unc-44 Ankyrin and stn-2 gamma-syntrophin regulate sax-7 L1CAM function in maintaining neuronal positioning in Caenorhabditis elegans

Abstract

The L1 family of single-pass transmembrane cell adhesion molecules (L1CAMs) is conserved from Caenorhabditis elegans and Drosophila to vertebrates and is required for axon guidance, neurite outgrowth, and maintenance of neuronal positions. The extracellular region of L1CAMs mediates cell adhesion via interactions with diverse cell-surface and extracellular matrix proteins. In contrast, less is known regarding the function of the intracellular domains in the L1CAM cytoplasmic tail. Previously, we identified a role of the C. elegans L1CAM homolog, SAX-7, in maintaining neuronal and axonal positioning. Here, we demonstrate that this function is dependent on three conserved motifs that reside in the SAX-7 cytoplasmic tail: (1) the FERM-binding motif, (2) the ankyrin-binding domain, and (3) the PDZ-binding motif. Furthermore, we provide molecular and genetic evidence that UNC-44 ankyrin and STN-2 gamma-syntrophin bind SAX-7 via the respective ankyrin-binding and PDZ-binding motifs to regulate SAX-7 function in maintaining neuronal positioning.

Figure 1.—

Immunofluorescence analysis shows positive SAX-7 immunostaining in the different sax-7 mutant backgrounds, except for sax-7(eq1). SAX-7 staining is strong in the nervous system; arrow points to the ventral nerve cord while arrowhead points to the pharyngeal posterior bulb. Bar, 50 μm.

Figure 2.—

P_sur-5∷sax-7 is similarly expressed as endogenous sax-7 in wild-type animals and rescues the positional defects in the GABA neuronal soma and commissural axons in sax-7(eq1) animals just as well as genomic sax-7 does. (A) A schematic of the two SAX-7 protein isoforms, SAX-7L and SAX-7S. The extracellular region contains immunoglobulin (Ig) and fibronectin type III (FNIII) repeats while the cytoplasmic tail contains the FB, AB, and PB motifs. (B) Quantitation of displaced cholinergic and GABA neurons and commissural axons in wild-type, sax-7(eq1), and transgenic sax-7(eq1) animals. Three independent transgenic lines for each construct were analyzed. Error bar shows standard error of the proportions of three sample sets where in each set n = 100. (C) Immunofluorescence analyses of transgenic sax-7(eq1) animals show that transgenic SAX-7 is similarly expressed and localized as endogenous SAX-7 in wild-type animals in multiple tissues, including the nervous system (i), body-wall muscle (ii), and hypodermis (iii), the three tissues where sax-7 activity is required for optimal positioning maintenance of GABA neurons. Respective images of wild-type and transgenic sax-7(eq1) animals are taken with the same exposure times. SAX-7 is present in (i) the nerve ring (large arrow) and ventral nerve cord (small arrow), (ii) the cell membrane of the elliptical-shaped body-wall muscle cells (arrows), and (iii) the cell membrane of the hypodermal seam cells (the arrows in the enlarged insets show SAX-7 localization on the membrane of two seam cells). Bar, 20 μm.

Figure 3.—

Quantitation of displaced GABA neurons in sax-7(eq1) transgenic animals expressing SAX-7 variants with targeted mutations in the FB, AB, and PB motifs in the SAX-7 cytoplasmic tail reveals the functional importance of these conserved domains. Each sax-7 construct is introduced into sax-7(eq1) animals (stippled bars) to test the ability of these constructs to rescue neuronal displacement. Each sax-7 construct is introduced also in wild-type animals (solid bars) to confirm that these constructs do not function in a dominant fashion. Next to the rescue data for each variant SAX-7 construct is a schematic of the SAX-7 protein containing the corresponding mutation. Dashed lines indicate deletion of the respective motif. Three independent transgenic lines for each construct in both sax-7 and wild-type backgrounds were analyzed. Error bar shows the standard error of the proportions of three sample sets where in each set n = 100. Statistical significance was assessed by t-test where ***P < 0.5 × 10⁻⁸, **P < 0.1 × 10⁻⁴, and *P < 0.5 × 10⁻⁴, as compared to sax-7(eq1); Ex[Psur-5∷sax-7L] animals. The statistical significance between the rescue ability of SAX-7:;SFIGQA and SFIGQE was assessed by the Student's t-test and is as stated in the figure.

Figure 4.—

sax-7 and unc-44 genetically interact in neuronal positioning maintenance. (A) Quantitation of animals exhibiting displaced GABA neurons in sax-7, unc-44, and unc-119 mutant backgrounds. Error bar shows the standard error of the proportions of three sample sets where in each set n = 100. (B) UNC-44 and SAX-7 localizations are not interdependent. (Left) UNC-44 immunostaining. (Right) SAX-7 immunostaining in wild-type, unc-44(e362), and sax-7 (eq1) adult animals as well as arrested embryos homozygous for the nDf41 chromosomal deficiency, which removes the unc-44 gene; as expected, these embryos do not show UNC-44 staining. Images of UNC-44 and SAX-7 immunostaining in respective animals of different genetic backgrounds were taken with the same exposure times. Insets show corresponding UNC-44 and SAX-7 localization to the membrane boundaries of cells. Bar, 20 μm.

Figure 5.—

SAX-7 and UNC-44 molecularly interact as determined in a recruitment/binding assay in HEK293 cells and in yeast. (A) UNC-44∷GFP is primarily localized in the cytoplasm of transformed HEK293 cells. (B) Cotransfection with neurofascin∷SAX-7CT (labeled SAX-7) causes UNC-44∷GFP to be redistributed to the plasma membrane, colocalizing with neurofascin∷SAX-7CT. (C) However, loss of the AB motif in neurofascin∷SAX-7CT (labeled SAX-7ΔAB) dramatically reduces this redistribution of UNC-44∷GFP so that UNC-44∷GFP largely remains in the cytoplasm. Bar, 5 μm. (D) Cell growth on selective media (L/T) shows yeast cells that are successfully transformed with the SAX-7 and UNC-44 constructs. Cell growth on selective media (L/T/H or L/T/H/A) reveals positive interaction between UNC-44 and SAX-7CT in yeast. In contrast, no cell growth is seen on selective media in cells transformed with UNC-44 and SAX-7CTΔAB, revealing the requirement for the AB motif for the UNC-44/SAX-7 interaction.

Figure 6.—

sax-7 and stn-2 interact genetically in neuronal positioning maintenance. (A) A schematic of the genomic structure of the stn-2 gene (solid boxes represent coding sequence and lines represent introns) and the predicted protein structure. The position of the stn-2 deletion alleles, tm1869 and ok2417, are shown relative to the gene and protein structure. (B) Quantitation of animals exhibiting displaced GABA and cholinergic neurons in sax-7, stn-2, and stn-1 mutant backgrounds. Error bar shows the standard error of the proportions of three sample sets where in each set n = 100.

Figure 7.—

stn-2 is expressed in neurons and striated muscles where STN-2 partially colocalizes with SAX-7. (A) On the basis of a GFP epifluorescence of a rescuing stn-2∷gfp reporter, STN-2∷GFP, is detected in neurons (large arrow in i), including the ventral nerve cord motor neurons (arrow in ii), and axonal processes (ventral nerve cord, arrowhead in ii; commissural axons, open arrowheads in iii; dorsal nerve cord, solid arrowheads in iii; lateral nerve cord, solid arrow in iii). Bar, 20 μm. (B) Immunostaining of SAX-7 in wild-type animals reveals that SAX-7 is localized to muscle membrane boundaries (arrows) and sarcomeres (arrowheads) in body-wall muscles. The small arrow is pointing to a commissural axon that also shows high SAX-7 expression. Bar, 5 μm. (C) In sarcomeres, SAX-7 and STN-2 partially colocalize, as shown by co-immunostaining of SAX-7 and GFP in stn-2(tm1839) animals expressing stn-2∷gfp. Bar, 5 μm.

Figure 8.—

A model of intracellular interactions with the SAX-7 cytoplasmic tail. The model speculates that UNC-44 ankyrin and STN-2 γ-syntrophin link SAX-7 to the actin cytoskeleton. It is not known if UNC-44 and STN-2 bind simultaneously to SAX-7 as depicted in the model. Studies in both vertebrates and C. elegans reveal that γ-syntrophins are likely components of the DPC, thus suggesting that other DPC proteins, such as DYB-1 dystrobrevin and DYS-1 dystrophin, may also function together with SAX-7 to maintain neuronal positioning.