(A) Oli- cells and primary oligodendrocytes were transfected with full-length hnRNP A2 and immunostained for hnRNP A2 or MBP

Images were acquired by confocal microscopy, and either a single slice (Oli-) or the complete stack (primary oligodendrocytes) is depicted. HnRNP A2–containing granules are present in the processes of Oli- cells as well as primary oligodendrocytes. Insets show an enlarged view of the boxed sections. Bars, 10 μm. (B) A granule-free supernatant was analyzed by Western blotting for hnRNP E1 and A2 proteins after RNase A treatment or L1-Fc binding. Western blot bands were analyzed densitometrically from 8 and 15 experiments for RNase treatment and L1-Fc binding, respectively. The control values (RNase A and C-Fc) were set to 1 and the mean relative increase of hnRNP E1 and A2 in the granule-free fraction was plotted in response to RNase A treatment or L1-Fc binding. Error bars indicate SEM; significance was tested with tests: *, P ≤ 0.05; **, P ≤ 0.01. = 8 (RNase A) and = 15 (L1-Fc). (C) The model illustrates the proposed events: During initial axon–glial contacts, neuronal L1 binds glial F3 (1), leading to an activation of Fyn (2), which phosphorylates hnRNP A2 (3). This leads to a release of hnRNP A2 and E1 from the granule and liberation of MBP mRNA (4) at the axon–glial contact site, allowing localized synthesis of the MBP protein (5) required for generation of the myelin sheath. The dotted lines illustrate potential alternative activation pathways of Fyn kinase mediated by L1 binding.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

(A) Differentiated Oli- cells or primary oligodendrocytes were treated with the indicated concentrations of L1-Fc

Equal amounts of cell lysates were analyzed by Western blotting using the indicated antibodies. Numbers on top indicate L1-Fc concentration in nM. (B, left) Oli- cells were incubated with control Fc (C-Fc, human IgG) or L1-Fc in the presence of control or Fyn siRNA. Tyrosine-phosphorylated proteins were immunoprecipitated (P-Tyr IP) and analyzed by Western blotting for hnRNP A2 (P-Tyr A2). Levels of tyrosine-phosphorylated A2 are strongly increased in L1-Fc–treated cells compared with control Fc–treated cells, and this effect is reduced in cells treated with Fyn siRNA. Total lysates (before IP) were analyzed by Western blotting with hnRNP A2, Fyn, and GAPDH antibodies and demonstrated unchanged levels of total hnRNP A2 and a reduction of Fyn protein by Fyn siRNA. GAPDH served as a loading control. (B, right) The diagram represents the data from three such experiments. Protein bands of tyrosine-phosphorylated hnRNP A2 were densitometrically quantified and the values of control Fc–treated cells were set to 1. The relative increase of tyrosine-phosphorylated hnRNP A2 in L1-Fc–treated cells in the presence of control and Fyn siRNA was plotted. Error bars indicate SEM; = 3.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

(A) Oli- cells were transfected with control EGFP or a constitutively active Fyn (Fyn) construct

Tyrosine-phosphorylated proteins were immunoprecipitated (P-Tyr IP) from cell lysates (lysate) and analyzed for hnRNP A2 and hnRNP E1/E2. HnRNP A2 immunoprecipitates from cells transfected with active Fyn, whereas hnRNP E1/E2 is absent. Mouse brain lysate and antibody beads alone served as blotting controls. Note that the hnRNP A2 antibody also recognizes the splice variants hnRNP B1 and B0a. HC and LC, heavy and light chain of mouse anti-phosphotyrosine antibody used in the immunopreciptiation. Black lines indicate that intervening lines have been spliced out. (B) Endogenous hnRNP A2 was immunoprecipitated from Oli- cells and subjected to an in vitro kinase assay using purified recombinant Fyn kinase. The top shows a phosphotyrosine blot and a band at 36 kD only in the presence of recombinant Fyn. This was identified as hnRNP A2 by reblotting with an hnRNP A2 antibody (bottom). Numbers to the right of the gel blots indicate molecular mass in kD. (C) HnRNP A2 coimmunoprecipitates with Fyn from Oli- cells transfected with wild-type Fyn, whereas a control protein (α-tubulin) does not.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

(A) L1-Fc was bound to Oli- cells in the presence of control or F3 siRNA and detected with an anti–human Fc Cy2 antibody (left)

F3 knockdown was analyzed by Western blotting of the cells used for L1-Fc binding. Mouse brain lysate was used as control and GAPDH served as a loading control (right). Bars, 10 μm. (B) Differentiated Oli- cells were incubated with 75 nM of control Fc (human IgG, C-Fc) or L1-Fc and 75 nM L1-Fc in the presence of monoclonal F3 or O4 (control) antibodies. Binding was quantified by cell ELISA (see Materials and methods; = 6). (C) Oli- cells were treated with 0 or 25 nM L1-Fc after treatment with control or F3 siRNA. Binding was quantified by cell ELISA and the ratio of 25 nM Li-Fc–treated cells/0 nM L1-Fc–treated cells was plotted to express the relative amount of bound L1-Fc in control and F3 siRNA-treated cells. Note that because of a different experimental setup, the reduction of F3 protein levels was not as efficient as in the experiment shown in A ( = 9). Error bars indicate SEM. Significance was assessed by tests: *, P < 0.02; **, P < 0.01.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

(A) An A2RE-containing region of the 3′ UTR of MBP14 mRNA was cloned downstream of the luciferase coding sequence (CDS) and used as a translational reporter in the DualGlo luciferase assay

(B) Oli- cells were nucleofected with an A2RE containing luciferase reporter, a luciferase control, and either wild-type Fyn (Fyn WT), constitutively active Fyn (Fyn), or EGFP. was normalized to luciferase activity for every measurement in all experiments ( = 3). (C) Total RNA was isolated from the cells used in the luciferase assay shown in B. qRT-PCR was performed to compare A2RE luciferase mRNA from Fyn (WT and Fyn)-transfected cells with EGFP-transfected cells ( was used for normalization). Error bars indicate SEM. Significance was assessed with tests: *, P < 0.05; **, P < 0.01; ns, not significant.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

Identification of p130Cas as an oligodendroglial tyrosine-phosphorylated protein in the presence of active Fyn.

<p>(A) Tyrosine-phosphorylated proteins were immunoprecipitated from Oli-<i>neu</i> cells overexpressing active Fyn. A Coomassie-stained SDS-Polyacrylamide gel with first and second elution (E1 and E2), antibody control (control) and MW marker (M) is shown. The indicated band (p130Cas) was excised and analyzed by mass spectrometry. Molecular weights in kilodalton are indicated on the right. (B) Primary structure of p130Cas. The identified peptides are shown in bold red and cover 20% of the protein sequence. (C) p130Cas expression at different developmental stages in cultured oligodendrocytes (DIV, days in vitro, 1, 2, 4, 6, 8) was analyzed by Western blots with antibodies indicated on the left. CNP and MOG represent early and late maturation markers, respectively, and GAPDH serves as a loading control.</p

The effect of p130Cas on spreading and migration of oligodendroglial cells.

<p>(A) Oli-<i>neu</i> cells were treated with p130Cas or control siRNA. 24 hours later, the cells were detached from their culture vessel and 30 min after re-plating they were subjected to immunofluorescence analysis of cell spreading. Cells were classified as either not spread (arrowhead), showing lamellipodia (asterisk) or lamella (arrow). (B) Statistical evaluation of A. Data represents the mean ± SEM from 3 independent experiments; ** p<0.01 (Student’s <i>t</i> test). (C) Oli-<i>neu</i> cells were treated as in A. Here, immunofluorescence analysis of process thickness was carried out 4 hours after re-plating. Three examples per condition are shown. Areas of measurement are outlined by white dashed rectangles. (D) Statistical evaluation of C. The thickest process for each individual cell was measured and plotted on a chart (from high to low diameters) and the average ratio ± SEM from 3 independent experiments is presented; ** p<0.01 (Student’s <i>t</i> test). (E) Oli-<i>neu</i> cells were treated with siRNA as in A. After 24 hours, they were re-plated in Boyden chambers and allowed to migrate for 6 hours in the presence of 5 ng/ml bFGF before fixation and migration analysis were carried out. Data are expressed as a percentage of basal migration, i.e. the migration of Oli-<i>neu</i> without chemoattractant. Data represent the mean ± SEM from 3 independent experiments; * p<0.05 (Student’s <i>t</i> test). (F) Knockdown of p130Cas in Oli-<i>neu</i> cells. The siRNA-treated cells as analyzed in A-E were lysed and p130Cas levels were assessed by Western blotting. GAPDH serves as loading control.</p

The effect of p130Cas knockdown on spreading and migration in Oli-<i>neu</i> does not result from changes in cellular viability.

<p>(A) Oli-<i>neu</i> cells were treated with p130Cas or control siRNA. 24 hours later, the cells were detached from their culture vessel and 6 h after re-plating a TUNEL assay was performed to test for apoptosis. Data show the mean ± SEM from 3 independent experiments; ns, not significant (Student’s <i>t</i> test). (B) Knockdown of p130Cas in Oli-<i>neu</i> cells. The siRNA-treated cells as analyzed in A were lysed and p130Cas levels were assessed by Western blotting. α-tubulin serves as loading control. (C) Oli-<i>neu</i> cells were treated with p130Cas or control siRNA. 24 h later, the cells were detached from their culture vessel and 0.5 h and 6 h after re-plating MTT and LDH assays were carried out to test for cell viability and membrane integrity, respectively. Data show the mean ± SEM from 3 independent experiments; ns, not significant (Student’s <i>t</i> test). (D) Knockdown of p130Cas in Oli-<i>neu</i> cells. The siRNA-treated cells as analyzed in C were lysed and p130Cas levels were assessed by Western blotting. α-tubulin serves as loading control.</p

p130Cas is involved in oligodendroglial apoptosis.

<p>(A) Primary OPCs were treated with control or p130Cas siRNA. 48 hours later, cells were lysed and tested for levels of p130Cas and cleaved Caspase 3 (indicative of apoptosis) by Western analysis. GAPDH serves as loading control. (B) Primary OPCs were treated with siRNA as in A. Additionally, some cells were treated with the apoptosis inducing agent staurosporine as positive control. After 42 hours, the cells were subjected to a TUNEL assay to test for apoptotic cells. Data show the mean ± SEM from 11 independent experiments; ** p<0.01 (Student’s <i>t</i> test). (C) Relative cell numbers from B were counted as additional indication of cell death. Data show the mean ± SEM from 11 independent experiments; ** p<0.01, *** p<0.001 (Student’s <i>t</i> test).</p