Charalambous A et al. (2011), Split-inteins for simultaneous, site-specific c...

XB-ART-44828

J Nanobiotechnology 2011 Sep 15;9:37. doi: 10.1186/1477-3155-9-37.

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Split-inteins for simultaneous, site-specific conjugation of quantum dots to multiple protein targets in vivo.

Charalambous A , Antoniades I , Christodoulou N , Skourides PA .

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Proteins labelled with Quantum Dots (QDs) can be imaged over long periods of time with ultrahigh spatial and temporal resolution, yielding important information on the spatiotemporal dynamics of proteins within live cells or in vivo. However one of the major problems regarding the use of QDs for biological imaging is the difficulty of targeting QDs onto proteins. We have recently developed a DnaE split intein-based method to conjugate Quantum Dots (QDs) to the C-terminus of target proteins in vivo. In this study, we expand this approach to achieve site-specific conjugation of QDs to two or more proteins simultaneously with spectrally distinguishable QDs for multiparameter imaging of cellular functions. Using the DnaE split intein we target QDs to the C-terminus of paxillin and show that paxillin-QD conjugates become localized at focal adhesions allowing imaging of the formation and dissolution of these complexes. We go on to utilize a different split intein, namely Ssp DnaB mini-intein, to demonstrate N-terminal protein tagging with QDs. Combination of these two intein systems allowed us to simultaneously target two distinct proteins with spectrally distinguishable QDs, in vivo, without any cross talk between the two intein systems. Multiple target labeling is a unique feature of the intein based methodology which sets it apart from existing tagging methodologies in that, given the large number of characterized split inteins, the number of individual targets that can be simultaneously tagged is only limited by the number of QDs that can be spectrally distinguished within the cell. Therefore, the intein-mediated approach for simultaneous, in vivo, site-specific (N- and C-terminus) conjugation of Quantum Dots to multiple protein targets opens up new possibilities for bioimaging applications and offers an effective system to target QDs and other nanostructures to intracellular compartments as well as specific molecular complexes.

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Species referenced: Xenopus
Genes referenced: fn1 gnao1 pxn

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	Figure 1. In vivo conjugation of QDs to the C- or N-terminus of target proteins via intein mediated protein splicing. (A) Schematic representation of site-specific Ssp DnaE split intein-mediated conjugation of QDs to the C-terminus of the PH domain of Akt. (B) Schematic representation of site specific Ssp DnaB mini intein-mediated conjugation of QDs to the N-terminus of mem-EGFP.
	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 IC-QDot585)(in red) and RNA encoding Paxillin-EGFP-DnaE IN (in green) or (B) probe (DnaE IC-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 IC-QDot585 ii) Paxillin-EGFP-DnaE IN RNA iii) DnaE IC-QDot585 + Paxillin-EGFP-DnaE IN RNA, D: i) Uninjected ii) QDot585-DnaB IN iii) DnaB IC-memEGFP RNA iv) QDot585-DnaB IN + DnaB IC-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 iBoxImaging 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).
	Figure 3. Paxillin-QD conjugates associate with newly formed focal adhesion complexes and are released once the complexes are disassembled. Xenopus embryos were injected at the 2-cell stage with the probe (DnaE IC-QDot525 or 585) and RNA encoding Paxillin-EGFP-DnaE IN. Animal cap cells were dissociated from stage 8 Xenopus embryos, induced with activin, and plated onto fibronectin coated slides. (A) Time lapse images (time-interval: 30 sec) show paxillin-QD conjugates associating with newly formed focal adhesion complexes at the filopodia and lamellipodia of mesodermal cells during their migration on fibronectin substrates (see arrows). (B) Time lapse images (time-interval: 10 sec) show paxillin-QD conjugates being released from focal adhesion complexes as they disassemble during migration of mesodermal cells on fibronectin substrates (see arrows).
	Figure 4. Increased QD size imposes constraints on the translocation efficiency of Paxillin-EGFP-QD conjugates to the focal adhesion complexes. Co-localization of QDots525, QDots565 and QDots655 with Paxillin-EGFP on focal adhesion complexes. Note that unlike QDot525, the QDot655 are not recruited as effectively to the focal adhesion complexes.
	Figure 5. In vivo conjugation of QDs to the N-terminus of mem-EGFP via intein mediated protein splicing. (A) Co-localization of QDot605 with mem-EGFP on the cell membrane. Fluorescence images of stage 10 Xenopus embryos microinjected with the probe (QDot605-DnaB IN) shown in red, in one blastomere at the two-cell stage, and then injected with RNA encoding the target protein (DnaB IC-memEGFP) shown in green, in three of four blastomeres. Yellow shows the overlap between red QDot605 and green EGFP indicating successful QD-protein conjugation in a live embryo. (B) In embryos injected with the probe (QDot605-DnaB IN) alone, in the absence of RNA encoding the target protein (DnaB IC-memEGFP), QDs do not target the cell membrane but remain diffusely localized in the cytosol.
	Figure 6. Evaluation of commercially available streptavidin coated QDs. Commercially available streptavidin coated QDot605 (from Invitrogen) were incubated with biotinylated DNA (lane 1) and non biotinylated DNA (lane 2) at a molar ratio of 1:100, for 30 minutes at room temperature. Following the conjugation reaction the DNA-QD mixtures were run on a 1% agarose gel to assess the percentage of QDs capable of efficient biotin-streptavidin conjugation. QDot605 were imaged using the ethidium bromide filter set on the UVP iBoxImaging System. As shown, the QDs used in our experiments exhibit great variability in terms of their biotin binding ability (see arrows). Arrow 1 indicates QDs that are unable to bind biotin.
	Figure 7. Simultaneous targeting of QDs to two different proteins via Ssp DnaE and Ssp DnaB intein mediated splicing without cross reactivity. Fluorescence images of stage 10 Xenopus embryos injected with (A) the probes QDot585-DnaB IN and DnaE IC-QDot705 and the corresponding RNAs encoding DnaB IC-memEGFP and Akt-EGFP-DnaE IN, (B) the probe QDot655-DnaB IN and RNA encoding Akt-EGFP-DnaE IN or the probe DnaE IC-QDot655 and RNA encoding DnaB IC-memEGFP. Both QDot585 and QDot705 translocated to the cell membrane in cells derived from the embryo injected with the complementary probes where they colocalized with memEGFP and Akt-EGFP. In contrast, Akt-EGFP and mem-EGFP clearly target to the cell membrane whereas the non-complementary probes, remain diffuse in the cytoplasm, implying that the two inteins do not cross react.

References [+] :

Aranko, In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins. 2009, Pubmed