SYD-1 function requires SAX-3.

<p>(A) The <i>syd-1(ju2)</i> null mutation does not enhance guidance defects in <i>sax-3(ky123)</i> null mutants. (B) The <i>syd-1(ju2)</i> null mutation enhances guidance defects in <i>sax-3(ky200)</i> hypomorphic mutants. For experiments with the <i>sax-3(ky123)</i> null mutants, we scored only severe HSN defects (the axon never reaches the ventral nerve cord). For the <i>sax-3(ky200)</i> hypomorphic mutants, we did not observe any severe HSN axon guidance defects, but only mild defects (the axon trajectory is defective but eventually reaches the ventral nerve cord). Therefore, for experiments with the <i>sax-3(ky200)</i> hypomorphic mutants, we scored mild axon guidance defects. For all experiments, n≥200. Bracket indicates statistically significant difference, Z test for proportions (*p<0.01).</p

SYD-1 function requires SAX-3.

<p>(A) The <i>syd-1(ju2)</i> null mutation does not enhance guidance defects in <i>sax-3(ky123)</i> null mutants. (B) The <i>syd-1(ju2)</i> null mutation enhances guidance defects in <i>sax-3(ky200)</i> hypomorphic mutants. For experiments with the <i>sax-3(ky123)</i> null mutants, we scored only severe HSN defects (the axon never reaches the ventral nerve cord). For the <i>sax-3(ky200)</i> hypomorphic mutants, we did not observe any severe HSN axon guidance defects, but only mild defects (the axon trajectory is defective but eventually reaches the ventral nerve cord). Therefore, for experiments with the <i>sax-3(ky200)</i> hypomorphic mutants, we scored mild axon guidance defects. For all experiments, n≥200. Bracket indicates statistically significant difference, Z test for proportions (*p<0.01).</p

MIG-2 functions with SAX-3 to regulate axon guidance.

<p>HSN guidance defects were observed in <i>mig-2</i> null mutants and in <i>sax-3</i> null mutants. HSN guidance defects were not enhanced in <i>sax-3 mig-2</i> double null mutants, indicating that SAX-3 and MIG-2 function together to regulate HSN axon guidance. The <i>mig-2</i> null mutant enhances HSN guidance defects in <i>unc-40</i> null mutants. Alleles are <i>syd-1 (ju2)</i>, <i>mig-2(ok2273)</i>, <i>sax-3(ky123)</i>, <i>unc-40(e1430)</i>. HSN axon guidance was scored as defective if the axon failed to reach the ventral nerve cord. For all experiments, n≥200. Brackets indicate statistically significant difference, Z test for proportions (***p<0.0001).</p

Interactions between the UNC-6 and SLT-1 signaling pathways in the HSN neuron.

<p>(A) Example of normal HSN axon guidance. (B) Example of defective HSN axon guidance. (C) Synergistic genetic interaction between null mutations in the genes that encode the UNC-6 and SLT-1 guidance cues. (D) The UNC-40 and SAX-3 pathways can function in parallel to each other. For the data shown in this panel, the <i>sax-3(ky200)</i> temperature sensitive allele was used to circumvent lethality. (E) The SAX-3 receptor can function independently of the SLT-1 ligand. A null mutation in the gene that encodes the SLT-1 guidance cue results in only rare guidance defects. However, a null mutation in the gene that encodes SAX-3, the receptor for SLT-1, results in 20% guidance defects. Guidance defects are not enhanced in <i>sax-3 slt-1</i> double null mutants. (F) The UNC-40 receptor can function independently of the UNC-6 guidance cue. A null mutation in the gene that encodes the UNC-40 receptor causes HSN guidance defects at a penetrance that is similar that caused by a null mutation in the gene that encodes the UNC-6 guidance cue. However, loss of UNC-40 function enhances guidance defects in the absence of UNC-6 function, indicating that UNC-40 can function independently of UNC-6. For the data shown in panel D, we analyzed progeny of worms maintained at 20°C and shifted to 24° just prior to egg laying. For all other panels, strains were grown at 20°C. Scale bars are 10 μm. Anterior is to the right. Ventral is down. Alleles shown in all panels, except where noted in panel D, are <i>unc-6(ev400)</i>, <i>slt-1(eh15)</i>, <i>sax-3(ky123)</i>, <i>unc-40(e1430)</i>. HSN axon guidance was scored as defective if the axon failed to reach the ventral nerve cord. For the data shown in panel D, n>39. For all other data, n≥200. Brackets indicate statistically significant difference, Z test for proportions (***p<0.0001, **p<0.001).</p

SYD-1 mediates UNC-6 independent UNC-40 signaling.

<p>(A) SYD-1 functions in parallel to UNC-6, but not UNC-40. The <i>syd-1(ju2)</i> null mutation enhances HSN guidance defects in <i>unc-6</i> null mutants. However, the <i>syd-1</i> null mutation does not enhance guidance defects in an <i>unc-40</i> null mutant. (B) SYD-1 functions with UNC-40 independently of UNC-6. Guidance defects in an <i>unc-40</i>; <i>syd-1; unc-6</i> triple null mutant are not enhanced relative to <i>unc-40</i>; <i>unc-6</i> or <i>syd-1</i>; <i>unc-6</i> double null mutants. (C) The <i>syd-1</i> null mutation enhances HSN guidance defects in <i>ev546</i> and <i>tm5504</i> hypomorphic <i>unc-40</i> mutants. Alleles for panels A and B are <i>unc-6(ev400)</i>, <i>syd-1(ju2)</i>, <i>unc-40(e1430)</i>. Alleles for panel C are <i>unc-40(ev546)</i>, <i>unc-40(tm5504)</i> and <i>syd-1(ju2)</i>. HSN axon guidance was scored as defective if the axon failed to reach the ventral nerve cord. For all experiments, n≥200. Brackets indicate statistically significant difference, Z test for proportions (***p<0.0001, *p<0.01).</p

The function of the <i>syd-1</i> gene in axon guidance is mediated by the SYD-1C isoform.

<p>(A) Schematic of the three isoforms of SYD-1 and location of three mutations. The <i>ju82</i> mutation is a nonsense mutation the affects the SYD-1A and SYD-1B isoforms. The <i>ju2</i> mutation is a nonsense mutation that affects all three isoforms. The <i>tm6234</i> mutation is a deletion that affects all three isoforms. (B) The SYD-1C isoform is necessary and sufficient to mediate the role of SYD-1 in axon guidance. Guidance defects in <i>unc-6</i> null mutants are enhanced by the <i>ju2</i> and <i>tm6234</i> mutations, but not by the <i>ju82</i> mutation. Guidance defects in a <i>syd-1</i>; <i>unc-6</i> double null mutant are rescued by expressing SYD-1C in the HSN neuron with an <i>unc-86</i>::<i>syd-1c</i> transgene. (C) SYD-1C binds to the cytoplasmic domain of UNC-40 fused to GST (GST::UNC-40) and also to the cytoplasmic domain of SAX-3 fused to GST (GST::SAX-3), but not to GST alone. An unrelated protein, luciferase, does not bind to GST, GST::UNC-40, or GST::SAX-3. For reference, an amount of protein (SYD-1C or luciferase) equal to 20% of the amount of protein in the binding assay was run on the gel (20% Input). HSN axon guidance was scored as defective if the axon failed to reach the ventral nerve cord. For all experiments, n≥200. Bracket indicates statistically significant difference, Z test for proportions (**p<0.001).</p

SYD-1 promotes axon guidance by negatively regulating the MIG-2 GTPase.

<p>(A) The RhoGAP-like domain of SYD-1 can bind to the active form of MIG-2. Binding between the C-terminal RhoGAP-like domain of SYD-1 (His-SYD-1) and GST-MIG-2 was substantially enhanced after incubation with GTPγS (GST-MIG-2 + GTPγS) relative to incubation with GDP (GST-MIG-2 + GDP). No binding was observed between His-SYD-1 and GST. (B-C) SYD-1 negatively regulates MIG-2 activity. (B) The <i>syd-1(ju2)</i> null mutation enhances HSN guidance defects in partially activated <i>mig-2(gm38)</i> mutants. However, the null mutation in <i>syd-1</i> does not enhance HSN guidance defects in the fully activated <i>mig-2(gm103)</i> mutants. (C) Transgenic expression of SYD-1C suppresses axon guidance defects in the partially activated <i>mig-2(gm38)</i> mutants. HSN axon guidance was scored as defective if the axon failed to reach the ventral nerve cord. For all experiments, n≥200. Brackets indicate statistically significant difference, Z test for proportions (***p<0.0005, **p<0.005).</p

Mechanism of Intramembrane Cleavage of Alcadeins by γ-Secretase

<div><p>Background</p><p>Alcadein proteins (Alcs; Alcα, Alcβand Alcγ) are predominantly expressed in neurons, as is Alzheimer's β-amyloid (Aβ) precursor protein (APP). Both Alcs and APP are cleaved by primary α- or β-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by γ-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTFβ is initially cleaved at the ε-site to release the intracellular domain fragment (AICD) and consequently the γ-site is determined, by which Aβ generates. The initial ε-site is thought to define the final γ-site position, which determines whether Aβ40/43 or Aβ42 is generated. However, initial intracellular ε-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final γ-site position is determined remains unclear in Alcs.</p><p>Methodology</p><p>Using HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of γ-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal γ-cleavage site and Alc ICD possessing N-terminal ε-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ε-site position of all Alcα, Alcβ and Alcγ, and analyzed the relationship between the initially determined ε-site position and the final γ-cleavage position.</p><p>Conclusions</p><p>The initial ε-site position does not always determine the final γ-cleavage position in Alcs, which differed from APP. No additional γ-cleavage sites are generated from artificial/non-physiological positions of ε-cleavage for Alcs, while the artificial ε-cleavage positions can influence in selection of physiological γ-site positions. Because alteration of γ-secretase activity is thought to be a pathogenesis of sporadic Alzheimer's disease, Alcs are useful and sensitive substrate to detect the altered cleavage of substrates by γ-secretase, which may be induced by malfunction of γ-secretase itself or changes of membrane environment for enzymatic reaction.</p></div

Alteration of Alcs γ-cleavage when physiological ε-cleavage sites are replaced with non-physiological/pseudo-ε-cleavage sites.

<p>A. Positions of the physiological major and minor (ε1 and ε2) and pseudo- (ε1p and ε2p) ε-cleavage sites (upper left) are shown along with the physiological major and minor γ-cleavage sites (γ1 and γ2). The shaded amino acid sequence indicates a putative membrane-embedded region. Non-physiological/pseudo-ε-cleavage sites were designed by shifting one residue toward the N terminal of the physiological ε-cleavage sites. Representative MS spectra of p3-Alcα secreted by HEK293 cells expressing Alcα CTF, Alcα CTF-ε1, Alcα CTF-ε2, Alcα CTF-ε1p, or Alcα CTF-ε2p are shown (lower left panels). The major species p3-Alcα2N+35 with γ1 site (γ1/35, closed arrowheads) and minor species p3-Alcα2N+38 with γ2 site (γ2/38, open arrowheads) are indicated. The peak area of p3-Alcα2N+38 was compared with that of p3-Alcα2N+35, and the ratios (p3-Alcα2N+38/p3-Alcα2N+35) are indicated as γ2/γ1 (right panel). The spectra of minor species p3-Alcα38 are enlarged in windows in which intensities of 200, 300, and 400 on the y-axis correspond to 0.02, 0.03 and 0.04 in the original panels. <b>B.</b> Positions of the physiological major and minor (ε1, ε2, and ε3) and pseudo- (ε1p, ε2p, and ε3p) ε-cleavage sites (upper left) are shown along with the physiological major and minor γ-cleavage sites (γ1 and γ2). Representative MS spectra of p3-Alcβ secreted by HEK293 cells expressing Alcβ CTF, Alcβ CTF-ε1, Alcβ CTF-ε2, Alcβ CTF-ε3, Alcβ CTF-ε1p, Alcβ CTF-ε2p, or Alcβ CTF-ε3p are shown (lower left). The major species p3-Alcβ40 withγ1 site (γ1/40, closed arrowheads) and minor species p3-Alcβ37 with γ2 site (γ2/37, open arrowheads) are indicated. The peak area of p3-Alcβ37 was compared with that of p3-Alcβ40, and the ratios (p3-Alcβ37/p3-Alcβ40) are indicated asγ2/γ1 (right panel). <b>C.</b> Positions of the physiological major and minor (ε1, ε2, and ε3) and pseudo- (ε1p, ε2p, and ε3p) ε-cleavage sites (upper left) are shown along with the physiological major and minor γ-cleavage sites (γ1 and γ2). Representative MS spectra of p3-Alcγ secreted by HEK293 cells expressing Alcγ CTF, Alcγ CTF-ε1, Alcγ CTF-ε2, Alcγ CTF-ε3, Alcγ CTF-ε1p, Alcγ CTF-ε2p, or Alcγ CTF-ε3p are shown (lower left). The major p3-Alcγ31 with γ1 site (γ1/31, closed arrowhead) and minor p3-Alcγ34 with γ2 site (γ2/34, open arrowhead) are indicated. The peak area of p3-Alcγ34 was compared with that of p3-Alcγ31, and the ratios (p3-Alcγ34/p3-Alcγ31) are indicated as γ2/γ1 (right panel). (<b>A–C</b>) The ratios of products from the pseudo-site were compared to those from the respective physiological sites. Statistical analysis was performed by Student's t test (mean ± S.E., n = 4, *<i>P</i><0.05).</p

Alteration of ε-cleavage is not necessarily prerequisite to determine a specific γ-cleavage site in Alcα.

<p>A<b>.</b> Representative MS spectra of p3-Alcα secreted by HEK293 cells expressing Alcα CTF, Alcα CTF-ε1, or Alcα CTF-ε2 with either wild-type PS1 (wt) or a FAD-linked PS1 mutant (A434C, L166P, or R278T). The p3-Alcα species in cell culture media were immunoprecipitated and subjected to MALDI-TOF/MS analysis. Closed arrowheads indicate the major product with γ1 site (p3-Alcα2N+35, “γ1/35”), while open arrowheads indicate the minor product with γ2 site (p3-Alcα2N+38, “γ2/38”). The spectra of the minor p3-Alcα38 product are enlarged in windows in which intensity of 300 on the y-axis corresponds to 0.03 in the original panels. <b>B.</b> The peak area of p3-Alcα2N+38 (minor species) was compared with that of p3-Alcα2N+35 (major species), and the minor to major ratios (p3-Alcα2N+38/p3-Alcα2N+35) are indicated asγ2/γ1. Statistical analysis was performed using one-way analysis of variance followed by the Tukey-Kramer multiple comparison test (means ± S.E., n = 4). Significance in comparison to the ratio of Alcα CTF was not observed in cells expressing wild-type PS1 (wt) or FAD-linked PS1 mutants. The columns of “wt” and “A434C” are enlarged in window.</p