14-3-3 Proteins Regulate a Cell-Intrinsic Switch from Sonic Hedgehog-Mediated Commissural Axon Attraction to Repulsion after Midline Crossing

SummaryAxons must switch responsiveness to guidance cues during development for correct pathfinding. Sonic Hedgehog (Shh) attracts spinal cord commissural axons ventrally toward the floorplate. We show that after crossing the floorplate, commissural axons switch their response to Shh from attraction to repulsion, so that they are repelled anteriorly by a posterior-high/anterior-low Shh gradient along the longitudinal axis. This switch is recapitulated in vitro with dissociated commissural neurons as they age, indicating that the switch is intrinsic and time dependent. 14-3-3 protein inhibition converted Shh-mediated repulsion of aged dissociated neurons to attraction and prevented the correct anterior turn of postcrossing commissural axons in vivo, an effect mediated through PKA. Conversely, overexpression of 14-3-3 proteins was sufficient to drive the switch from Shh-mediated attraction to repulsion both in vitro and in vivo. Therefore, we identify a 14-3-3 protein-dependent mechanism for a cell-intrinsic temporal switch in the polarity of axon turning responses

RhoA Proteolysis Regulates the Actin Cytoskeleton in Response to Oxidative Stress.

The small GTPase RhoA regulates the actin cytoskeleton to affect multiple cellular processes including endocytosis, migration and adhesion. RhoA activity is tightly regulated through several mechanisms including GDP/GTP cycling, phosphorylation, glycosylation and prenylation. Previous reports have also reported that cleavage of the carboxy-terminus inactivates RhoA. Here, we describe a novel mechanism of RhoA proteolysis that generates a stable amino-terminal RhoA fragment (RhoA-NTF). RhoA-NTF is detectable in healthy cells and tissues and is upregulated following cell stress. Overexpression of either RhoA-NTF or the carboxy-terminal RhoA cleavage fragment (RhoA-CTF) induces the formation of disorganized actin stress fibres. RhoA-CTF also promotes the formation of disorganized actin stress fibres and nuclear actin rods. Both fragments disrupt the organization of actin stress fibres formed by endogenous RhoA. Together, our findings describe a novel RhoA regulatory mechanism

RhoA fragments and cleavage-resistant promote the formation of actin stress fibres.

<p><b>A)</b> Serum-starved Swiss 3T3 fibroblasts were transfected with Flag-tagged RhoA 1–56 and 57–193, which correspond to RhoA-NTF and -CTF respectively, as well as WT-, Q63L- and the cleavage-resistant L57A/Q63L-RhoA. Cells were stained with anti-Flag M2 antibody (green), Alexa-Fluor 546 phalloidin (red) and Hoechst (blue) to label RhoA, the actin stress fibres and the nucleus respectively. Scale bar, 20 μm. <b>B)</b> Classification of the actin phenotype in transfected Swiss 3T3 cells. Significance was determined by the Chi-square test. *, <i>p</i> < 0.05; ** <i>p</i> < 0.005; ***, <i>p</i> < 0.0005; ****, <i>p</i> < 0.0001. <i>n</i> > 40 cells from 7 independent experiments. <b>C)</b> Quantification of the ratio of actin-covered area divided by the total surface of the transfected cells represented as the mean +/- S.E.M. Significance was established by one-way ANOVA from <i>n</i> > 40 cells collected from 7 independent experiments. *, <i>p</i> < 0.05; ** <i>p</i> < 0.005; ***, <i>p</i> < 0.0005; ****, <i>p</i> < 0.0001. <b>D)</b> Representative side view of a z-stack of a Swiss 3T3 cell overexpressing RhoA-CTF showing the localization of nuclear actin rods (red) relative to the nucleus (blue) and RhoA 57–193 (green). Scale bar, 10 μm.</p

Identification of a 10 kDa amino-terminal RhoA fragment.

<p><b>A)</b> Western blot analysis of COS-7 cell lysates transfected with Flag-tagged WT-RhoA or with 2Xmyc-tagged WT-RhoA using an anti-Flag M2 or anti-c-myc antibody reveals the presence of FL-RhoA and RhoA-NTF bands. <b>B, C)</b> Western blot of cell lysates following treatment with the proteosome inhibitors MG132 <b>(B)</b> or epoxomicin <b>(C)</b>. <b>D)</b> Expression of wild-type (WT), constitutively active (Q63L and G14V), dominant-negative (T19N) as well as non-prenylated wild-type (C190A), active (Q63L/C190A) and inactive (T19N/C190A) RhoA constructs in COS-7 cells analyzed by western blot using the Flag M2 antibody. Western blot panels illustrating FL-RhoA only are exposed to evaluate loading of full-length protein while panels with FL-RhoA and RhoA-NTF are a longer exposure of the same blot to visualize RhoA-NTF.</p

Serine proteases, caspases and calpain regulate RhoA proteolysis.

<p><b>(A-F)</b>. Lysates from COS-7 cells transfected with Flag-tagged WT-RhoA were analyzed by western blot with anti-Flag M2 antibody following treatment with protease inhibitors. Transfected cells were treated for 3h with the serine protease inhibitor AEBSF <b>(A)</b>, 24h hours with the pan-caspase inhibitor z-VAD-fmk <b>(C)</b>, or 3 hours with the calpain inhibitor calpeptin <b>(E)</b> and the levels of RhoA-NTF were quantified by densitometry <b>(B, D, F)</b>. Water and DMSO were vehicle controls. <b>(G-H</b>). 2Xmyc WT-RhoA <b>(G)</b> or Flag RhoA 1–56 <b>(H)</b> were immunoprecipitated from transfected COS-7 cells and treated for 45 minutes with recombinant μ-calpain in the presence or absence of 14 μM calpeptin. <b>I)</b> Diagram illustrating the mechanism underlying RhoA proteolysis.</p

Endogenous RhoA proteolysis is enhanced by oxidative stress.

<p><b>A, B)</b> Lysates from COS-7 cells were transfected with Flag-tagged WT-RhoA <b>(A)</b> or T19N-RhoA <b>(B)</b> and western blotted with anti-Flag M2 antibody. Cells were exposed to H₂O₂ for 1 hour, 24 hours prior to lysis. <b>C)</b> COS-7 cells transfected with Flag-tagged WT-RhoA were immunoprecipitated with the Rho Y486 antibody from Abcam. Lysates and immunoprecipitates were probed with anti-RhoA Y486 or anti-FLAG M2 antibodies. <b>D)</b> Immunoprecipitation of RhoA-NTF from untransfected COS-7 cells treated with H₂O₂ for 1 hour at 24 hour prior to lysis or from various healthy adult rat tissues, including the heart, brain and lungs, using the Rho Y486 antibody. Arrow: RhoA-NTF. <b>E-F)</b> Analysis of the relative abundance of FL-RhoA and RhoA-NTF in cell lysates compared to a dose curve of recombinant WT-RhoA by Western blot analysis with the Rho Y486 antibody. The graph in F quantifies the average concentration of FL-RhoA as well as RhoA-NTF upon treatment with 1000 μM H₂O₂.</p