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First published online 16 October 2007
doi: 10.1242/jcs.009852

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RPTP is required for rigidity-dependent inhibition of extension and differentiation of hippocampal neurons

¹ Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, New York, NY 10027, USA
² Institute for Molecular Pathology, Teilumbygningen Frederik V vej 11, 6. sal, University of Copenhagen, 2100-Copenhagen, Denmark

View larger version (34K): [in this window] [in a new window]   Fig. 1. RPTP is required for the reinforcement of FN-specific integrin-cytoskeleton bonds in neuronal growth cones. A laser tweezer (represented by red circle) was used to place FN-VN-coated beads at the edge of the growth cones. (A,B) In non-reinforced beads (A), the linkage between the bead and the cytoskeleton was broken by the trap force and the beads were pulled back into the trap after initial rearward movement (breaking event). If reinforcement occurred, the linkage was not broken and beads moved out of the optical trap towards the axon hillock (B). Bars, 4 µm. (C,D) FN-bead reinforcement was decreased by half but bead binding was unaffected in RPTP–/– neurons relative to controls. By contrast, VN-bead reinforcement and binding were unaltered on RPTP–/– neurons (C,D). Mean square displacement (MSD) was calculated for the initial 10 seconds of rearward movement for each condition. The results (mean ± s.e.) were statistically significant (P<0.01). (E,F) On average, MSD was higher for FN-coated beads bound to RPTP–/– growth cones compared with RPTP+/+ growth cones (E); no significant difference was observed for VN-coated beads (F).

View larger version (22K): [in this window] [in a new window]   Fig. 2. v6 integrins are required for the reinforcement of FN-cytoskeleton bonds at the leading edge of the growth cones. (A,B) Function-blocking antibodies against 51 and v3 integrins had no effect on binding and the reinforcement of FN-coated beads, but blocking of the v and v6 integrins reduced binding and reinforcement to the background levels (A,B). Localization of RPTP and v6 integrin in growth cones was ECM-specific. Both RPTP and v6 were localized to the leading edge of growth cones in neurons plated on FN-coated glass (C,D). On LN, RPTP was localized to the growth cones, whereas v6 integrin was expressed at low levels along the axons and in the growth cones (E,F). Insets provide image of the entire neuron. Bars 5 µm. (G) Fluorescence intensities of the RPTP and v6 signals were quantified, the data were normalized against nuclear fluorescence intensity and are presented as the mean ± s.e. for at least 20 representative neurons.

View larger version (40K): [in this window] [in a new window]   Fig. 3. Neurite extension on FN depends on RPTP and matrix rigidity. (A,B) Primary neurons isolated from the hippocampi of RPTP+/+ and RPTP–/– mice were plated on FN-coated polyacrylamide gels of decreasing rigidities, incubated for 48 hours, fixed and visualized using phalloidin. Anti-Tau immunofluorescence was used as axonal marker. Typical (A) stage 2 and (B) stage 3 neurons are shown. Bars, 10 µm. (C,D) Control neurons showed faster differentiation on soft than on rigid substrates; RPTP–/– neurons, however, showed a high level of differentiation irrespective of matrix rigidity (C). Similarly, control neurons extended longer neurites on soft than on rigid surfaces, whereas RPTP–/– neurons extended long neurites on both soft and rigid surfaces (D). Results shown in C and D (mean ± s.e.) were statistically significant (P<0.01). (E,F) Phalloidin staining of RPTP–/– and RPTP+/+ neurons. Neurites of RPTP–/– neurons appeared more meandering than the neurites of control neurons. Bars, 15 µm.

View larger version (17K): [in this window] [in a new window]   Fig. 4. LN rigidity response is RPTP independent in neurons. (A) Axon differentiation is inhibited by increasing rigidities of LN-coated substrates in both control and knockout neurons. (B) Neurite extesion is stimulated by soft LN-coated substrates, and loss of RPTP had no effect on this behavior. The results (mean ± s.e.) were statistically significant (P<0.01).

View larger version (33K): [in this window] [in a new window]   Fig. 5. Rigidity response in the growth cones is SFK-dependent, and tyrosine phosphorylation of p130Cas requires RPTP activity and rigid matrix. (A-G) RPTP+/+ 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+/+ 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+/+ neurons. In growth cones of RPTP–/– 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 and rigid matrices. In RPTP+/+ neurons on soft matrix, and in RPTP–/– 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.

View larger version (39K): [in this window] [in a new window]   Fig. 6. Proposed molecular mechanism of FN-specific reinforcement and rigidity response in hippocampal neurons. In RPTP+/+ neurons, matrix rigidity triggers force-dependent activation of the RPTP, followed by activation of Fyn, which consequently phosphorylates stretch-sensitive p130Cas. This results in the recruitment of the focal-contact proteins causing the reinforcement of the growth-cone–substrate links. This reinforcement has a negative effect on the neurite extension. On the soft matrices, the force exerted by the actin-myosin network in response to matrix rigidity, does not reach a threshold critical for the reinforcement of the FN-cytoskeleton bonds and subsequent focal-contact formation. Thus, the neurite extension is stimulated on soft matrices.

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