Figure 2

In vivo conjugation of QDs to the C-terminus of Paxillin-EGFP via intein mediated protein splicing. Co-localization of QDot₅₈₅ with Paxillin-EGFP on focal-adhesions of mesodermal cells during migration. Stage 2 Xenopus embryos were injected with (A) probe (DnaE I_C-QDot₅₈₅)(in red) and RNA encoding Paxillin-EGFP-DnaE I_N (in green) or (B) probe (DnaE I_C-QDot₅₈₅) alone. Fluorescence images of animal cap cells dissociated from stage 8 Xenopus embryos, induced with activin, and plated onto fibronectin coated slides. (A) Yellow shows overlap between red QDot₅₈₅ and green EGFP indicating successful QD-protein conjugation in vivo. (B) In the absence of Paxillin-EGFP, QDs do not target focal adhesions but remain diffusely localized in the cytosol. (C &D) Biochemical characterization of protein-QD conjugates. Xenopus embryos were injected as follows, C: i) Uninjected ii) DnaE I_C-QDot₅₈₅ ii) Paxillin-EGFP-DnaE IN RNA iii) DnaE IC-QDot₅₈₅ + Paxillin-EGFP-DnaE IN RNA, D: i) Uninjected ii) QDot₅₈₅-DnaB I_N iii) DnaB I_C-memEGFP RNA iv) QDot₅₈₅-DnaB I_N + DnaB I_C-memEGFP RNA, lysed at stage 10 and loaded onto a 0.5% agarose gel in this order, from left to right. QDot₅₈₅ were visualized with the ethidium bromide emission filter under UV excitation and EGFP was imaged with a band pass 500/50 filter set on UVP iBox Imaging System. The ligation products appear as a single band under the GFP and QD filters, only in lysates of Xenopus embryos injected with RNA + QD probe (vertical white arrows). Bands corresponding to Paxillin-EGFP and memEGFP proteins not conjugated to QDs are detectable under the GFP filter, in lysates of Xenopus embryos injected with RNA only and QD probes + RNA, but not QDs only (horizontal arrows). Bands corresponding to QD probes are detectable under the QD filter, in lysates of Xenopus embryos injected with the QD probes only or the QD probes + RNA, but not RNA only, (horizontal arrows).