Henson JH et al. (2021), The nanoscale organization of the Wnt signaling...

ECB-ART-49156

PLoS One 2021 May 26;165:e0248197. doi: 10.1371/journal.pone.0248197.

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The nanoscale organization of the Wnt signaling integrator Dishevelled in the vegetal cortex domain of an egg and early embryo.

Henson JH , Samasa B , Shuster CB , Wikramanayake AH .

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Canonical Wnt/β-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the Frizzled:LRP5/6 receptor complex with β-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica transmission electron microscopy (TEM) to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D structured illumination microscopy (SIM) imaging of isolated egg cortices demonstrated the graded distribution of Dsh in the VCD, whereas higher resolution stimulated emission depletion (STED) imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first cleavage actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for the localized activation of the cWnt pathway at the sea urchin vegetal pole. These observations in sea urchins may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.

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	Fig 2. Platinum replica TEM of Dsh and actin in the VCD in cortices isolated from unfertilized eggs.Low (A,C) to medium (B,D) magnification images of cortices shows Dsh-specific colloidal gold (colored gold in B-G) staining of patches in the plane of the membrane (arrows in A). Tangled knots of short actin filaments appear in the cores of microvilli (MV in B) and numerous elongate actin filaments running parallel to the plane of the membrane are present. Cortical granules appear as shriveled structures (CG in A) and other membranous structures are also present. The meshwork that appears in the background of the images corresponds to the vitelline envelope. The white box in C appears at higher magnification in E in which Dsh aggregates are colored magenta and identifiable actin filaments in green. Dsh positive patches do not associate with MV core actin assemblages but do come in close proximity to submembranous actin filaments. (F,G) High magnification images indicate that Dsh patches consist of aggregates of pedestal-like structures—each labeled with a single colloidal gold particle—that can be grouped into one or more clusters. L. pictus cortices = A,B,F,G; S. purpuratus cortices = C,D,E. Bar length indicated in the images.
	Fig 3. Platinum replica TEM demonstrates that Dsh puncta correspond to patches of aggregates of pedestal-like structures.(A-F) A gallery of high magnification images of Dsh labeled structures demonstrates that they consist of aggregates of pedestal-like structures in a variety of groupings. The top of each pedestal is labeled with a single colloidal gold particle suggesting that this area may correspond to the location of the C terminus of the Dsh protein. (G) The density of the Dsh puncta in TEM images from 3 separate cortices over three experiments (grey box) falls in the range of densities seen in the immunofluorescence images (black boxes reproduced from graph in Fig 1J). (H) The area of the Dsh aggregates from five cortices each from two separate experiments shows no statistically significant difference between the two species. (I,J) Comparison of regions of the same cortex in which Dsh labeling is present (I) and is not present (J) shows that the patches associated with the Dsh labeling are not visible in the unlabeled regions, suggesting that these structures are specific to the Dsh puncta. L. pictus cortices = A,B,D,E; S. purpuratus cortices = C,F,I,J. Scale bars = 200 nm.
	Fig 4. The Dsh VCD array is resistant to Triton detergent extraction.(A-H) Control unfertilized egg cortex from L. pictus (A-D) stained for Dsh (magenta) and actin (green) showing Dsh array and cortical granules in phase contrast. The Triton extracted cortex (E-H) demonstrates the persistence of the Dsh array following detergent extraction despite the loss of cortical granules seen in phase contrast. (I-N) Membrane staining with the fixable dye FM1-43 (green) shows that in control unfertilized egg cortices (I-K) that the Dsh (magenta) array is present along with a variety of membranous structures. In detergent extracted cortices (L-N) the Dsh array is still present even though specific membrane staining is lost. Scale bars = 10 μm.
	Fig 5. The Dsh VCD persists following disruption of actin filaments or inhibition of myosin II contraction.Control unfertilized egg cortex from L. pictus (A-C) contains the expected Dsh array (magenta) along with microvillar actin (green). In cortices from eggs treated with the actin filament disrupting drug LatA (D-F) the Dsh array persists (D) whereas the actin staining is greatly reduced (E). Both Dsh (G) and actin (H) staining appear unaffected in cortices isolated from unfertilized eggs treated with the MLCK inhibitor ML-7 (G-I) in order to inhibit myosin II contraction. Scale bar = 10 μm; magnifications of A-I are equivalent.
	Fig 6. The Dsh VCD in cortices isolated from first cleavage embryos is bisected by the cytokinetic contractile ring.Widefield microscopy of cortices isolated from first division S. purpuratus embryos indicate that the Dsh punctate VCD array (A,E,I,N; magenta in C,G,L,Q) is often bisected by the cytokinetic contractile ring which labels for activated myosin II (P-MyoRLC; B,F,J,O; green in C,G,L,Q) and F-actin (K,P; blue in L,Q). Transmitted light DIC (D,H at equivalent magnification) and phase contrast (insets M,R at reduced magnification) images of individual cortices are provided for context. Scale bar = 10 μm, magnifications of A-L and N-Q are all equivalent.
	Fig 7. Comparison of the Dsh VCD in cortices isolated from unfertilized eggs versus first cleavage embryos.Widefield microscopy of the Dsh VCD in unfertilized (UF) egg (A,B) and first cleavage (CL) embryo (D,E) S. purpuratus cortices highlights the differences in the two arrays. Panels B and E correspond to higher magnification versions of the 5x5 μm white boxes in panels A and C. Panel D shows P-MyoRLC staining of the actomyosin contractile ring in the CL cortex seen at higher magnification in C. The maximum density of Dsh puncta in the central VCD in CL cortices is statistically less dense than in the central VCD of UF egg cortices (F), and the puncta appear not as uniform in structure or distribution (A,B,C,E). Scale bars = 10 μm.

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Angerer, Animal-vegetal axis patterning mechanisms in the early sea urchin embryo. 2000, Pubmed, Echinobase