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First published online 4 July 2006
doi: 10.1242/jcs.03045

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-Actinin-dependent cytoskeletal anchorage is important for ICAM-5-mediated neuritic outgrowth

Henrietta Nyman-Huttunen^*, Li Tian^*, Lin Ning and Carl G. Gahmberg

Division of Biochemistry, Faculty of Biosciences, PO Box 56 (Viikinkaari 5), 00014 University of Helsinki, Finland

View larger version (32K): [in a new window]   Fig. 1. (A) A schematic picture of the ICAM-5 molecule with the amino acid sequence of the cytoplasmic domain and the peptides used in the ICAM-5--actinin interaction studies. The short peptides contain an additional C-terminal cysteine for coupling purposes. The peptides used in SPR analysis were biotinylated. PM, plasma membrane. (B) A GST-ICAM-5 cytodomain fusion protein binds -actinin. (a) A GST-cytoICAM-5 fusion protein immobilized on glutathione Sepharose beads was incubated with a Paju cell lysate, and interacting proteins characterized by western blotting, in this case an anti--actinin antibody. (b) Binding of purified -actinin to GST-cytoICAM-5, visualized by blotting. (C) Binding of purified -actinin to membrane proximal ICAM-5 cytoplasmic peptides. (a) Purified -actinin was incubated with the ICAM-5 cytodomain peptides (described in detail in Materials and Methods and in Fig. 1A) affinity matrices and the eluates were analyzed by SDS-PAGE together with purified -actinin, and immunoblotted with anti--actinin antibody. (b) Based on results shown above, the main -actinin-binding domain in the ICAM-5 cytodomain is located at residues 857-861 (box). (D) Binding of talin and filamin to ICAM-5857-861 peptide. Paju cell lysates were incubated with the ICAM-5 peptide and the scrambled control peptide, and the eluates were run on SDS-PAGE and immunoblotted for talin (a) and filamin (b). No binding was observed. (E) Binding of -actinin to GGGKKGEY peptide as determined by SPR. Binding levels of -actinin at steady state of interaction with GGGKKGEY as response units (RU) after subtraction of control peptide sensorgrams from each GGGKKGEY binding sensorgram, respectively.

View larger version (46K): [in a new window]   Fig. 2. Co-immunoprecipitation of ICAM-5 with -actinin. (A) -actinin was immunoprecipitated from Paju-ICAM-5 cell lysates with a specific anti--actinin antibody and the precipitates were subjected to SDS-PAGE and immunoblotted with anti-ICAM-5 antibody to detect possible co-immunoprecipitation of ICAM-5. The successful precipitation of -actinin was ensured by immunoblotting with anti--actinin antibody (not shown). (B) ICAM-5 was immunoprecipitated from rat brain homogenates and the precipitates were run on SDS-PAGE and immunoblotted for ICAM-5 and after stripping for -actinin.

View larger version (13K): [in a new window]   Fig. 3. Schematic presentation of full-length ICAM-5, C-terminal truncated constructs (ICAM-5-GPI, ICAM-5-TM), and point mutant (ICAM-5-KK/AA) used in the cell transfections. The asterisks indicate the sites of point mutations. EC, extracellular; TM; transmembrane; CP, cytoplasmic part.

View larger version (90K): [in a new window]   Fig. 4. The distribution of ICAM-5, -actinin and F-actin in different transfected Paju cell lines. The Paju-neo (A-C,a-c), Paju-ICAM-5 (D-F,d-f), Paju-ICAM-5-TM (G-I,g-i), Paju-ICAM-5-GPI (J-L,j-l), and Paju-ICAM-5-KK/AA cells (M-O,m-o) were double-stained for ICAM-5 (green) and -actinin (red, left panel) or F-actin (red, right panel) as described in Materials and Methods, and analyzed by confocal microscopy. The regions where the two proteins colocalize appear in yellow. Bars, 10 µm.

View larger version (131K): [in a new window]   Fig. 5. Colocalization of ICAM-5 with -actinin in cultured rat hippocampal neurons. The 7-day-old (A-F) or 14-day-old neurons (G-L) were stained for ICAM-5 (green) and -actinin (red), and analyzed by confocal microscopy. Both ICAM-5 and -actinin showed a punctated expression pattern along the dendrites. The colocalization of the two molecules in apical dendritic filopodia (indicated by arrows, image F) at day 7, and in dendritic spines at day 14 (indicated by arrowheads, image L) are shown under high magnification. Bars, 10 µm.

View larger version (41K): [in a new window]   Fig. 6. Effect of ICAM-5 contructs on neuritic outgrowth. (A) The Paju-ICAM-5 (a-d, I-IV), Paju-ICAM-5-TM (e-h) and Paju-ICAM-5-KK/AA cells (i-l) were activated by 100 nM PdBu to induce neurite outgrowth. The cells were triple-stained for -actinin (green), actin (red), ICAM-5 (magenta), and visualized by confocal microscopy. Concentrations of ICAM-5, -actinin and F-actin at cell-anchorage sites (a-d) and growth cones of neurites (I-IV) in Paju-ICAM-5 cells were seen. Bars, 20 µm. (B) Histogram displaying neurite outgrowth in PdBu-activated Paju-ICAM-5, Paju-ICAM-5-TM and Paju-ICAM-5-KK/AA cells (neurite length presented as micrometers). The differences between the neuritic length in Paju-ICAM-5 cells compared with Paju-ICAM-5-TM and Paju-ICAM-5-KK/AA cells are statistically significant (P<0.001). Standard deviations are shown.

View larger version (119K): [in a new window]   Fig. 7. Effect of ICAM-5 cytoplasmic peptides on cell morphology. Paju-ICAM-5 (A,C,E) and Paju (B,D,F) cells were incubated with penetratin-coupled ICAM-5857-861 and ICAM-5-K857-K858/A-A peptides at 20 µM concentrations. After 2 hours of incubation the cells were washed and bright-field pictures were taken to visualize the effects of the peptides on cell morphology. Bars, 20 µm. The relative percentage of round-shaped Paju-ICAM-5 cells (treated with ICAM-5857-861 peptide) was 95.2%, and the difference is statistically significant (P<0.001) when compared with the cells treated with ICAM-5-K857-K858/A-A peptide or without treatment (data not shown).