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. 2009 Jun 15;23(12):1393–1398. doi: 10.1101/gad.519409

Figure 4.

Figure 4.

Xcad-11 function is mediated by small GTPases and GEF-Trio. (A) Whole-mount ISH. Injection of ca Rac1 and two GEF subunits of hTrio together with Xcad-11MO rescued CNC migration, demonstrated by twist ISH. (B) Statistics of reconstitution experiments with 10 pg of ca cdc42, Rac1, or RhoA. Coinjection with Xcad-11MO rescued CNC migration into pharyngeal pouches in 76%, 62%, or 66%, respectively. Twenty picograms of Trio or single GEF domains restored disturbed invasion in pharyngeal pouches in 52% (Trio), 55% (Gef1), 65% (Gef2), or 88% (Gef 1 + 2). (C) In Xcad-11-depleted CNC explants, coinjection of ca cdc42, Rac1, or RhoA, as well as with Trio or the two GEF subunits, restored formation of lamellipodia and filopodia. Bar, 10 μm. (D) Statistics of gain-of-function analysis with soluble Xcad-11 constructs; dn GTPases cdc42, Rac1, and RhoA; as well as a combination of all three GTPases together. Overexpression of the cytoplasmic domain of Xcad-11 with or without the β-cat-binding site resulted in a strong reduction of CNC migration (30% and 28% migrating CNC). The three dn GTPases cdc42, Rac1, and RhoA showed migration in 65%, 55%, and 44% of the embryos, respectively. A combination of all three dn GTPases together resulted in 46% migrating CNC. (E) Coimmunoprecipitation of Trio with different Xcad-11 constructs. (Bottom left panel) Precipitation of Trio with HA antibody from transfected COS cells. Trio was detected in Western blot. Western blot for Xcad-11 showed successful coprecipitation of Xcad-11 (top left) as well as of Xcad-11ΔeΔβ-cat (top right). In the case of Xcad-11ΔeΔIMD, no coprecipitation was detected. (Right half of panels) Input.