114 was cytoplasmic tail was necessary and sufficient for axonal targeting no longer somatodendritically enriched, but became axonally of LDLR chimeras. enriched instead (A/D PI=2.4±0.3 s.e.m.; n=16 cells; Fig. 4C). This finding indicates that: (1) somatodendritic sorting information is cis- Glycines, but not serines, are required for the targeting dominant for sorting at the TGN; and (2) that axonal cytoplasmic capacity of the glycine-rich axonal signal signals are only executed in the TGN in the absence of active In order to determine whether the glycine residues or the serine somatodendritic signals. residues, or both, were necessary for axonal targeting capacity of the glycine-rich motif, we introduced point mutations into The axonal cytoplasmic information mapped to a small region LexNct45-59 either replacing serines with alanines (S-A: of NgCAM SAAGAGAGAGVGAPG) thereby removing potential LexNct1-114 and LexNct45-114 were then used as starting phosphorylation sites or glycines with arginines (G-R: templates to further map the axonal targeting region in the SASRSRSRSRVRSPG) to introduce charged residues to create cytoplasmic tail of NgCAM. Successive truncations from the C LexNct45-59(S-A) and LexNct45-59(G-R). Total surface expression terminus were generated in LexNct45-114, terminating at positions of the mutants was the same as for LexNct45-59 (not shown), but 97, 78 and 59 of the cytoplasmic tail. All of these constructs showed axonal targeting of LexNct45-59(G-R) was abolished (Fig. 6). The similar axonal enrichment (Fig. 5A). The axonal enrichment of the A/D PI of LexNct45-59(S-A) was not significantly different from smallest region mapped (LexNct45-59) is shown in Fig. 5B (green; LexNct45-59. Surprisingly then, the serine residues are not essential arrowheads). A chimera containing residues 66-114 of the NgCAM for the activity of the axonal targeting motif; however, the signal cytoplasmic tail (LexNct66-114), however, lost axonal enrichment does not tolerate positive charges. 1520 Journal of Cell Science 121 (9)

Fig. 5. Residues 45-59 of the cytoplasmic tail of NgCAM contain a necessary and sufficient axonal targeting signal. (A) The location of the cytoplasmic axonal targeting signal of NgCAM was mapped by sequential truncations and deletions. The constructs depicted diagrammatically were expressed in neurons and the axon/dendrite polarity index of the surface pool determined. ***P<0.0001, *P<0.01; Journal of Cell Science significantly different from LΔCT. (B,C) Examples of one axonally enriched construct LexNct45-59 (B) and one uniform construct LexNct66-114 (C) are shown. Surface distribution of the chimeras was detected with an anti-Lex antibody without permeabilization (green). DsRed was co-expressed (red) to delineate all neuronal processes. The soma region containing the dendrites (arrows) are shown in the top row, whereas the distal regions of the long axons (arrowheads) are shown in the bottom row for both B and C.

The axonal cytoplasmic signal of NgCAM is recognized as an Lct27Nct45-59 and Lct27Nct66-114 (Fig. 7A), and expressed them apical signal in MDCK cells by transient transfection in MDCK cells. As described before When NgCAM is expressed in MDCK cells, it localizes to the (Matter et al., 1992), Lct27 localized to the basolateral surface (Fig. apical surface (Anderson et al., 2005) (Fig. 7B) and reaches it 7C). By contrast, Lct27Nct45-59 accumulated highly at the apical overwhelmingly by transcytosis (Anderson et al., 2005; Hua et al., domain (Fig. 7D), with lower levels detectable on the lateral 2006). We therefore wondered whether the cytoplasmic axonal surfaces. By contrast, Lct27Nct66-114 was restricted to the signal we identified in NgCAM was active as an apical signal in basolateral domain, indistinguishable from Lct27 by itself (Fig. 7E). MDCK cells as well. As LΔCT was apically enriched in MDCK Together, these observations suggested that the axonal targeting cells on its own (Hunziker et al., 1991), chimeras based on LΔCT signal (cytoplasmic tail residues 45-59) of NgCAM also promoted could not be used for trafficking studies in MDCK cells. Therefore, apical targeting. we tested two chimeras made with Lct27 as the backbone. We added either NgCAM cytoplasmic tail 45-59 (which still possesses axonal Discussion targeting information) or NgCAM cytoplasmic tail 66-114 (which Previously we suggested that L1/NgCAM follows a transcytotic does not contain axonal targeting information) to generate route to the axonal/apical domains (Wisco et al., 2003; Yap et al., Somatodendritic and axonal signals in L1/NgCAM 1521

2008; Anderson et al., 2005). In this work, we identify the signal (Fig. 1). The same sequence mediates basolateral sorting cytoplasmic tail signals that orchestrate trafficking along the in MDCK cells (Anderson et al., 2005). The YRSLE motif successive steps of the transcytotic pathway, including a sufficient corresponds to the classical tyrosine-based sorting motif somatodendritic signal of the YxxØ class and a cytoplasmic axonal (consensus: YxxØ where x is any amino acid and Ø is a bulky targeting signal in a novel class. Additionally, we establish that the hydrophobic amino acid). Significantly, the YRSLE motif has same signal can mediate axonal and apical transport in neurons and previously been shown to act as an endocytosis motif and bind MDCK cells, respectively. the clathrin adaptor AP-2 (Kamiguchi et al., 1998). Both somatodendritic targeting and endocytosis of NgCAM are Mapping of a somatodendritic targeting signal therefore mediated by the same motif. The transcytotic model proposes that NgCAM first becomes We also investigated the somatodendritic sorting capacity of targeted to the somatodendritic domain. We identified the the two basolateral sorting motifs in LDLR. Both of these sequences surrounding the alternatively spliced exon RSLE as a basolateral signals are tyrosine-dependent signals, but fall into sufficient somatodendritic sorting signal, confirming our previous distinct classes from YxxØ. The proximal signal falls into the indirect results using a tyrosine 33 point mutant of NgCAM FxNPxY class, whereas the distal signal is atypical and requires (Wisco et al., 2003). The tyrosine preceding the RSLE motif was two tyrosines for activity (QDGYSYPSR). A triple mutant found to be necessary for the activity of this somatodendritic replacing the three tyrosines with alanines in both motifs loses somatodendritic targeting (Jareb and Banker, 1998). Unexpectedly, the proximal signal on its own had poor capacity for somatodendritic targeting (Fig. 4). The proximal signal is strictly dependent on the epithelial adaptor AP-1B in MDCK cells (Fields et al., 2007). AP-1B is highly expressed in many, but not all, epithelia and only expressed at very low levels in embryonic brain (Ohno et al., 1999). It is therefore likely that neurons express insufficient levels of AP-1B for efficient somatodendritic targeting via the proximal motif. The distal signal, however, was a potent somatodendritic signal (Fig. 4). Adaptors other than AP-1B, possibly AP-4 (Yap et al., 2003), therefore could mediate somatodendritic targeting of YxxØ and GYSY somatodendritic signals. The FxNPxY motif, however, is a poor somatodendritic signal in neurons and apparently does not make efficient use of neuronal adaptor complexes.

Mapping of a cytoplasmic axonal targeting signal

Journal of Cell Science Two domains of NgCAM contain sufficient information for axonal transport, the extracellular domain and the cytoplasmic tail (Figs 2, 5). The extracellular axonal signal was mapped to the FNIII repeats (Sampo et al., 2003). It has previously been reported that the cytoplasmic tail of NgCAM was dispensable for targeting because a chimera between the extracellular domain of CD8α and the cytoplasmic tail of NgCAM was not targeted preferentially to the axon (Sampo et al., 2003). By contrast,

Fig. 6. Glycine but not serines are required for activity of the CT45-59 axonal signal. (A) The A/D PI of NgCAM (n=23), LΔCT (n=24), LexNct45-59 (n=38) and its mutants LexNct45- 59(G-R) (n=41) and LexNct45-59(S-A) (n=30) (as indicated on left) were determined. Error bars correspond to s.e.m. ***P<0.0001. (B,C) The localization of LexNct45-59(G-R) (B) and LexNct45-59(S-A) (C) is shown. Surface distribution of the chimeras was detected with an anti-Lex antibody without permeabilization (green). DsRed was co-expressed (red) to delineate all neuronal processes. The soma regions containing the dendrites are shown in the top row, whereas the distal regions of the long axons are shown in the bottom row for both B and C. 1522 Journal of Cell Science 121 (9)

chimeras accumulated as highly on axons as did full-length NgCAM, suggesting that the NgCAM extracellular domain contributes to the efficiency of axonal targeting. Likewise, the cytoplasmic axonal signal improves axonal targeting (NgCAM versus NgCAMCT43). We mapped the location of the cytoplasmic axonal signal to CT residues 45-59 (Fig. 5), encompassing the glycine-rich region SASGSGAGSGVGSPG. As the signal contains also numerous serine residues, it seemed possible that the signal might be regulated by phosphorylation. However, as mutating all serine residues to alanines had no effect, phosphorylation does not seem to play a role. Instead, disruption of the hydrophobic nature of this segment by introducing charged residues in places of glycine residues inactivated the signal (Fig. 6). Importantly, we show here that the same cytoplasmic sequence in NgCAM promotes both axonal and apical accumulation (Fig. 7), pointing to conservation of signals and possibly machinery for the transcytotic pathway in the two cell types. The L1 family members NrCAM and neurofascin are found restricted to axon initial segments and nodes of Ranvier, rather than localized all along axons (Hedstrom and Rasband, 2006; Salzer, 2003). Sequence alignment of the cytoplasmic tails of chick NgCAM, NrCAM and neurofascin revealed that both NrCAM and neurofascin in chick share the YRSLE motif with NgCAM, but only NgCAM contains the glycine-rich axonal motif (Fig. 8). It is thus likely that different L1 family members use different targeting pathways and machinery from L1/NgCAM to localize to their correct domains (Boiko et al., 2007; Dzhashiashvili et al., 2007).

Cytoplasmic axonal signals: a comparison Fig. 7. NgCAM tail 45-59 is sufficient for sorting to the apical membrane in Other axonal targeting signals have been mapped in several other MDCK cells. (A) Diagrammatic depiction of LDLR-based NgCAM transmembrane proteins. These signals can be found in the cytoplasmic tail chimeras. Chimeras contain the proximal (amino acid residues β 5-27) basolateral sorting signal of LDLR that is co-linear with the endocytosis extracellular domain in some proteins ( APP) (Simons et al., 1995), + motif and part of the NgCAM cytoplasmic tail as indicated. (B) Fully in the cytoplasmic domain [K channels (Chung et al., 2006; Gu polarized MDCK cells were transiently transfected with cDNA encoding et al., 2003; Rivera et al., 2005)] or in both [agrin (Neuhuber and NgCAM and incubated at 37°C for 30 hours. Cells expressing NgCAM were Journal of Cell Science Daniels, 2003); NgCAM, this work and Sampo et al. (Sampo et al., surface stained with anti-NgCAM antibody (8D9). Subsequently, cells were 2003)]. In the case of the Kv1 class of K+ channels, the axonal fixed, permeabilized and incubated with secondary antibodies labeled with β Alexa 594. The locations of the apical, basal and lateral domains are indicated targeting motif maps to the binding site of its auxiliary subunit Kv by arrows for easier orientation. (C-E) Polarized MDCK cells were transiently which provides the link to microtubule binding proteins (EB1) and transfected with cDNAs encoding Lct27 (C), Lct27Nct45-59 (D) and motors (KIF3). It is possible, but we think unlikely, that other axonal Lct27Nct66-114 (E), and incubated at 37°C for 30 hours. The transfected proteins also bind to Kvβ for axonal targeting. Rather, each axonal constructs were visualized by surface staining with anti-LDLR antibodies (C7). After surface staining, cells were fixed, permeabilized and stained for an protein might use distinct signals to bind to unique adaptors that endogenous basolateral marker protein using the anti-gp58 antibody. Cells enable complexing with common sets of microtubule-regulating were then incubated with secondary antibodies labeled with Alexa 488 (gp58) proteins and motors. Multiple KIFs other than KIF3 have been and Alexa 594 (Lct27 constructs). Specimens were analyzed by confocal implicated in axonal cargo transport, for example KIF5 [βAPP microscopy, and representative x-z sections are shown. (Nakata and Hirokawa, 2003); Kv1.3 (Rivera et al., 2007)] and KIF4 [L1 (Peretti et al., 2000)]. However, the adaptors linking L1 to its presumptive motor KIF4 are not known and no candidate genes we observed axonal targeting of chimeras constructed from the were identified in a yeast two-hybrid screen using the glycine-rich ectodomain of LDL receptor and the cytoplasmic tail of NgCAM axonal signal of NgCAM as bait (C.C.Y., unpublished). or from the ectodomain of CD4 and the cytoplasmic tail of NgCAM In several proteins, the axonal signal maps to an endocytosis (Fig. 2). It is unclear why the CD8α chimera does not polarize to signal that is active preferentially in the somatodendritic domain. the axon, but folding or accessibility of tail signals might be Axonal accumulation for these proteins is therefore achieved by influenced by the nature of the extracellular domain. None of the selective removal from the somatodendritic surface by endocytosis

Fig. 8. Sequence alignment of the cytoplasmic tail of chick NgCAM with chick neurofascin and chick NrCAM. Identical sequences are boxed and shaded. The NgCAM axonal targeting motif is underlined. The somatodendritic targeting signal is in red. Somatodendritic and axonal signals in L1/NgCAM 1523

and selective retention on the axon (Garrido et al., 2003a). The In hippocampal neurons, however, NgCAM with a mutation in the axonal signal identified here in the NgCAM cytoplasmic tail does YRSLE motif did not accumulate in dendrites but was instead routed not map to the known endocytosis signal (YRSLE) in NgCAM and directly into the axon (Wisco et al., 2003). Furthermore, the does not act as a somatodendritic endocytosis signal (C.C.Y., YRSLE motif acts as a sufficient somatodendritic targeting signal unpublished). in hippocampal neurons (Fig. 1). Sorting differences depending on neuronal cell type have also been reported for some K+ channels Signal hierarchy (Rivera et al., 2005). Taken together, these observations raise the As the NgCAM cytoplasmic tail is sufficient to direct a heterologous interesting possibility that different neurons may differ in the ways protein to the axon via transcytosis (LexNct1-114; Fig. 3), it contains they process sorting signals of membrane proteins and might be all the signals needed to execute all three steps: a somatodendritic able to route proteins along distinct pathways in a regulated fashion and endocytosis signal co-linear with YRSLE and an axonal (Winckler, 2004). targeting determinant in CT45-59. Therefore, axonal localization of NgCAM does not depend on a single ‘axonal targeting motif’, Materials and Methods but on the sequential read-out and execution of multiple sorting Antibodies motifs in different intracellular compartments. Hybridomas producing anti-LDLR (C7) antibodies were purchased from American The transcytotic pathway raises important questions of how a Type Culture Collection. Hybridomas producing antibodies directed against gp58 (6.23.3) were generated in the laboratory of Dr Kai Simons (Max Planck Institute, multi-signal protein traverses a multi-step pathway in a sequential Dresden, Germany). The 8D9 anti-NgCAM hybridoma was obtained from NIH fashion. The best studied transcytosing receptor is the pIgR, which Hybridoma Bank. Anti-CD4 antibody was a generous gift from Dr Benedicte Dargent carries dimeric IgA from the basolateral to the apical side of (INSERM Marseille). epithelial cells (Mostov et al., 2003). For pIgR, ligand binding Generation of LDLR and CD4-based NgCAM cytoplasmic tail stimulates transcytosis several-fold in epithelial cells (van chimeras Ijzendoorn et al., 2002) and in neurons (de Hoop et al., 1995). The CD4 plasmid was a kind gift from Dr Benedicte Dargent (INSERM Marseille), Phosphorylation of a serine residue in the basolateral sorting motif while LDLR plasmid was from Dr Ira Mellman. NgCAMCT43 has been described might silence it and permit apical delivery from endosomes previously (Anderson et al., 2005). To generate LDLR and CD4-based NgCAM chimeras, a SalI site was inserted downstream of the transmembrane region of LDLR (Casanova et al., 1990; Luton et al., 1998). Alternatively, kinetic or CD4 or in the cytoplasmic tail of LDLR at position 27 using QuickChange Site- studies combined with modeling suggested that pIgR might enter directed Mutagenesis Kit (Stratagene). Various length of NgCAM cytoplasmic tails the transcytotic pathway by bulk-flow (Sheff et al., 1999), rather were amplified by PCR with forward primers that contained a SalI site and reverse primers with a stop codon followed by a XbaI site. The amplified DNAs were ligated than by a signal-mediated process. with LDLR- or CD4-containing extracellular domain and transmembrane region at We mapped an axonal targeting signal in the cytoplasmic tail the SalI site and subcloned into pCB6BS vector to generate various LDLR and CD4 of NgCAM that can provide access to the transcytotic pathway. chimeras. For constructing LΔCT5-27-based plasmids, the cytoplasmic region of LDLR from residues 5 to 27 was looped out using Altered Sites Mutagenesis Kit Additionally, we show that the axonal targeting information in (Promega), and a SalI site was inserted downstream of the last residue of the CT45-114 can be read and executed not only in the endosome cytoplasmic tail for the purpose of ligating in frame with NgCAM cytoplasmic region. (i.e. transcytotic pathway, LexNct1-114), but also in the TGN (i.e. All point mutations produced in this study were generated using Site-directed direct pathway) if somatodendritic signals are removed Mutagenesis Kit. The sequences of all PCR products were confirmed by automated sequence analysis. Adenoviruses were prepared for several of the chimeras after

Journal of Cell Science (LexNct45-114; Fig. 3). In MDCK cells, NgCAM remains subcloning into pShuttle according to the manufacturer’s instructions (see Wisco et basolateral if the activity of tyrosine kinases is inhibited (Anderson al., 2003). et al., 2005). Regulated phosphorylation of the YRSLE motif might thus silence this sorting motif in one compartment and keep Generation of point mutations in the glycine-rich motif To generate point mutations in the glycine-rich motif of the cytoplasmic region of it active in another. Indeed, work by Lemmon’s group has NgCAM from residue 45 to 59, plasmid LexNct45-59 was used as a template and demonstrated that the YRSLE motif can be phosphorylated by a amplified by PCR with reverse primers containing point mutations, where either all src family kinase, which leads to inhibition of AP-2 binding glycines were replaced by arginines or alanines were substituted for serines to create LexNct45-59 (G-R) and LexNct45-59 (S-A). The sequences of both constructs were (Schaefer et al., 2002). Furthermore, the YRSLE motif is bound confirmed bidirectionally by sequencing analysis. by additional proteins (Dickson et al., 2002), the binding of which might also be regulated by phosphorylation. Thus, phosphorylation Cell culture probably plays a role in regulating the activity of signals in Primary cultures of hippocampal neurons were grown as described by Wisco et al. (Wisco et al., 2003) and cultured for 8-11 days. The data in Fig. 6 were obtained NgCAM. from a changed culture protocol which we adopted late during the course of this Previously, we suggested that NgCAM tail contains crucial work. In this new protocol, glial-conditioned media was used to feed the neuronal binding sites for src kinases in the cytoplasmic stretch following cultures. This led to longer lived and more robust cultures that polarize NgCAM and chimeras more efficiently, as manifested in higher A/D PI. MDCK cells were cultured the YRSLE motif as NgCAMCT43 was not phosphorylated in vitro in MEM containing 7% (v/v) fetal bovine serum, 2 mM L-glutamine and 100 μg/ml and remained expressed at the basolateral membrane (Anderson et penicillin/streptomycin. To allow for polarization, cells were seeded on polycarbonate al., 2005). Interestingly, we found in this study that NgCAMCT43 membrane filters at a density of 4ϫ105 cells per 12 mm filter (0.4-μm pore size; is axonal in neurons. The exact role of phosphorylation in regulating Corning-Costar Transwell units) and cultured for 3 days with daily changes of the medium in the basolateral chamber. the activity of targeting signals in NgCAM in neurons therefore requires additional work in the future. Transfections/infections The filter-grown MDCK cells were transiently transfected with cDNAs encoding Multiple regulated pathways to the axon? LDLR-CT27, NgCAM, or LDLR-CT27/NgCAM constructs added to the apical chamber using LipofectAMINE (Invitrogen) according to the manufacturer’s protocol In previous studies by Vance Lemmon and co-workers, and incubated at 37°C for 30 hours. Subsequently, cell-surface staining was performed accumulation of L1 in neurites of DRG neurons (rather than just as described previously (Fields et al., 2007). All immunofluorescence preparations in somata) was shown to be dependent on the YRSLE motif were analyzed using a Zeiss confocal microscope (Microsystem LSM 510; Carl Zeiss MicroImaging) with an Axiovert 100 microscope (Carl Zeiss MicroImaging) and a (Kamiguchi and Lemmon, 1998) suggesting that the cytoplasmic Plan-Apochromat 63ϫ objective. Images were enhanced and combined using Adobe YRSLE sequence acts as an axonal targeting motif in DRG neurons. Photoshop. 1524 Journal of Cell Science 121 (9)

Neuronal cultures at DIV 9-12 were transfected using Lipofectamine2000 with 1 Casanova, J. E., Breitfeld, E. E., Ross, S. A. and Mostov, K. E. (1990). Phosphorylation μg DNA, 3 μl Lipofectamine2000 for 60-90 minutes, washed and incubated for 16- of the polymeric immunoglobulin receptor required for its efficient transcytosis. Science 20 hours. A plasmid encoding DsRed was mixed with the chimera-encoding plasmids 248, 742-745. and co-transfected. The extent of polarization to axons or dendrites (axon/dendrite Chang, M. C., Wisco, D., Ewers, H., Norden, C. and Winckler, B. (2006). Inhibition of polarity index) was determined as in Wisco et al. (Wisco et al., 2003) for staining sphingolipid synthesis affects kinetics but not fidelity of L1/NgCAM transport along without permeabilization. For some experiments, recombinant adenoviruses were used direct but not transcytotic axonal pathways. Mol. Cell. Neurosci. 31, 525-538. as described before (Wisco et al., 2003). Hippocampal cultures grown on glass Chuang, J. Z. and Sung, C. H. (1998). The cytoplasmic tail of rhodopsin acts as a novel coverslips were moved to 12-well plates in 500 μl conditioned medium in the presence apical sorting signal in polarized MDCK cells. J. Cell Biol. 142, 1245-1256. Chung, H. J., Jan, Y. N. and Jan, L. Y. (2006). Polarized axonal surface expression of of 1 mM kynurenic acid and 1-5 μl of purified adenovirus was added per well. After μ neuronal KCNQ channels is mediated by multiple signals in the KCNQ2 and KCNQ3 4 hours, 500 l of conditioned medium was added and cells incubated for 24-36 C-terminal domains. Proc. Natl. Acad. Sci. USA 103, 8870-8875. hours. 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 Brefeldin A block/release in hippocampal neurons in culture. J. Cell Biol. 130, 1447-1459. Brefeldin A block/release experiments were carried out as described (Wisco et al., Dickson, T. L., Mintz, C. D., Benson, D. L. and Salton, S. R. J. (2002). Functional 2003). Briefly, cells were infected as described above. After 4 hours, Brefeldin A binding interaction identified between the axonal CAM L1 and members of the ERM (Epicentre Technologies) was added to 0.5 to 0.75 μg/ml final concentration. After family. J. Cell Biol. 157, 1105-1112. 12-18 hours, coverslips were washed twice in conditioned medium and transferred Dotti, C. G. and Simons, K. (1990). Polarized sorting of viral glycoproteins to the axon to fresh dishes. Coverslips were removed and fixed at indicated times and stained and dendrites of hippocampal neurons in culture. Cell 62, 63-72. by the sandwich technique described below in which surface and intracellular pools Dzhashiashvili, Y., Zhang, Y., Galinska, J., Lam, I., Grumet, M. and Salzer, J. L. (2007). of the same protein can be visualized with two different fluorophores. The Nodes of Ranvier and axon initial segments are ankyrin G-dependent domains that sandwich staining allowed counting cells that were infected (i.e. positive for assemble by distinct mechanisms. J. Cell Biol. 177, 857-870. Fields, I. C., Shteyn, E., Pypaert, M., Proux-Gillardeaux, V., Kang, R. S., Galli, T. and intracellular NgCAM), but where the protein had not reached the surface yet (i.e. Folsch, H. (2007). v-SNARE cellubrevin is required for basolateral sorting of AP-1B- negative for surface staining). We observe that the kinetics of surface transport are dependent cargo in polarized epithelial cells. J. Cell Biol. 177, 477-488. somewhat variable between cultures and appear slower for older cultures (12DIV) Folsch, H. (2005). The building blocks for basolateral vesicles in polarized epithelial cells. than for younger (8 DIV). All experiments were performed at least three independent Trends Cell Biol. 15, 222-228. times. Folsch, H., Pypaert, M., Maday, S., Pelletier, L. and Mellman, I. (2003). The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct Immunofluorescence membrane domains. J. Cell Biol. 163, 351-362. Cells were fixed in 2% paraformaldehyde/3% sucrose/PBS in 50% conditioned Galvan, C., Camoletto, P. G., Dotti, C. G., Aguzzi, A. and Ledesma, M. D. (2005). medium at room temperature for 30 minutes, quenched in 10 mM glycine/PBS for Proper axonal distribution of PrP(C) depends on cholesterol-sphingomyelin-enriched 10 minutes. Alternatively, 2% paraformaldehyde/3% sucrose/0.125% membrane domains and is developmentally regulated in hippocampal neurons. Mol. Cell. glutaraldehyde/PBS was used in some experiments. The fixation conditions used do Neurosci. 30, 304-315. not introduce holes into the overwhelming majority of cells. Coverslips were then Gan, Y., McGraw, T. E. and Rodriguez-Boulan, E. (2002). The epithelial-specific adaptor blocked in 5% horse serum/1% BSA/PBS ±0.05% saponin for 30 minutes. Antibodies AP1B mediates post-endocytic recycling to the basolateral membrane. Nat. Cell Biol. were diluted in 1% BSA/PBS ±0.05% saponin and incubated for 1-2 hours. Coverslips 4, 605-609. Garrido, J. J., Fernandes, F., Giraud, P., Mouret, I., Pasqualini, E., Fache, M. P., Jullien, were mounted in Vectashield (Vector labs) and viewed on a Zeiss Axiophot with a ϫ F. and Dargent, B. (2001). Identification of an axonal determinant in the C-terminus 40 objective lens. Images were captured with the Orca cooled CCD camera of the sodium channel Na(v)1.2. EMBO J. 20, 5950-5961. (Hamamatsu) using Openlab software (ImproVision) and processed identically in Garrido, J. J., Fernandes, F., Moussif, A., Fache, M. P., Giraud, P. and Dargent, B. Adobe Photoshop. For surface staining, live cells were incubated for 5 minutes at (2003a). Dynamic compartmentalization of the voltage-gated sodium channels in axons. room temperature in primary antibody before fixation and secondary antibody Biol. Cell 95, 437-445. incubation. Garrido, J. J., Giraud, P., Carlier, E., Fernandes, F., Moussif, A., Fache, M. P., Debanne, D. and Dargent, B. (2003b). A targeting motif involved in sodium channel clustering Separate staining of surface and intracellular population of at the axonal initial segment. Science 300, 2091-2094. chimera proteins Gu, C., Jan, Y. N. and Jan, L. Y. (2003). A conserved domain in axonal targeting of Kv1 Journal of Cell Science To visualize the internal and surface population of NgCAM separately, a protocol (Shaker) voltage-gated potassium channels. Science 301, 646-649. om, K. L. and Rasband, M. N. from V. Lemmon’s group was used with modifications (Kamiguchi et al., 1998). Hedstr (2006). Intrinsic and extrinsic determinants of ion channel localization in neurons. J. Neurochem. 98, 1345-1352. Briefly, cells were fixed, blocked and incubated with primary antibody as described Hodson, C. A., Ambrogi, I. G., Scott, R. O., Mohler, P. J. and Milgram, S. L. (2006). above. The primary antibody was detected with a FITC-GaM Fab (Jackson Polarized apical sorting of guanylyl cyclase C is specified by a cytosolic signal. Traffic Immunologicals). Any unbound primary antibody was subsequently blocked with 7, 456-464. unconjugated GaM-Fab (1 mg/ml) for 1 hour and fixed for 10 minutes. Cells were Hua, W., Sheff, D., Toomre, D. and Mellman, I. (2006). Vectorial insertion of apical and then permeabilized in 0.05% saponin/1% BSA/PBS for 30 minutes and incubated basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D with a second round of primary antibody. This second round of antibody was detected live cell imaging. J. Cell Biol. 172, 1035-1044. with a RhodamineRed Goat-anti-Mouse secondary antibody (Jackson Hunziker, W., Harter, C., Matter, K. and Mellman, I. (1991). Basolateral sorting in Immunologicals). This protocol leads to separable staining of the internal and external MDCK cells requires a distinct cytoplasmic domain determinant. Cell 66, 907-920. populations in all cells, except very highly overexpressing cells. In those cases, the Jacob, R. and Naim, H. Y. (2001). Apical membrane proteins are transported in distinct surface could not be completely blocked with unconjugated Fab even at much higher vesicular carriers. Curr. Biol. 11, 1444-1450. concentrations. Jareb, M. and Banker, G. (1998). The polarized sorting of membrane proteins expressed in cultured hippocampal neurons using viral vectors. Neuron 20, 855-867. Kamiguchi, H. and Lemmon, V. (1998). A neuronal form of the cell adhesion molecule Work in the Winckler laboratory was supported by a March of Dimes L1 contains a tyrosine-based signal required for sorting to the axonal growth cone. J. Basil O’Connor Award and NIH NS045969 from NINDS. Work in the Neurosci. 18, 3749-3756. Fölsch laboratory was supported by a grant from the NIH (GM070736). Kamiguchi, H., Long, K. E., Pendergast, M., Schaefer, A. W., Rapoport, I., We gratefully acknowledge the generosity of Dr Benedicte Dargent Kirchhausen, T. and Lemmon, V. (1998). The neural cell adhesion molecule L1 interacts with the AP-2 adaptor and is endocytosed via the clathrin-mediated pathway. J. Neurosci. (Université de la Méditerranée, Marseille, France) for providing crucial 18, 5311-5321. reagents. We thank Kevin Liao and Johanna Cannon for culturing Lai, H. C. and Jan, L. Y. (2006). The distribution and targeting of neuronal voltage-gated neurons. ion channels. Nat. Rev. Neurosci. 7, 548-562. Ledesma, M. 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