Dudley CE et al. (2022), SNAP- and Halo-tagging and dye introduction pro...

XB-ART-59311

STAR Protoc 2022 Sep 16;33:101622. doi: 10.1016/j.xpro.2022.101622.

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SNAP- and Halo-tagging and dye introduction protocol for live microscopy in Xenopus embryos.

Dudley CE , van den Goor L , Miller AL .

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Traditional fluorescent proteins exhibit limitations in brightness and photostability that hinder optimal characterization of the dynamic cellular behavior of proteins of interest. SNAP- and Halo-tagging are alternatives to traditional fluorescent protein tagging utilizing bright, stable chemical dyes, which may improve signal-to-noise ratio. However, there has been limited use of this approach in vivo in developing organisms. Here, we present a protocol for implementing SNAP- and Halo-tagging in gastrula-stage Xenopus laevis embryos for live confocal microscopy. For complete details on the use and execution of this protocol, please refer to Varadarajan et al. (2022).

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Species referenced: Xenopus laevis

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	Figure 1. Workflow for expression of pCS2+/SNAPf- and Halo-tagged constructs in Xenopus embryospCS2+/SNAPf- and Halo-vectors that were codon optimized for Xenopus can be acquired from Addgene. The sequence of interest is cloned into the multiple cloning site (MCS), located upstream/downstream of the SNAPf/Halo-tag. Tagged constructs are in vitro transcribed to generate mRNA that is microinjected into 2–4 cell stage Xenopus embryos.
	Figure 2. Dye introduction methods for labeling SNAPf- or Halo-tagged proteins expressed in intact Xenopus embryos(Top) SNAP/Halo dyes can be microinjected with SNAPf- or Halo-tagged mRNAs. (Bottom) After microinjecting SNAPf- or Halo-tagged mRNAs, embryos can also be bathed in SNAP/Halo dyes overnight (O/N, 15–22 h) for next-day imaging.
	Figure 3. SNAP-tagged proteins can be visualized in both wild type (WT) and albino gastrula-stage Xenopus laevis embryosImages of WT (left) and Albino (right) Xenopus laevis embryos co-microinjected with E-cadherin-SNAPf mRNA (50 pg) and SNAP-Cell 647 SiR dye (10 μM). Scale Bars: 20 μm.
	Figure 4. Microinjecting or bathing embryos in dye can label E-cadherin-SNAPf in vivo(A) Images of Xenopus embryos expressing E-cadherin-SNAPf (50 pg) labeled via dye microinjection with SNAP-Cell 647 SiR (10 μM), SNAP-Cell TMR Star (25 μM), or SNAP-Cell Oregon Green (5 μM).(B) Images of Xenopus embryos expressing E-cadherin-SNAPf (50 pg) labeled via dye bath with SNAP-Cell 647 SiR (4 μM) or SNAP-Cell Oregon Green (3.3 μM).Scale Bars: 20 μm.
	Figure 5. Microinjecting or bathing embryos in dye can label Halo-ZO-1 in vivo(A) Images of Xenopus embryos expressing Halo-ZO-1 (100 pg) labeled via dye microinjection with Janelia Fluor 646 HaloTag (20 μM), Janelia Fluor 549 HaloTag (5 μM), or Oregon Green HaloTag (5 μM).(B) Images of Xenopus embryos expressing Halo-ZO-1 (100 pg) labeled via dye bath with Janelia Fluor 646 HaloTag (1.3 μM), Janelia Fluor 549 HaloTag (1.3 μM), and Oregon Green HaloTag (3.3 μM).Scale Bars: 20 μm.
	Figure 6. Microinjecting dye can label SNAPf-/Halo-Vinculin in vivo(A) Images of Xenopus embryos expressing Halo-Vinculin (10 pg) labeled via dye microinjection with Janelia Fluor 646 HaloTag (5 μM) and Janelia Fluor 549 HaloTag (5 μM).(B) Image of Xenopus embryo expressing SNAPf-Vinculin (10 pg) labeled via dye microinjection with SNAP-Cell 647 SiR (20 μM).Scale Bars: 20 μm.
	Figure 7. Halo-tag improves the visualization of difficult to image proteins compared with traditional fluorescent tags(A–E) Images of Xenopus embryos expressing (A) Vinculin-mNeon (10 pg), (B) Vinculin-3XGFP (30 pg), (C) Vinculin-GFP (10 pg), (D) mNeon-Vinculin (10 pg), and (E) Halo-Vinculin (10 pg) labeled with microinjected Janelia Fluor 646 HaloTag (5 μM).Scale Bars: 20 μm and 5 μm.
	Figure 8. SNAP- and Halo-tagged proteins can be visualized simultaneously, through dye microinjections or dye baths, in gastrula-stage Xenopus laevis embryos(A) Xenopus laevis gastrula-stage embryos expressing BFP-membrane (15 pg) (blue), Halo-ZO-1 (100 pg) labeled with microinjected Oregon Green HaloTag (10 μM) (green), and E-cadherin-SNAPf (50 pg) labeled with microinjected SNAP-Cell 647 SiR (5 μM) (red).(B) Xenopus laevis gastrula-stage embryos expressing BFP-membrane (15 pg) (blue), Halo-ZO-1 (100 pg) labeled via dye bath with Janelia Fluor 549 HaloTag (1.3 μM) (green), and E-cadherin-SNAPf (50 pg) labeled via dye bath with SNAP-Cell 647 SiR (4 μM) (red).Scale Bars: 20 μm.
	Figure 9. Dye and mRNA concentration optimization is necessary to reduce non-specific signal(A–E) Images of E-cadherin-SNAPf (50 pg) labeled with SNAP-Cell TMR Star ((A) 1.2 μM, (B) 5 μM, (C) 10 μM, (D) 20 μM, (E) 40 μM) through dye microinjections.(E′) E-cadherin-SNAPf (100 pg) labeled with 40 μM SNAP-Cell TMR Star.Scale Bars: 20 μm.
	Figure 10. Non-desiccated SNAP-Cell TMR-Star dye aliquots can lose brightness following long-term storageE-cadherin-SNAPf (50 pg) labeled with SNAP-Cell TMR Star (40 M) (newly acquired, top) (long term storage, bottom). Scale Bars: 20 μm.

References [+] :

Arnold, Anillin regulates epithelial cell mechanics by structuring the medial-apical actomyosin network. 2019, Pubmed, Xenbase