Sema6A reverse signaling is stimulated by multimerisation.

<p>(<b>A</b>) Scheme representing how the induced multi-aggregation of the cytosolic domain of Sema6A activates signaling pathways. A chimeric construct was engineered by putting in frame the entire cytosolic domain of Sema6A (Sema6A-cyt) together with 3 self-aggregation FKBP domains, and GFP. The chimeric peptide targets the plasma membrane by the myristoylating sequence located at the N-terminal. The application of the drug FK1012 triggers the chemically induced multimerisation (CIM) of Sema6A-cyt via the self-aggregation of FKBP. (<b>B</b>) Cerebellar neurons transfected with MF3-GFP or MF3-Sema6A-cyt-GFP (MF3-Sema6A-cyt), and treated with 500nM FK1012 (“CIM”). GFP-positive aggregates are indicated (arrowheads). Scale bar = 20μm. (<b>C</b>) Axonal shaft of cerebellar neurons transfected with myc-Sema6A-FL or MF3-Sema6A-cyt-GFP (MF3-Sema6A-cyt), and treated with PlxnA2-EC or CIM respectively. Scale bar = 5 μm. (<b>D</b>) and (<b>E</b>) Graphs summarising the myc-Sema6A-FL aggregation in myc-Sema6A-FL-expressing neurons treated with PlxnA2-EC at different time points. The aggregation is represented as the average distance between Sema6A clusters, or the average size of Sema6A clusters; <i>n</i> = 50–100 cells per time point. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test. (<b>F</b>) MF3-Sema6A-cyt-expressing granular neurons were cultured on NIH3T3 cell layers, and treated with CIM. Scale bar = 50 μm. (<b>G</b>) Graph representing the axonal length of MF3-Sema6A-cyt-expressing cerebellar neurons treated with CIM for 24 and 48h; <i>n</i> = 50–100 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test.</p

Sema6A exerts constitutive and PlxnA2-dependent cell-autonomous functions.

<p>(<b>A-F</b>) NIH3T3 cells expressing GFP alone (<b>A and B</b>), myc-Sema6A-FL (Sema6A; <b>B and E</b>), or myc-Sema6A-K393D (Sema6A-K393D; <b>C and F</b>) were treated with purified AP-Fc (AP; <b>A-C</b>) or PlxnA2-EC-Fc (PlxnA2-EC; <b>D-F</b>). Scale bar = 20 μm. (<b>G</b>) Graph represents the cell area in cells transfected with GFP, Sema6A or Sema6A-K393D and treated with AP or PlxnA2-EC; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> <0.001; Student’s <i>t</i>-test.</p

Sema6A cytoplasmic domain interacts with Abl and Mena.

<p>(<b>A</b>) Immunoprecipitations from untreated or PlxnA2-EC-treated COS-7 cells previously transfected with different combinations of myc-Sema6A, Abl-V5 and PlxnA2. Immunoprecipitations (Ip) were performed employing an anti-V5 antibody. Antibodies against V5, myc and PlxnA2 were used in the immunoblots (Ib). (<b>B</b>) Immunoprecipitations from untreated or PlxnA2-EC treated COS-7 cells previously transfected with different combinations of myc-Sema6A, Mena-V5 and PlxnA2. Immunoprecipitations (Ip) were performed employing an anti-myc antibody. Antibodies against V5, myc and PlxnA2 were used in the immunoblots (Ib). (<b>C</b>) and (<b>D</b>) Interactions were quantified by immunoblotting and densitometric analysis. Histograms represent quantification of interaction normalized to the amount of immunoprecipitated protein (Abelson-V5 for Sema6A-Abelson or Sema6A-myc for Sema6A-Mena) from three independent experiments. Students t-test *p<0,05 indicating signifiance. S6A-Abl/S6A-Abl+PlA2 <i>p</i> = 0.013, S6A-Abl-PlA2/S6A-Abl-PlA2+PlA2 <i>p</i> = 0.034. Errors bars represent standard error.</p

Cell-contraction induced by PlxnA2-Sema6A interaction depends on Abl signaling.

<p>(<b>A-F</b>) NIH3T3 cells expressing GFP (<b>A</b> and <b>D</b>), Sema6A-FL (<b>B</b> and <b>E</b>), and Sema6A-∆Abl (<b>C</b> and <b>F</b>) were treated with purified AP-Fc (AP; <b>a-c</b>) or PlxnA2-EC-Fc (PlxnA2-EC; <b>D-F</b>). Scale bar = 20 μm. (<b>G</b>) Scheme illustrates that the cell contraction is via Abl signaling. (<b>H</b>) Graph represents the contraction of the cell area in NIH3T3 transfected with GFP, Sema6A or Sema6A-∆Abl and treated with AP or PlxnA2-EC; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; Student’s <i>t</i>-test.</p

PlxnA2 interacts with Sema6A <i>in cis</i> and <i>in trans</i>.

<p>(<b>A</b>) Scheme summarising Sema6A-AP bindings. (<b>B-F</b>): COS-7 cells were transfected with the empty vector (mock; <b>B</b>), Sema6A-FL (<b>C</b>), PlxnA2-FL (<b>D</b>), PlxnA2-FL together with Sema6A-FL (<b>E</b>) or PlxnA2-A396E (<b>F</b>); and treated with Sema6A-AP. (<b>G</b>) Scheme summarising PlxnA2-AP bindings. (<b>H-K</b>): COS-7 cells were transfected with the empty vector (mock; <b>H</b>), Sema6A-FL (<b>I</b>), PlxnA2-FL (<b>J</b>) or PlxnA2-FL together with Sema6A-FL (<b>K</b>) and treated with PlxnA2-AP. (<b>L</b>) PlxnA2 immunoprecipitation (IP) from protein samples of COS-7 cells transfected with Sema6A-FL, PlxnA2-FL or Sema6A-FL together PlxnA2-FL. Antibodies against PlxnA2 and Sema6A were used in the immunoblots. (<b>M</b>) Sema6A IP from protein samples of COS-7 cells transfected with Sema6A-FL, PlxnA2-FL or Sema6A-FL together with PlxnA2-FL. (<b>N</b>) PlxnA2 IP from protein samples of mouse cerebellums or EGL explants. (<b>O</b>) PlxnA2 IP from protein samples of COS-7 cells transfected with PlxnA2-FL, Sema6A-∆cyt or Sema6A-∆cyt together with PlxnA2-FL. (<b>P</b>) PlxnA2 IP from protein samples of COS-7 cells transfected with PlxnA2-A396D, Sema6A-FL or Sema6A-FL together with PlxnA2-A396D. (<b>Q</b>) PlxnA2 IP from protein samples of COS-7 cells transfected with PlxnA2-FL, Sema6A-K393D or Sema6A-K393D together with PlxnA2-FL.</p

Sema6A signaling reduces the axonal length of granular neurons.

<p>(<b>A-C</b>) GFP-expressing granular neurons were cultured on Control cells (<b>A</b>), Sema6A Cells (<b>B</b>), PlxnA2 Cells (<b>C</b>) and PlxnA2-A396E Cells (A396E Cells, <b>D</b>). Scale bar = 20 μm. (<b>E</b>) Graph represents the axonal length of granular neurons grown on different NIH3T3 layers; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test. (<b>F</b>) Graph represents the axonal length of <i>PlxnA2</i>^<i>+/+</i> (dark columns) and <i>PlxnA2</i>^<i>-/-</i> (light columns) granular neurons grown on NIH3T3 cells. Neurons were transfected with GFP alone or together with PlxnA2-FL, and cultured on control (Ctrl) or on Sema6A cell layers; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; Student’s <i>t</i>-test. (<b>G</b>) Graph represents the axonal length of <i>Sema6A</i>^<i>+/-</i> (dark columns) and <i>Sema6A</i>^<i>-/-</i> (light columns) granular neurons grown on NIH3T3 cells. Neurons were transfected with GFP alone or together with Sema6A-FL, and cultured on control (Ctrl) or on PlxnA2 cell layers; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; Student’s <i>t</i>-test. (<b>H-J</b>) Granular neurons transfected with GFP alone (<b>H</b> and <b>I</b>) or together with Sema6A-∆cyt (<b>J</b>), and grown on control or on PlxnA2 cells. Scale bar = 20 μm. (<b>K</b>) Graph represents the axonal length of <i>Sema6A</i>^<i>+/-</i> (dark columns) and <i>Sema6A</i>^<i>-/-</i> (light columns) granular neurons. Neurons were transfected with GFP alone or together with Sema6A-∆cyt, and cultured on control (Ctrl) or on PlxnA2 cell layers; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; Student’s <i>t</i>-test.</p