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. 2021 Mar 11;19(3):e3001020. doi: 10.1371/journal.pbio.3001020

Fig 5. U-ExM coupled with NHS-ester/Tubulin staining reveals the position of the ATR.

Fig 5

(A) Merged representative confocal image of an expanded ookinete stained for α/β-tubulin (magenta) and NHS-ester (grey). The magenta arrow indicates the position of the ATR. Scale bar: 1 μm. (B) Insets of the apical region from (A) highlighting the position of the ATR relative to the general ookinete structural features. (C) EM image of the apical region of an ookinete. Black arrowhead points to the apical protrusion. Scale bar: 250 nm. (D, E) Section (D) or maximum intensity projection (E) of the apical region of an expanded ookinete stained for NHS-ester from an entire image stack. Black arrowhead points to the apical protrusion. Scale bar: 250 nm. (F) Schematic representation of the apical region highlighting the apical protrusion, the collar, and subpellicular microtubules. (G) Section of a stack of an expanded ookinete stained with NHS-ester (grey) and α/β-tubulin (magenta). Dotted square indicates the position of the zoom shown in H. Scale bar: 500 nm. (H) Zoom in from image in G. Dotted line with the open arrowhead indicates the position of the ATR, the black arrowhead shows the apical protrusion, the magenta arrowhead points to the ATR, and the black arrow to the position of the plot profile (I). Scale bar: 250 nm. (I) Plot profile intensity over length in nm showing the position of the ATR relative to the apical protrusion. Note that the ATR position is in line with the end of the collar and/or the IMC, below the apical protrusion. The underlying data can be found in S1 Data. (J) Three-dimensional rendering of an NHS-ester/tubulin overlay (grey and magenta, respectively) of an expanded ookinete. Black and magenta arrowheads indicate the apical protrusion and ATR, respectively. ATR, apical tubulin ring; EM, electron microscopy; IMC, inner membrane complex; NHS, N-hydroxysuccinimide; U-ExM, ultrastructure expansion microscopy.