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doi: 10.1242/10.1242/jcs.00173

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Structure of laminin substrate modulates cellular signaling for neuritogenesis

¹ Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil
² Departamento de Anatomia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil
³ Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil
⁴ Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil

View larger version (31K): [in a new window]   Fig. 2. Neurite outgrowth on neutral or acidic laminin matrices. Dissociated cortical cells were incubated for 24 hours over laminin matrices obtained at neutral (A) or acid pH (B). Immunocytochemistry for neuronal cells was carried out using a polyclonal anti-Tau reagent as the primary antibody and Cy3-labeled anti-rabbit as the secondary antibody. Panels C-F show quantitative analyses of data obtained in panels A (pH 7, open bars) and B (pH 4, closed bars). Analyses were carried out considering only the 30 longest neurites (C) or 100 neurons randomly chosen (D-F). Three asterisks correspond to P<0.0001. Bar, 100 µm.

View larger version (18K): [in a new window]   Fig. 4. Comparison between individual neurons on neutral and acidic matrices. A shows an isolated neuron after 24 hours on a laminin matrix obtained at neutral pH. B shows a neuron after 6 hours on a laminin matrix obtained at acid pH. Cells were immunostained for Tau. Bar, 25 µm.

View larger version (108K): [in a new window]   Fig. 9. Neurite outgrowth from explants of newborn rat brains. Brain explants were obtained from the cerebral cortex of postnatal day 2. rats and plated over laminin matrices obtained at neutral (A, C and E) or acidic pH (B, D and F). Explants were developed after 24 hours for DAPI (C,D) and for the neuronal marker Tau (E,F). A and B show corresponding phase-bright images. Bar, 100 µm.

View larger version (65K): [in a new window]   Fig. 8. Effect of protein kinase inhibitors on neuritogenesis. Cortical cells were plated over laminin matrices polymerized under either neutral (A, C, E and G) or acidic conditions (B, D, F and H) and incubated for 24 hours in the absence of inhibitors (A and B) or in the presence of 0.1 µM H-89 (C and D) or 0.15 nM staurosporin (E and F). The scale bar corresponds to 100 µm. The lowest panels show average neurite lengths (quantified as the 30 longest neurites) in each case. Three asterisks correspond to P<0.001.

View larger version (103K): [in a new window]   Fig. 3. Neurite outgrowth from explants plated on neutral or acidic laminin matrices. Brain explants were incubated for 24 hours on laminin matrices obtained at neutral (A,B) or acidic pH (C,D). Explants were developed for the neuronal marker ß tubulin III (A,C) and for total cells, using DAPI (B,D). Arrows point to typical aggregates of cell bodies occurring on the neutral matrix. The inset in A shows short neurites emerging from a rare region of the explant where aggregation of cell bodies did not occur. Bar, 100 µm.

View larger version (103K): [in a new window]   Fig. 6. Double staining for laminin and neuronal cells. Cortical cells were plated over laminin polymerized in neutral buffer. After 24 hours immunocytochemical analyses for laminin (rabbit polyclonal) and for the neuronal marker, ß tubulin (mouse monoclonal), were performed. A shows the laminin matrix and B neuronal cell bodies accumulating on regions corresponding to large laminin aggregates. The scale bar corresponds to 50 µm.

View larger version (57K): [in a new window]   Fig. 7. BrdU incorporation by cells plated on neutral or acidic laminin matrices. Neurons were plated on neutral (A, C and E) or acidic laminin matrices (B, D and F). After 2 hours BrdU was added and kept in culture for the following 22 hours, after which cells were fixed, permeabilized and analyzed for the incorporation of BrdU. A and B show cells that incorporated BrdU, C and D show the same fields analyzed for the nuclear marker, DAPI and E and F show the BrdU and DAPI images overlaid using the software Photoshop. Bar, 100 µm. G represents the quantitative analysis of data in panels A to F. One asterisk indicates P<0.05.

View larger version (113K): [in a new window]   Fig. 1. Self-assembly of laminin in neutral or acidic buffer. Frozen aliquots of laminin were dissolved in either Tris-HCl, pH 7 or in sodium acetate, pH 4, both containing 2 mM CaCl2. Dilutions were made either in a pre-silanized cuvette (graphic) or in drops of buffer previously placed onto non-silanized coverslips (pictures). The graphic shows fluorescence spectra obtained for laminin in neutral (continuous line) and acidic buffer (dotted line) using excitation at 275 nm. The inset shows light-scattering intensity, using incidental light at 400 nm, for neutral (lower trace) and acidic condition (upper trace). Panels A to D show immunocytochemical analyses of laminin matrices formed on glass coverslips at pHs 7 (A,B) or 4 (C,D). Note that the apparent difference in the amount of bound protein in panels A and C is due to the impossibility of homogeneously focusing laminin aggregates throughout the neutral matrix. Insets show details of the corresponding main panels. Panels E to G show immunohistochemistry for laminin on whole-mount preparations of the retina of newborn rats (PO). A honeycomb pattern is typically seen on the periphery of the whole mount (E and F), whereas unorganized deposits characterize the center of the retina (G). The scale bars in panel A, main, and B correspond to 50 µm and apply to panels A, C and E or to panels B and D, respectively. The bar in the inset of panel A represents 10 µm and applies to all insets. In panel F, the bar corresponds to 100 µm in panels F and G.

View larger version (18K): [in a new window]   Fig. 5. Kinetics of neurite outgrowth on neutral and acidic laminin matrices. Cells were plated onto neutral (open symbols) or acidic laminin matrices (closed symbols) and fixed after 2, 6, 16 or 24 hours. After immunostaining for Tau, the average sizes of the 30 longest neurites were averaged and plotted against the time of incubation. Inset shows the absence of cell clumps within 2 hours of plating on the neutral matrix.