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First published online August 9, 2006
doi: 10.1242/10.1242/jcs.03094

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Competition and cooperation between tenascin-R, lecticans and contactin 1 regulate neurite growth and morphology

¹ Max-Delbrück-Center for Molecular Medicine, R.-Rössle-Str.10, 13092 Berlin-Buch, Germany
² Department of Experimental Pathology, Institute for Clinical Sciences, Lunds University, University Hospital, Lund, Sweden

View larger version (85K): [in a new window]   Fig. 1. Time lapse analysis of TN-R-induced microprocesses and enlarged growth cones. Tectal cells were cultivated on immobilized contactin 1, observed under time-lapse control, and phase-contrast images were taken before (upper panel), 2 hours after (middle panel) and 4 hours after (lower panel) the addition of TN-R (10 µg/ml). Bar, 10 µm. For evaluation of motility at a 1-minute interval, compare left with right image. Within a 4-hour observation period, TN-R stimulates the formation of large and motile growth cones and of highly dynamic microprocesses at the neurite shaft (arrows).

View larger version (26K): [in a new window]   Fig. 2. Lecticans inhibit the formation of microprocesses and of complex growth cones by TN-R. (A,B) Tectal cells were cultured on a contactin 1 substrate and treated for 6 hours with increasing amounts of (A) aggrecan (5-20 µg/ml) and (B) chondroitinase-ABC-treated aggrecan (20 µg/ml), brevican or neurocan in the absence or presence of TN-R (10 µg/ml) as indicated. For quantitative analysis of neuronal morphology see Materials and Methods. The percentage of neurons belonging to each morphological category was calculated and typical examples are shown. Error bars represent s.e.m. The blocking effect of aggrecan on TN-R-induced microprocess formation and growth cone enlargement is dose-dependent, mediated by the core protein and similar to that of other lecticans.

View larger version (25K): [in a new window]   Fig. 3. Antibodies to TN-R FNIII domains 4-A neutralize the inhibitory effect of aggrecan on TN-R-induced morphological changes. Tectal cells were cultivated on immobilized contactin 1 and treated for 6 hours with TN-R (10 µg/ml), aggrecan (20 µg/ml) and Fab fragments of pAb against TN-R FNIII domains 4-A (500 µg/ml) as indicated. For quantitative analysis see Fig. 2. The addition of antibodies against TN-R FNIII domains 4-A has no effect on TN-R-induced microprocess formation and growth cone enlargement, but strongly reduces the blocking effect of aggrecan.

View larger version (86K): [in a new window]   Fig. 4. Lecticans inhibit the interaction between TN-R and contactin 1. (A-F) COS7 cells expressing contactin 1 on their surface were incubated with contactin-1-coated microspheres in (A) the absence or (B-F) the presence of TN-R (10 µg/ml), and with (C) aggrecan (20 µg/ml), (D) brevican, (E) neurocan or (F) chondroitinase-ABC-treated aggrecan (20 µg/ml). Double fluorescence images obtained with a confocal microscope are shown. The left panels reveal expression of contactin 1 by indirect immunofluorescence. The right panels depict binding of contactin-1-coated microspheres in the same microscopic field. Bar, 100 µm. TN-R-mediated molecular-bridge formation between contactin 1 polypeptides is blocked by aggrecan and other lecticans. (G) Schematic illustration of the sandwich binding assay performed in the presence of TN-R and aggrecan.

View larger version (55K): [in a new window]   Fig. 5. Aggrecan binding to TN-R FNIII domains 4-A blocks the interaction between TN-R and contactin 1. COS7 cells expressing contactin 1 on their surface were incubated with contactin 1-coated microspheres in the presence of TN-R (10 µg/ml) (A) together with aggrecan (20 µg/ml) (B-D), Fab fragments of pAb to TN-R FNIII domains 4-A (500 µg/ml) (C) and TN-R fragment FNIII domains 4-A (100 µg/ml) (D). Double fluorescence images of contactin 1 expression (left) and contactin 1-coated microsphere binding (right) are shown as described in Fig. 4. Bar, 100 µm. The blocking effect of aggrecan on TN-R bridge formation between contactin 1 polypeptides depends on the interaction of aggrecan with TN-R FNIII domains 4-A.

View larger version (103K): [in a new window]   Fig. 6. Morphology and motility of microprocesses and growth cones are not acutely affected by aggrecan. Tectal cells were cultivated on immobilized contactin 1 and pretreated for 20 hours with TN-R (10 µg/ml). (A) Phase-contrast images were taken before (upper panel), 15 minutes after (middle panel) and 1 hour after (lower panel) the addition of aggrecan (20 µg/ml) under time-lapse control. Bar, 10 µm. For evaluation of motility within a 1-minute intervall, compare left with right image. (B) For quantification, growth cones were scored according to their size (see Materials and Methods) and the percentage of cells with an enlarged growth cone was calculated in the absence or presence of aggrecan (20 µg/ml) for 15 minutes or 1 hour as indicated. The protein in solution and the incubation periods are given at the bottom. Error bars indicate s.e.m. In contrast to Figs 2 and 3, growth cone size was scored regardless of the morphology of the neurite. Addition of aggrecan does neither affect the motility of microprocesses and growth cones of tectal cells after pretreatment with TN-R (see arrows in A) nor significantly reduce the high percentage of large and complex growth cones.

View larger version (130K): [in a new window]   Fig. 7. Aggrecan induces retraction of tectal cell neurites after TN-R pretreatment. (A-C) Tectal cells were cultured on a contactin 1 substrate and pretreated for 20 hours with TN-R (10 µg/ml). Control medium (A), aggrecan (20 µg/ml) (B) or chondroitinase-ABC-treated aggrecan (20 µg/ml) (C) were added under time-lapse control, and phase-contrast images were taken at the indicated time points. Bar, 10 µm. (D) For quantification, the percentage of neurites that retracted within 4 hours was calculated (see Materials and Methods). The protein in solution and the incubation periods are given below. Error bars represent s.e.m. Aggrecan and chondroitinase-ABC-treated aggrecan induce strong neurite retraction in cells pretreated with TN-R compared with control cells.

View larger version (52K): [in a new window]   Fig. 8. TN-R cooperates with immobilized brevican and contactin 1 on the tectal cell surface to induce neurite outgrowth. (A-F) Petriperm dishes were coated with brevican overnight and blocked, and tectal cells were cultivated for 40 hours in the (A) absence or (B,D-F) presence of TN-R (10 µg/ml) together with (D) Fab fragments of pAb against contactin 1 (500 µg/ml), (E) Fab fragments of pAb against TN-R FNIII domains 4-A (500 µg/ml) or (F) aggrecan (20 µg/ml). Bar, 50 µm. Cell attachment and neurite outgrowth on a brevican substrate are induced in the presence of TN-R and depend on the simultaneous interaction of TN-R with immobilized brevican and contactin 1 on the tectal cell surface. (C) Schematic illustration of the proposed sandwich that mediates neurite outgrowth.

View larger version (43K): [in a new window]   Fig. 9. Expression of contactin 1, TN-R and lecticans in the developing chick optic tectum and in tectal cell cultures. (A) Cryostat sections of embryonic day E9 (upper panel), E11 (middle panel), and E13 tectum (lower panel) were stained with antibodies against chondroitin sulfate, neurocan, aggrecan, TN-R or contactin 1. Laminae in cryostat sections were revealed by DRAQ 5 as a nuclear counterstain. For lamina nomenclature see Yamagata et al. (Yamagata et al., 1995). SO, stratum opticum; SGFS, stratum griseum et fibrosum superficiale; SGC, stratum griseum centrale; SAC, stratum album centrale; SGP, stratum griseum periventriculare; SFP, stratum fibrosum periventriculare; NE, neuroepithelium. Bar, 100 µm. Inverted images illustrate the distinct but overlapping expression pattern of contactin 1, TN-R, and lecticans in the chick optic tectum. (B) Cryostat sections of E13 tectum were double-stained with antibodies against neurocan and contactin 1 or TN-R as indicated. Double fluorescence images show their colocalization in the SGFS lamina H at higher magnification. Bar, 100 µm. (C) Tectal cells in low-density cultures were double-labeled with antibodies to neurocan and contactin 1 or TN-R as indicated. Double fluorescence images reveal a cell-associated staining pattern of contactin 1 and TN-R, whereas neurocan is distributed pericellular. Bar, 10 µm.

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