Subcellular localization of GFP-hCASK and GFP-hCASK mutants in HEK-393 cells.

<p>Images were obtained with a 63X Plan-apochromat 1.4 N.A oil lens. White arrows indicate representative intracellular aggregates. Insert shows higher magnification.</p

Structural modeling of four CASK mutations.

<p>Dotted lines indicate contacts. <i>A.</i> CAMK domain of CASK (3c0i.pdb) showing R28 and Y268. <i>B.</i> Native (Arg, cyan) and mutant (Leu, magenta) side-chains at position 28. <i>C.</i> Native (Tyr, cyan) and mutant (His, magenta) side-chains at position 268. <i>D.</i> SH3-GuK domain homology model showing Y728 and W919. SH3 region, yellow. GuK region, pink. <i>E.</i> Native (Tyr, cyan) and mutant (Cys, magenta) side-chains at position 728. <i>F.</i> Native (Trp, cyan) and mutant (Arg, magenta) side-chains at position 919.</p

Glycerol treatment eliminates intracellular aggregates.

<p>Six hours after transfection, media was exchanged for either fresh media alone or containing 10% glycerol. <i>A.</i> Images, 40X. Insert shows higher magnification. <i>B.</i> Using five representative 20X images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088276#pone.0088276.s007" target="_blank">Figure S7</a>) for each condition, individual cells were classified as free of or containing aggregates in Image J. Bars and error bars represent the average and standard deviation of three independent analyses. * and # indicate statistically significant differences from the wild-type images.</p

Domain location and conservation of five CASK mutations.

<p><i>A.</i> Five CASK XLMR mutations are shown in reference to CASK’s domain structure. <i>B.</i> A comparison of the five mutation sites in CASK orthologs from nine species. Conserved residues, white. Residues identical to the mutation, black. Residues that differ from the wild-type and mutant hCASK sequence are gradiently shaded to indicate their similarity to the native hCASK residue.</p

Characterization of aggregates.

<p>Images of HEK cells transfected with <i>A)</i> GFP-CASK-Y728C or <i>B)</i> GFP-CASK-W919R were obtained with a 63X Plan-apochromat 1.4 N.A oil lens. First column shows aggregated GFP-CASK protein. Panels labeled “mCherry” show cells that were co-transfected with GFP-CASK and mCherry, which remains cytosolic. Panels labeled “Thioflavin T” represent coverslips that were fixed and then stained with Thioflavin T, which shows enhanced fluorescence in the presence of amyloid fibrils. Panels labeled “Golgi-RFP” represent coverslips that were treated with CellLight® Golgi-RFP which labels the Golgi network. Third column shows an overlay, demonstrating that aggregates are cytosolic (mCherry, Golgi-RFP) but not amyloid in nature (Thioflavin T).</p

Predicted impact of mutations (structure-based).

<p>For mutations in CaMK domain (R28L and Y268H), the structure 3c0i.pdb was used. For mutations in the SH3-GuK domain (Y728C and W919R), the homology model based on 1 kgd.pdb and 1 kjw.pdb was used. Positive ΔΔG values suggest that the indicated mutation destabilizes CASK’s overall fold.</p

Functional CASK XLMR mutations (R28L, Y268H and P396S) do not disrupt interactions with liprin-α, Mint-1, or Veli.

<p>Lysates from HEK-293 cells co-transfected with GFP-CASK (wild-type or mutants R28L, Y268H, or P396S) and either liprin-α3 or FLAG-tagged Mint-1 were incubated with anti-GFP beads to pull down GFP-CASK and binding partners. To assess Veli interaction, no co-transfection was performed; native Veli was pulled down after incubation of lysates from GFP-CASK-transfected HEK-293 cells with anti-GFP beads to pull down GFP-CASK. Western blots of samples containing whole cell lysate (Input) or immunoprecipitates (Co-IP) were probed with anti-liprin-α3, anti-Veli, or anti-FLAG primary antibodies.</p