Phosphodiesterase-Ialpha/autotaxin's MORFO domain regulates oligodendroglial process network formation and focal adhesion organization

Abstract

Development of a complex process network by maturing oligodendrocytes is a critical but currently poorly characterized step toward myelination. Here, we demonstrate that the matricellular oligodendrocyte-derived protein phosphodiesterase-Ialpha/autotaxin (PD-Ialpha/ATX) and especially its MORFO domain are able to promote this developmental step. In particular, the single EF hand-like motif located within PD-Ialpha/ATX's MORFO domain was found to stimulate the outgrowth of higher order branches but not process elongation. This motif was also observed to be critical for the stimulatory effect of PD-Ialpha/ATX's MORFO domain on the reorganization of focal adhesions located at the leading edge of oligodendroglial protrusions. Collectively, our data suggest that PD-Ialpha/ATX promotes oligodendroglial process network formation and expansion via the cooperative action of multiple functional sites located within the MORFO domain and more specifically, a novel signaling pathway mediated by the single EF hand-like motif and regulating the correlated events of process outgrowth and focal adhesion organization.

Figures

Fig. 1

PD-Iα/ATX’s MORFO domain promotes oligodendroglial process outgrowth. (A) Cells of the immortalized oligodendroglial cell line CIMO were found to be responsive to the adhesion-antagonizing effect of PD-Iα/ATX’s MORFO domain. Means and standard errors of three independent experiments done in quadruplicates are shown. Student’s t-test analysis revealed an overall two-tailed significance level of p<0.05. (B) Scanning electron micrographs (SEM) taken at 500x (top panels) and 2000x (bottom panels) magnification and 48° tilt. CIMO cells plated in the presence of rPD-Iα/ATX-MORFO (right panels) extended numerous primary and secondary processes and possessed rounded somas. In contrast, these cells appeared flat and non-process bearing under control conditions (rLacZ; left panels).

Fig. 2

PD-Iα/ATX’s MORFO domain promotes the extension of a complex process network by post-migratory, premyelinating oligodendrocytes. (A) and (B) Oligodendrocyte progenitor cells were isolated from brains of 3 day-old rats. Cells were plated onto mixed (FN plus rControl (rControl) or FN plus rPD-Iα/ATX (rPD-Iα/ATX)) substrates and after 24 hrs soluble rControl or rPD-Iα/ATX protein was added. 48 hrs after plating cells were immunostained with the O4 antibody to visualize post-migratory, premyelinating oligodendrocytes. (A) Normalized values of the cells’ process indices (total amount of O4-positive process surface per cell) were plotted against normalized values of their complexity indices (ratio of process index and network area) on logarithmic scales (mean control value = 50; see dashed lines). Five independent experiments with at least 30 cells per condition are shown. In the upper right corner of each of the scatter plots a representative confocal image of an O4-positive oligodendrocyte, cultured under the indicated conditions, is depicted. Confocal images represent 2D maximum projections of stacks of 0.5 μm optical sections. Scale Bar: 20 μm. (B) Bar graph illustrating the percentage of cells with a process or complexity index greater than the calculated control mean (means and standard errors are shown). For both parameters, Student’s t-test analysis revealed an overall two-tailed significance level of p<0.05. (C) and (D) O4-positive oligodendrocytes were isolated from brains of 4 day-old rats. Cells were cultured for 48 hrs and treated with a control (siControl) or PD-Iα/ATX-specific (siPD-Iα/ATX) siRNA SMARTpool. 48 hrs after siRNA transfection cells were immunostained with the O4 antibody and analyzed as in (A) and (B). Means and standard errors are shown in B. For both parameters, Student’s t-test analysis revealed an overall two-tailed significance level of p<0.05. Scale Bar: 20 μm.

Fig. 3

PD-Iα/ATX’s MORFO domain promotes oligodendroglial process network formation in part via its EF hand-like motif. (A) Structure-function domains of full length PD-Iα/ATX (top) and detailed sequence information for the region around the EF hand-like motif (bottom). The MORFO domain and the loop region of the EF hand-like motif are indicated by a light and dark gray box, respectively. PD-Iα/ATX’s catalytic domain is represented by a diagonally striped box, its two somatomedinB domains by vertically striped boxes and the signal peptide crucial for secretion by a horizontally striped box. Protein sequences are noted for native and mutated (ΔEF and D→N) recombinant (r)PD-Iα/ATX-MORFO fusion proteins and for the peptides representing PD-Iα/ATX’s EF hand loop region in its native (EF peptide) and mutated (EF D→N peptide) form. Mutated amino acid residues are underlined. Conserved amino acid residues characteristic for EF hand loops are indicated by an asterisk above the native sequence. (B) Bar graph representing the areas occupied by the cells’ process networks (network area = white area in insets to E, upper panel). Mean control network areas were set to 100% and experimental network area values adjusted accordingly. In the bar graph, means and standard errors are shown. Stars indicate overall two-tailed significance levels of pt-test). (C) 2D graph representing process index (X-axis) and complexity index (Y-axis) of all cells in the presence of native or mutated (ΔEF and D→N) recombinant (r)PD-Iα/ATX-MORFO fusion proteins, as well as native (EF peptide) or mutated (EF D→N peptide) peptides. Control indices were set to 100% and experimental index values adjusted accordingly. In the graph, means and standard errors are shown. (D) and (E) Representative examples of oligodendrocytes cultured in the presence of control, EF or EF D→N peptide (D) and rPD-Iα/ATX-MORFO, rPD-Iα/ATX-MORFO D→N, rPD-Iα/ATX-MORFO ΔEF or control protein (E). Cells were isolated and treated as in the experiments to Fig. 2A and B. Confocal images represent 2D maximum projections of stacks of 0.5 μm optical sections. Scale Bar: 20 μm (note that the magnification is higher in D than in E).

Fig 4

Paxillin-containing focal adhesions are present at the leading edge of oligodendroglial protrusions and their number is diminished in the presence of PD-Iα/ATX’s MORFO domain. (A) CIMO cells were plated in the presence of rControl protein for 1 hr. Under these conditions focal adhesions located toward the leading edge of cellular protrusions were detectable by immunocytochemical staining for paxillin. (B) Similar to CIMO cells paxillin-containing focal adhesions (see arrows) were detected in OLG-growth cones of primary oligodendrocytes nucleofected with a plasmid encoding paxillin-EGFP. (C) CIMO cells were plated in the presence of rPD-Iα/ATX-MORFO for 1 hr and immunostained for paxillin. Compared to control conditions (A) a decrease in the number of paxillin-containing focal adhesions was noticed. (D) Quantification of focal adhesions after immunostaining of CIMO cells plated on the different substrates. Bar graph depicts the number of paxillin-containing focal adhesions per cell as % of control (mean of control = 100%). (E) Quantification of PD-Iα/ATX’s lysoPLD activity. Full length rPD-Iα/ATX, native (rPD-Iα/ATX) and mutated (rPD-Iα/ATX-T210A), was purified from stably transfected Cos-7 cells and lysoPLD activity was determined using the fluorogenic assay described by Ferguson et al. (2006). A typical example done in triplicates is depicted in the graph. Numbers on the Y-axis represent fluorescent intensities. (F) CIMO cells were plated onto fibronectin-coated glass coverslips in the presence of 0.1 μg/ml control (Control), native rPD-Iα/ATX (rPD-Iα/ATX) or enzymatically inactive rPD-Iα/ATX (rPD-Iα/ATX-T210A) protein. Paxillin-containing focal adhesions per cell were determined as in D. In all bar graphs, means and standard errors are shown. Stars indicate an overall two-tailed significance level of pt-test). Arrows in (A) indicate smallest sized focal adhesions counted (approximately 1 μm2) for the bar graphs in (D) and (F). Confocal images in (A), (B) and (C) represent optical sections approximately 250 nm in depth and close to the basal surface. Scale Bars: 20 μm in (A) and (C); 10 μm in (B).

Fig. 5

PD-Iα/ATX’s MORFO domain mediates a reduction in the number of detectable paxillin-containing focal adhesions via its EF hand-like motif. In addition, PD-Iα/ATX’s MORFO domain reduces the number of detectable α-actinin- but not vinculin-containing focal adhesions. (A–C) CIMO cells were plated for 1 hr in the presence of fusion protein or peptide (see label below each bar and Fig. 3A) and cells were immunolabeled for paxillin (A), vinculin (B) or α-actinin (C). The graphs illustrate the percentage of paxillin-, vinculin- or α-actinin-containing focal adhesions per cell as % of control (mean of control = 100%). Means and standard errors are shown. The stars indicate overall two-tailed significance levels of pt-test).

Figure 6

Proposed model of the functional properties of PD-Iα/ATX’s MORFO domain toward post-migratory, premyelinating oligodendrocytes. As described previously (Fox et al., 2004), PD-Iα/ATX’s MORFO domain induces adhesion-antagonism (symbolized by the distance between the integrin receptor (α and β) and the ECM constituent fibronectin (FN)) through a mechanism likely involving a yet to be identified cell surface receptor (white rectangle). In addition, the MORFO domain causes dephosphorylation at the tyrosine residue 925 (925) of focal adhesion kinase (FAK) without significantly affecting the phosphorylation of the tyrosine residue 397 (397P). Thus, FAK appears to remain associated with an integrin-containing complex at the cell surface. The data presented here further demonstrate that PD-Iα/ATX’s MORFO domain, likely via its EF hand-like motif, promotes a loss of paxillin and α-actinin from focal adhesions, thereby weakening the link between the ECM and the cytoskeleton and promoting the outgrowth of in particular higher order processes/branches. Vinculin distribution appears largely unaffected, suggesting that similar to FAK it remains associated with a protein complex located at the cell surface. Vinculin is not known to directly bind to either FAK or integrins. Thus, it is likely bound to the cell surface-associated complex via a yet to be identified adaptor protein (white rectangle with rounded edges). PD-Iα/ATX’s MORFO domain as a whole promotes not only the outgrowth of higher order processes but also stimulates process elongation and thus the formation of a highly complex and expanded process network.