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The Xenopus embryo provides an advantageous model system where genes can be readily transplanted as DNA or mRNA or depleted with antisense techniques. Here, we present a protocol to culture and image the cell biological properties of explanted Xenopus cap cells in tissue culture. We illustrate how this protocol can be applied to visualize lysosomes, macropinocytosis, focal adhesions, Wnt signaling, and cell migration. For complete details on the use and execution of this protocol, please refer to Tejeda-Muñoz et al. (2022).
Figure 1. Dissociation of animal caps for lysosomal studies(A) Images from a movie of animal cap excised at blastula stage 8.5 plated in fibronectin and filmed for 15 h; note that cells divide in these conditions. Scale bar, 500 μm.(B–C′) Animal cap cells serve as a model for lysosomal studies using LAMP1-RFP or CD63-RFP as markers; the DIC visible light channel shows yolk platelets present in the embryonic cells. See also Methods video S1. Scale bar, 10 μm.
Figure 2. Xenopus animal cap cells showing that microinjection of 2 pg xWnt8 mRNA at the 2–4 cell stage greatly stabilizes CD63 lysosomes(A–A‴) Control animal cap injected with DNA encoding membrane GFP and the MVB marker CD63; uninjected cells serve as an internal control.(B–B‴). Co-injection of xWnt8 mRNA results in a striking stabilization of CD63, a marker of the intraluminal vesicles presents in multivesicular bodies and lysosomes. Canonical Wnt is a potent regulator of lysosome function. The images on the right panels show the merged red, green, blue and visible light channels. Scale bar, 10 μm.
Figure 3. Xenopus animal cells display lysosomes and macropinocytosis activation after LiCl treatment, which mimics Wnt signaling by inhibiting GSK3 inhibition(A and A′) Control cells treated with SiR-Lysosome.(B and B′) LiCl treatment increases active lysosomes. Mimicking Wnt signaling with LiCl shows that lysosomes become more active.(C and C′) Untreated cells show low uptake of the macropinocytosis marker TMR-Dextran 70 kDa.(D and D′) LiCl treatment increases macropinocytosis (arrowhead). Scale bar, 10 μm.
Figure 4. Focal adhesions in animal cap cells(A) Diagram showing actin cytoskeletal cables, membrane integrins, and focal adhesions in the leading edge of a migratory cell, as well as the β-catenin destruction complex that regulates canonical Wnt signaling.(B) Xenopus animal cap cells showing focal adhesions in the leading edge and actin cables marked by TES-GFP. Focal adhesions are critical for cell adhesion and migration.(C) A DNA plasmid encoding the F-actin tracer LifeAct (Gonagen) (in red) was microinjected into the animal pole at the 4 cell-stage (20 pg/injection). Scale bar, 10 μm. See also Methods video S2.
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