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First published online 14 April 2008
doi: 10.1242/jcs.022442

Journal of Cell Science 121, 1514-1525 (2008)
Published by The Company of Biologists 2008
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Pathway selection to the axon depends on multiple targeting signals in NgCAM

Chan Choo Yap1, Rita L. Nokes2,*, Dolora Wisco1,*, Eric Anderson{ddagger}, Heike Fölsch2 and Bettina Winckler1,§

1 University of Virginia Medical School, Department of Neuroscience, 409 Lane Road, Charlottesville, VA 22908, USA
2 Northwestern University, Department of Biochemistry, Molecular Biology and Cell Biology, 2205 Tech Drive, Evanston, IL 60208, USA


Figure 1
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  		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 L{Delta}CT, 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.

 

Figure 2
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  		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 {Delta}CT backbones.

 

Figure 3
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  		Fig. 3. The presence of multiple cytoplasmic tail signals is required for transcytotic routing of NgCAM chimeras. The site of initial surface delivery of LexNct1-114 (A), Lct27Nct45-114 (B) and LexNct45-114 (C) was determined using the Brefeldin A (BFA) block release assay. The percentage of neurons displaying the chimera on the surface uniformly (triangles), axonally enriched (squares) or somatodendritically enriched (circles) was determined for multiple time points after release from BFA. Additionally, the percentage of cells was scored in which no surface staining was detectable, but intracellular staining was present (broken line). The chimera LexNct45-114, which contains the cytoplasmic axonal signal of NgCAM but lacks the somatodendritic and endocytosis motifs, is delivered directly to the axonal plasma membrane (C). If the somatodendritic/endocytic signal surrounding Tyr33 is present [as in LexNct1-114 (A)], the first surface appearance was frequently somatodendritically enriched, suggestive of transcytosis. Inclusion of the LDLR-derived somatodendritic/endocytic signal in Lct27Nct45-114 (B) also led to initial somatodendritic surface appearance, suggesting transcytotic routing. One representative experiment (of a minimum of three) is shown for each construct.

 

Figure 4
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  		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.

 

Figure 5
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  		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; significantly different from L{Delta}CT. (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.

 

Figure 6
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  		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), L{Delta}CT (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.

 

Figure 7
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  		Fig. 7. NgCAM tail 45-59 is sufficient for sorting to the apical membrane in MDCK cells. (A) Diagrammatic depiction of LDLR-based NgCAM cytoplasmic tail chimeras. Chimeras contain the proximal (amino acid residues 5-27) basolateral sorting signal of LDLR that is co-linear with the endocytosis motif and part of the NgCAM cytoplasmic tail as indicated. (B) Fully polarized MDCK cells were transiently transfected with cDNA encoding NgCAM and incubated at 37°C for 30 hours. Cells expressing NgCAM were surface stained with anti-NgCAM antibody (8D9). Subsequently, cells were fixed, permeabilized and incubated with secondary antibodies labeled with Alexa 594. The locations of the apical, basal and lateral domains are indicated by arrows for easier orientation. (C-E) Polarized MDCK cells were transiently transfected with cDNAs encoding Lct27 (C), Lct27Nct45-59 (D) and Lct27Nct66-114 (E), and incubated at 37°C for 30 hours. The transfected constructs were visualized by surface staining with anti-LDLR antibodies (C7). After surface staining, cells were fixed, permeabilized and stained for an endogenous basolateral marker protein using the anti-gp58 antibody. Cells were then incubated with secondary antibodies labeled with Alexa 488 (gp58) and Alexa 594 (Lct27 constructs). Specimens were analyzed by confocal microscopy, and representative x-z sections are shown.

 

Figure 8
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  		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.

 

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© The Company of Biologists Ltd 2008