doi: 10.1242/10.1242/jcs.00173
Structure of laminin substrate modulates cellular signaling for neuritogenesis
Elisabete Freire1,3,
Flávia C. A. Gomes2,
Rafael Linden4,
Vivaldo Moura Neto2 and
Tatiana Coelho-Sampaio3,*
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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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© The Company of Biologists Ltd 2002
