J Cell Biol
August 4, 2014;
Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1.
The directed movement of cells is critical for numerous developmental and disease processes. A developmentally reiterated form of migration is radial intercalation; the process by which cells move in a direction orthogonal to the plane of the tissue
from an inner layer to an outer layer. We use the radial intercalation of cells into the skin
of Xenopus laevis embryos as a model to study directed cell migration within an epithelial tissue
. We identify a novel function for both the microtubule
-binding protein CLAMP and members of the microtubule
-regulating Par complex during intercalation. Specifically, we show that Par3
promote the apical positioning of centrioles, whereas CLAMP stabilizes microtubules along the axis of migration. We propose a model in which the Par complex defines the orientation of apical migration during intercalation and in which subcellular localization of CLAMP promotes the establishment of an axis of microtubule
stability required for the active migration of cells into the outer epithelium
J Cell Biol
[+] show captions
References [+] :
Figure 1. Apical positioning of centrioles and intercalation requires the Par complex. (A) Schematic representation of MCC intercalation. (B and C) Apical localization (arrowheads) of Par3-GFP (B) and aPKC-GFP (C) at different stages of intercalation (early and late). (D) Schematic of the quantification of centriole positioning in MCCs. (E and F) Representative z stack cross-section images (E) and quantification (F) of centriole position in control, DN-Par3, and aPKC-KD MCCs. (G) Mosaic image showing wild-type MCC that has intercalated (arrowhead) and a DN-PAR3-GFP (arrow) MCC that failed to intercalate. (H) Images illustrating the three different phenotypes used for scoring MCC intercalation. (I) Quantification of intercalation in MCCs expressing GFP, Par3-GFP, aPKC-GFP, Par-3 MO, DN-Par3-GFP, or aPKC-KD-GFP. For all experiments, cells from a total of at least five embryos from at least two independent experiments were quantified unless specified otherwise. Quantification in F is based on at least 10 cells each from a total of at least five embryos from at least two independent experiments. Quantifications of DN-Par3 and aPKC-KD phenotypes in F and I are statically significantly different from controls (P < 0.0001, see Table S1). Side projection refers to side views of projection along the x axis in all figures. See Fig. S1. Bars, 5 µm.
Figure 2. Stabilized apical MTs are required for radial intercalation of MCCs and ICs. (A and B) Anti-acetylated tubulin antibody staining of wild-type MCCs (A, arrowheads) and of a mosaic tissue showing a control and a DN-Par3-GFP–expressing MCC (B, arrowheads) during intercalation. Maximum-intensity projection (top) and corresponding cross section (bottom) are shown. (C and D) Images (C) of MCCs stained with an antibody marking acetylated α-tubulin in DMSO (left, arrowhead), which is lost following treatment with 1 μM Nocodazole (right, arrowhead), along with quantification of intercalation (D). (E and F) Images of IC marker AE1 in DMSO (left)- and Nocodazole (right)-treated ICs (E), along with quantification of intercalation (F). Quantifications of the Nocodazole phenotypes in D and F are statistically significantly different from controls (P < 0.0001, see Table S1). Bars, 5 µm.
Figure 4. CLAMP is required cell autonomously for intercalation. (A) Mosaic embryos coinjected with CLAMP-MO and αtub-GFP (arrows) stained with anti-acetylated tubulin and phalloidin. (B) Mosaic embryos coinjected with CLAMP-MO and membrane-RFP stained with anti–β-tubulin and phalloidin (wild-type MCCs still intercalate, arrows). (C) Mosaic embryo coinjected with CLAMP-MO and membrane-RFP (blue), showing MCC intercalation defects (arrows). (D) Quantification of intercalation in MCCs injected with control MO, CLAMP MO, or CLAMP MO rescued with xCLAMP or hCLAMP. (E and F) Representative image (E) and quantification (F) of intercalation defect in ICs (green, AE1 staining) containing CLAMP MO (arrowheads) versus wild-type (arrow, lack of red). (G and H) Image (G) and quantification (H) of centriole position in CLAMP morphant MCCs (the arrow highlights the centriole cluster). (I and J) Representative image (I) and quantification (J) of mosaic embryos showing the loss of CLAMP staining in DN-Par3–expressing MCCs (arrowheads) compared with wild-type (arrow). Quantification in H is based on at least 10 cells, each from a total of at least five embryos from at least two independent experiments. Quantifications of intercalation defects in CLAMP morphants in D and F are significantly different from controls, as are morphant embryos from rescue (P < 0.0001, see Table S1). A χ2 test shows no statistical significant difference in H (P = 0.236, see Table S1). Quantification of reduction in apical CLAMP levels in DN-Par–expressing cells is significantly different from controls (P < 0.01, see Table S1). See Fig. S3. Error bars represent standard deviations. Bars, 5 µm.
Figure 5. Model for the steps involved in regulating MT stability during radial intercalation. We propose that the Par complex mediates both apical positioning of centrioles (1) and asymmetric accumulation of CLAMP (2). CLAMP asymmetry leads to asymmetric stabilization of MTs (2) along the axis of migration that promotes intercalation (3).
Breuzard, Molecular mechanisms of Tau binding to microtubules and its role in microtubule dynamics in live cells. 2013, Pubmed