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. 1999 Mar 15;19(6):1965–1975. doi: 10.1523/JNEUROSCI.19-06-01965.1999

Fig. 5.

Fig. 5.

Dose-dependent CNS myelin-induced collapse of motor neuron growth cones is abolished by expressingV12rac1 or V14rhoA. A, Motor neurons were grown on FN for 2 d and then treated with CNS myelin (open bars) or a protein extract obtained from CNS myelin (hatched bars). In the case of CNS myelin, concentration of 20 μg/ml or higher achieved significant numbers of collapsed growth cones (*p < 0.01), whereas as little as 10 μg/ml CNS myelin protein extract induced significant growth cone collapse (**p < 0.01). Importantly, a basal level of collapsed growth cones existed in these motor neuron cultures. B, Motor neurons were infected at the time of plating with recombinant adenovirus carrying mutants of small GTPases and grown for 3 d. Cultures were treated with 100 μg/ml CNS myelin (stippled and filled bars) or with PBS (open bars). The percentage of collapsed growth cones was determined as a function of small GTPase mutants that were expressed. Only expression of constitutively active rac1 (V12rac1, filled bar) and rhoA (V14rhoA, filled bar) inhibited CNS myelin-induced growth cone collapse (*p < 0.01). Expression of other small GTPase mutants or lacZ was ineffective (stippled bars). It is noteworthy that a fraction of motor neuron growth cones exhibited a collapsed morphology regardless of proteins expressed (open bars). C, Motor neurons were plated on FN-coated dishes containing stripes of CNS myelin (100 μg/ml), infected with recombinant adenovirus carrying mutants of small GTPases, and grown for 3–4 d. The length of neurites grown entirely on CNS myelin stripes or grown into CNS myelin stripes was measured. Neurite length per 500 μm2 CNS myelin is plotted as a function of small GTPase mutants expressed. Expression of either V12rac1 or V14rhoA (open bars) resulted in considerable neurite outgrowth on CNS myelin (*p < 0.01) compared with lacZ or the other small GTPase mutants (filled bars). Con, PBS; lacZ, reporter gene coding β-galactosidase; V12rac1, constitutively active rac1; N17rac1, dominant negative rac1; V12cdc42, constitutively active cdc42; N17cdc42, dominant negative cdc42;V14rhoA, constitutively active rhoA.