Extended Data Fig. 7: Microtubule acetylation patterns in cells in confinement.
From: Compression-dependent microtubule reinforcement enables cells to navigate confined environments
(a) Simplified graphical schematic of constriction microchannel patterns; Circular, continuous, and ratcheting. (b) Immunofluorescence of 1205Lu melanoma cells labelled for acetylated α-tubulin (AcK40, magenta), tyrosinated α-tubulin (white) and nuclei (DAPI, cyan) navigating through indicated microchannel designs. Scale bar 5 µm. Images are displayed as maximum intensity projections of z-stacks. Single channels are displayed as contrast inverted images, demonstrating regardless of design, acetylated-α-tubulin forms a polarized network that contrasts tyrosinated-α-tubulin. A rear cytoplasmic compartment can be seen which flanks the rear of the nucleus (dotted line), predominantly filled with stable acetylated tubulin (red arrow heads) forming the microtubule cushion. (c) Representative timelapse images of a 1205Lu cell endogenously tagged for tubulin (eGFP-α-tubulin; orange hot) and coexpressing a nuclear market (SnapTag-H2B-JF646; cyan hot) undergoing nuclear occlusion and transmigration. (d) Maximum intensity projections of inverted greyscale tubulin images, orthogonal sections demonstrate the presence of a microtubule cage surrounding the nucleus. (e) 3D-Structured Illumination microscopy of acetylated tubulin (AcK40, magenta) and tyrosinated α-tubulin (Tyr, white) immunolabelled 1205Lu cells, nuclei labelled with DAPI, undergoing confined migration. Cells are expressing alternative CLASP1 and 2 hairpins from Fig. 4b.
