Inhibition of CaMKII and MEK1/2 reduces M6Ab early neurite outgrowth in nerve growth factor (NGF)-differentiated PC12 cells.

<p>(A) PC12 cells were transfected with the pcDNA3-M6Ab-GFP or pcDNA3-GFP plasmids. After transfection, PC12 cells were differentiated with 100 ng/ml NGF for 72 h in the presence of 10 µM KN-62 or 10 µM U1026 (Con; 0.1% DMSO). Cells were fixed, and images were taken with a Zeiss LSM510 laser scanning confocal microscope. (B) Quantification of the total number of neurites, total length of neurites, and filopodium-like processes in a 20-µm neurite length. Results are expressed as the mean ± SD of at least 40∼50 neurites. At least three independent experiments were analyzed. *Significant difference compared with the respective control of WT M6Ab overexpression (P<0.05). Scale bars, 10 µm.</p

Expression of M6Ab-GFP and M6Ab(S263D)-GFP driven by a neuron-specific HuC promoter in zebrafish embryos.

<p>(A) To express the wild-type and S263D mutant proteins of zebrafish M6Ab under the control of a neuron-specific HuC promoter, plasmids pHuC-GFP, pHuC-M6Ab-GFP, pHuC-M6Ab(S263D)-GFP, and pHuC-M6Ab-GFP/pHuC-CaMKII · 1(T287D)-DsRed were individually injected into zebrafish embryos at the one-cell stage. Zebrafish embryos at 48 h post-fertilization (hpf) with GFP fluorescence were selected for the image analysis. Images were taken using a Zeiss LSM510 laser scanning confocal microscope. Merged images of red and green fluorescence are shown in (e, e′, and e″), while only green fluorescence images are shown in (a, a′, a″, b, b′, b″, c, c′, c″, d, d′ and d″). Higher magnification of two regions marked with yellow boxes in panel a-e is shown in panels a′–e′ and a″–e″, respectively. (B) Quantification of zebrafish numbers with significant neurite outgrowth at 48 hpf. Scale bars, 10 µm.</p

Overexpression of zM6Ab can induce neurite outgrowth and filopodia in PC12 and COS-1 cells.

<p>(A) PC12 cells were transfected with the control pcDNA3-GFP or pcDNA3-M6Ab-GFP or pcDNA3-M6Ab-T166A-GFP plasmids. Twenty-four hours after transfection, cells were treated with nerve growth factor (NGF) (100 ng/ml) for 2 days. Cells were then fixed, and images were taken with a Zeiss LSM510 laser scanning confocal microscope. The insets are the 2× magnified images of the boxed areas. (B) Quantification of the total number of neurites, total length of neurites, and filopodium-like processes in a 20-µm neurite length. * indicates a significant difference compared with the respective control of GFP (P<0.05). (C) COS-1 cells were transfected with the control pcDNA3-GFP or pcDNA-M6Ab-GFP or pcDNA3-M6Ab-T166A-GFP plasmids. Cells were fixed, and images were taken with a Zeiss LSM510 laser scanning confocal microscope. Scale bars, 10 µm.</p

Effects of SU6656 on invadopodia formation and functioning.

<p>A, A431-III cells were plated on gelatin or Oregon Green® 488-conjugated gelatin and treated with DMSO or 5 µM SU6656 for 5 h to investigate the formation of invadopodia and matrix degradation. B, Quantification of cells associated with matrix degradation (upper panel). Quantification of the degradation area normalized against cell number (lower panel). C, Total cell lysates were prepared for immunoblotting analysis. Active Src and downstream target cortactin (Y421) were analyzed. D, Invasion assays were performed. *<i>p</i><0.05. P values are compared with control A431-III. Error bars present the standard error of the mean. Scale bar are 22 µm.</p

Phosphorylation of S263 is critical for regulation of filopodium formation in PC12 cells.

<p>(A) Partial amino acid sequences of the wide-type and mutant proteins of zebrafish M6Ab. (B) PC12 cells were transfected with pcDNA3-GFP-HA(a), pcDNA3-M6Ab-HA(b), pcDNA3-M6Ab(S263A)-HA(c), pcDNA3-M6Ab (S263D)-HA(d), pcDNA3-M6Ab(S274A/S277A)-HA(e), pcDNA3-M6Ab(S274D/S277D)-HA(f), pcDNA3-M6Ab(A3)-HA(g), or pcDNA3-M6Ab(D3)-HA(h) plasmids. Twenty-four hours after transfection, cells were treated with nerve growth factor (NGF) (100 ng/ml) for 2 days. Then transfected cells were fixed, and M6Ab-HA was detected using anti-HA antibodies for immunostaining. PC12 neurites are shown at a higher magnification (a′–h′). (C) Quantification of the total number of neurites, total length of neurites, and filopodium-like processes in a 20- · m neurite length. Results are expressed as the mean ± SD of at least 40∼50 neurites. At least three independent experiments were analyzed. *Significant difference compared with the respective control of WT-M6Ab overexpression (P<0.05). (D) Cell lysates from different transfected cells as indicated were extracted and immunoblotted with an anti-HA antibody or anti-Tubulin antibody. (E) MS/MS spectrum on [M+2H]²⁺ (m/z 718.32) ion for the peptides DIKpSKEEQELH from WT M6Ab protein. The product ion y₈ which carries a phosphate indicated that Serine 263 was phosphorylated. Residues bearing phosphate moieties are indicated with p. “b” and “y” ion series represent fragment ions containing the N- and C-termini of the peptide, respectively. Scale bars, 10 µm.</p

MMPs, especially MMP-9, were responsible for the invadopodia and degrading ability of A431-III cells.

<p>A, A431-III cells were plated on gelatin or Oregon Green® 488-conjugated gelatin and treated with DMSO or 25 µM GM6001 for 5 h to observe the formation of invadopodia and the matrix degrading ability. Tks5, invadopodia component protein, was used as a marker. B, Quantification of cells associated with matrix degradation (left panel). Quantification of degradation area normalized against cell number (right panel). C, Effect of GM6001 on MMPs’ activities and TIMPs’ expression were measured by zymography and western blot. D, The cells were treated with 40 nM MMP-9 siRNA or control siRNA. Knockdown efficiency was measured by qPCR (left) or gelatin zymography (right). E, A431-III cells (expressing control or MMP-9 knockdown siRNA) were plated on gelatin or Oregon Green® 488-conjugated gelatin to investigate the formation of invadopodia and the matrix degrading ability. F, Quantification of cells associated with matrix degradation (left panel) and degradation area normalized against cell number (right panel).*<i>p</i><0.05. Error bars present the standard error of the mean. Scale bar are 22 µm.</p

A431-III forms invadopodia and exhibits higher ability to degrade gelatin than A431-P.

<p>A, Upper panel: A431-P and A431-III cells were stained with cortactin (red), F-actin (green), and DAPI (blue). Arrowheads, examples of invadopodia that are identified as cortactin and actin-positive dots. Representative images taken of both cells. Lower panel: Both cells were plated on Oregon Green® 488-conjugated gelatin. Degraded ECM was identified as a dark area on the gelatin. B, Upper panel: Quantification of cells associated with matrix degradation. Lower panel: Quantification of the degradation area normalized against cell number. C, Invasion assays were performed. *<i>p</i><0.05. Error bars present the standard error of the mean. Scale bar are 22 µm. P (A431-P); III (A431-III).</p

Src kinase activity and phosphorylation of cortactin were responsible for invadopodia formation in A431-III cells.

<p>A, Expression of invadopodia regulators, core components and MMPs/TIMPs in A431-P and A431-III were analyzed by microarray. B, Expression of invadopodia regulators, components and MMPs/TIMPs were validated by qPCR. C, Total cell lysates were subjected for immunoblotting analysis. The active status of Src kinase and the phosphorylation of cortactin were determined.</p