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Figure 5


Fig. 5. Rigidity response in the growth cones is SFK-dependent, and tyrosine phosphorylation of p130Cas requires RPTP{alpha} activity and rigid matrix. (A-G) RPTP{alpha}+/+ neurons were plated on FN-coated substrates of varying rigidities and treated or not with the SFK inhibitor (10 µM SU6656) after cells had adhered to the substrate. (A,B) After a 48-hour incubation, there was no difference in treated RPTP{alpha}+/+ neurons on rigid versus soft surfaces. However, axon elongation and differentiation were inhibited in neurons treated with SFK compared with untreated controls. (C) Growth cones of treated neurons displayed decreased reinforcement of FN-coated beads in the laser tweezers experiments. (D,E) Immunostaining of Fyn revealed lower levels of edge accumulation on soft than on rigid matrices in RPTP{alpha}+/+ neurons. In growth cones of RPTP{alpha}–/– neurons there was a decreased edge accumulation regardless of rigidity. (F,G) Immunostaining of phosphorylated p130Cas (anti-phospho-Y615-Cas) showed high levels of phosphorylated p130Cas in the presence of RPTP{alpha} and rigid matrices. In RPTP{alpha}+/+ neurons on soft matrix, and in RPTP{alpha}–/– neurons – regardless of the matrix rigidity – the observed levels of phosphorylated p130Cas were significantly lower. (H) Fluorescence intensities of the Fyn and phosphorylated p130Cas signals were quantified, normalized against nuclear fluorescence intensity and are presented as the mean ± s.e. for at least 20 representative neurons.





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