PTPmu regulates N-cadherin-dependent neurite outgrowth

Abstract

Cell adhesion is critical to the establishment of proper connections in the nervous system. Some receptor-type protein tyrosine phosphatases (RPTPs) have adhesion molecule-like extracellular segments with intracellular tyrosine phosphatase domains that may transduce signals in response to adhesion. PTPmu is a RPTP that mediates cell aggregation and is expressed at high levels in the nervous system. In this study, we demonstrate that PTPmu promotes neurite outgrowth of retinal ganglion cells when used as a culture substrate. In addition, PTPmu was found in a complex with N-cadherin in retinal cells. To determine the physiological significance of the association between PTPmu and N-cadherin, the expression level and enzymatic activity of PTPmu were perturbed in retinal explant cultures. Downregulation of PTPmu expression through antisense techniques resulted in a significant decrease in neurite outgrowth on an N-cadherin substrate, whereas there was no effect on laminin or L1-dependent neurite outgrowth. The overexpression of a catalytically inactive form of PTPmu significantly decreased neurite outgrowth on N-cadherin. These data indicate that PTPmu specifically regulates signals required for neurites to extend on an N-cadherin substrate, implicating reversible tyrosine phosphorylation in the control of N-cadherin function. Together, these results suggest that PTPmu plays a dual role in the regulation of neurite outgrowth.

Figure 1

Expression of PTPμ in chick retina. E8 chick retina sections were immunohistochemically labeled with antibodies against (B) PTPμ, (C) L1, or (D) a bipolar neuron-specific antigen. (B) PTPμ labeling was present predominantly in regions that labeled positively for retinal ganglion cells (C) and bipolar neurons (D). Phase contrast image is shown in A. OFL, optic fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; PC, precursor cells; PE, pigmented epithelium. Scale bar, 50 μm.

Figure 2

Purification of PTPμ from brain. PTPμ was purified from adult rat brain by immunoaffinity methods. (A) The eluted PTPμ protein was separated by 5–15% gradient SDS-PAGE and silver stained. Both the full-length protein (200 kD) and the proteolytically processed extracellular (105 kD) and intracellular (100 kD) fragments were detected (arrows). (B) The purified PTPμ protein was examined by immunoblotting to confirm that the preparation was not contaminated by other cell–cell adhesion molecules that promote neurite outgrowth. N-cadherin, NCAM, L1, and two different preparations of PTPμ were purified and equal amounts of the proteins were separated by 6% SDS-PAGE. Immunoblots using antibodies to PTPμ (top left), NCAM (top right), L1 (bottom left), or N-cadherin (bottom right) are shown.

Figure 3

PTPμ promotes neurite outgrowth from chick retinal explants. Neural retina explants from E8 chick embryos were cultured for 2 d on N-cadherin–coated dishes (A–C) or 4 d on dishes coated with PTPμ (D–F). The fields shown in B and E are higher power images of the same fields shown in A and D, respectively. Long neurites grew out onto each substrate, but were more fasciculated on the PTPμ substrate (D and E) as compared with the N-cadherin substrate (A and B). Growth cones on N-cadherin were largely lamellipodial with several short filopodial processes (C), whereas growth cones on PTPμ were typically small and spiky in appearance with multiple long filopodial processes and a small lamellipodial region (F). Scale bars: (A) 100 μm, (B) 50 μm, (C) 10 μm.

Figure 6

PTPμ interacts with N-cadherin in chick retina. Lysates were made from embryonic day 4, 8, and 11 neural retinas. 5 μg total protein per lane was separated by 6% SDS-PAGE, transferred to nitrocellulose, and probed with antibodies against (A) PTPμ or (C) N-cadherin. (B and D) Immunoprecipitates from lysates of E8 retina were immunoblotted. Mouse IgG (lane 1), PTPμ (lanes 2 and 3) and N-cadherin (lane 4) antibodies were used for immunoprecipitation. The immunoprecipitates were subjected to 6% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to (B) PTPμ or (D) N-cadherin.

Figure 4

Neurite outgrowth on PTPμ is blocked by addition of antibodies against PTPμ. Neural retina explants from E8 chick embryos were cultured on a PTPμ (A–D), N-cadherin (E–G), or L1 (I–K) substrate in the presence of function-blocking antibodies against PTPμ (B, F, and J), N-cadherin (C and G), or L1 (D and K). The explants are on the left of each panel. The neurites in each dish were examined using dark-field optics. Antibodies against PTPμ inhibited neurite outgrowth on the PTPμ substrate (B), whereas they had no effect on neurite outgrowth on N-cadherin (F) or L1 substrates (J). Conversely, antibodies against N-cadherin inhibited neurite outgrowth on a N-cadherin substrate (G), but had no effect on neurite outgrowth on PTPμ (C). Similarly, antibodies against L1 inhibited neurite outgrowth on an L1 substrate (K), but had no effect on a PTPμ substrate (D). A dish coated with bovine serum albumin alone did not support neurite outgrowth (H), suggesting that, over time, the retinal explants did not secrete proteins that promote neurite outgrowth in this assay. Scale bar, 200 μm.

Figure 5

PTPμ promotes migration of several cell types from chick neural retina. Neural retina explants from E8 chick embryos were cultured on a PTPμ substrate for 4 d. The cells were fixed and immunolabeled with antibodies against a bipolar-specific antigen (A and B), a Müller glia-specific antigen (C and D), or L1 (E and F). Phase contrast (A, C, and E) and fluorescent images (B, D, and F) are shown. The long neurites that extended on the PTPμ substrate were from RGC neurons as shown by the labeling with an antibody against L1 (E and F), which is restricted to RGC axons in retina. Bipolar neurons (A and B) and Müller glia (C and D) were also observed to migrate onto PTPμ. Scale bar, 50 μm.

Figure 7

PTPμ colocalizes with N-cadherin in retinal neurites. Neural retina explants from E8 chick embryos were cultured overnight on laminin. The cells were fixed and double labeled with a polyclonal antibody against PTPμ (A and E) and a monoclonal antibody against either N-cadherin (B) or NCAM (F). These results show that PTPμ, N-cadherin, and NCAM are expressed in a continuous fashion along neurites and into growth cones. Live cells were incubated with a polyclonal antibody against PTPμ to cluster the PTPμ protein on the surface of the neurites into “patches” (C and G), and then the cells were fixed and double labeled with a monoclonal antibody against N-cadherin (D) or NCAM (H). N-cadherin (D), but not NCAM (H), colocalized to the PTPμ patch sites, suggesting an interaction with PTPμ within the neurites. As a control for nonspecific fluorescence from the secondary antibodies, fixed retina cultures were processed for immunocytochemistry as above except that the primary antibodies were omitted (I and J). The image shown in I is a control for images shown in A, C, E, and G, whereas the image shown in J is a control for images shown in B, D, F and H. The control images (I and J) were collected using identical settings on the confocal microscope as those used for the corresponding experimental images (A–H). Scale bar, 20 μm.

Figure 8

PTPμ expression is reduced in retinal cells infected with antisense PTPμ retrovirus. Dissociated chick retinal cells infected with control virus (lanes 1 and 2) or PTPμ antisense virus (lanes 3 and 4) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 3 μg/ml tetracycline were harvested 72 h after infection. 7.5 μg of each lysate was separated by 6% SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to (A) PTPμ, (B) N-cadherin, or (C) NCAM. (A) A substantial reduction in PTPμ expression was detected in lysates from PTPμ antisense– infected cells in the absence of tetracycline (lane 3) in comparison with lysates from control virus–infected cells (lane 1). Densitometric measurement of the PTPμ bands showed a 99% reduction of the 200-kD band and a 77% reduction of the 100-kD band, whereas a 95-kD band was unchanged. No difference in PTPμ expression was observed when cells infected with PTPμ antisense were cultured in the presence of tetracycline (lane 4). (B) In contrast, no difference in N-cadherin expression was detected in lysates from control virus (lanes 1 and 2) or PTPμ antisense virus (lanes 3 and 4) infected cells. (C) The blot probed for PTPμ expression was stripped and reprobed for a nonspecific protein, NCAM, to verify equal protein loading per lane.

Figure 9

PTPμ is required for N-cadherin–dependent neurite outgrowth. E4 chick retina explants were infected with control (A, C, and E) or PTPμ antisense (B, D, and F) virus and cultured on laminin (A and B), N-cadherin (C and D), or L1 (E and F) substrates for 48 h. The neurites in each dish were examined using dark-field optics. No difference in neurite length or density was observed in cultures infected with vector or PTPμ antisense virus when cultured on laminin (A and B) or L1 (E and F) substrates. In contrast, PTPμ antisense resulted in a dramatic reduction of both neurite length and density in cultures on a N-cadherin substrate (C and D). Scale bar, 400 μm for images shown in A and B. Scale bar, 200 μm for images shown in C–F.

Figure 10

Quantitation of the effect of PTPμ perturbation on neurite outgrowth. E4 chick retina explants were infected with control virus (gray bars) or PTPμ antisense virus (black bars) and cultured on laminin, N-cadherin, or L1 substrates for 48 h. PTPμ antisense virus caused a significant reduction in neurite length (A) and density (B) on N-cadherin, but not on laminin or L1 substrates. The retrovirus used is negatively regulated by tetracycline (tet-off system). Retinal explants plated on N-cadherin in the presence of both tetracycline and PTPμ antisense virus showed no reduction in either neurite length (A) or density (B) when compared with explants infected with control virus. For statistical analysis, see Table I. E4 chick retinas were infected with control virus (gray bars) or test virus encoding the c → s (C to S) mutant of PTPμ (cross-hatched bars) or PTPμGFP sense control (striped bars), and cultured on N-cadherin for 48 h. The c → s mutant caused a significant reduction in neurite length (C) and density (D), whereas the sense control had no effect on either length or density. For statistical analysis, see Table I.