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. 2011 Mar 23;31(12):4421–4433. doi: 10.1523/JNEUROSCI.5230-10.2011

Figure 7.

Figure 7.

Ckn exhibits physical and genetic interactions with Dock. A, Full-length Ckn interacts with Dock in yeast two-hybrid. Ckn-Full, Ckn-SAM, and Ckn-CT constructs were fused to Gal4-DNA activation domain (prey). Full-length Dock was fused to the Gal4-DNA binding domain (bait). Only full-length Ckn interacts with Dock. B, Mutant cDNAs for four ckn alleles were fused to the GAL4-DNA activation domain and tested against a Dock-Gal4-DNA binding domain bait plasmid. Neither Ckn-C nor Ckn-K bind Dock, whereas both Ckn-A and Ckn-Y maintain the interaction. C, A series of LexA-Dock fusions were tested for interaction with full-length Ckn-activation domain fusions. + +, Medium blue; −, white (no interaction) after 1 h. D, Schematic outlining the yeast three-hybrid interactions between Caskin, Lar, and Dock. Yeast were cotransformed with a Lar-cyto/Gal4-DNA binding domain fusion (bait) and a full-length Dock/Gal4-activation domain fusion (prey). Dock and Lar do not interact. After 2 d, the yeast were mated to yeast transformed with Ckn-WT, Ckn-A, Ckn-C, and Ckn-K. A nuclear localization signal (NLS) was added to the N terminus of Ckn. Only wild-type Ckn couples Lar and Dock. Ckn-Y could not be tested in this assay as it activates transcription in the absence of the Gal4-activation domain. E, Flag-tagged Dock coimmunoprecipitates Caskin-HA from S2 cells. F, A Dock-GST fusion protein precipitates Ckn-HA from elavGal4/UAS-Caskin-HA embryos, whereas GST alone does not. G–K, Two hemisegments of stage 16 embryos labeled with mAb 1D4 to mark motor axon projections. Anterior is to the left. Scale bar, 15 μm. G, ISNb nerve exhibits characteristic branched morphology in wild type. The black arrow indicates ISNd. H, Axons in dock3 cknK/dock3 + embryos are still en route to their targets at late stage 16. I, ISNb axons in dock3 cknK double homozygotes are loosely fasciculated and extend multiple projections. J–L, ISNb/d axons do not separate in ckn dock double mutants. Whereas in wild type, ISNb and ISNd axons are tightly bundled and cleanly separated (J), ISNb/d axons remain tangled in the double mutant (K). Schematic of the ISNb/d branch segregation defect is shown (L). M, N, The CNS midline of wild-type (M) and dock ckn (N) embryos labeled with ID4. Axons in the lateral two connectives are poorly fasciculated and stall in the double mutant.