Figure 3. Mechanisms of Nuclear Tunnels in Organizing Cellular Structures for Ribosome Biogenesis.

All fluorescence images are Expansion Microscopy images taken on an Airyscan microscope with a 63x objective. All scale bars are in pre-expansion units.

(A) i: Image of total lipid and total protein of a UCI082014 cell. Lipids were stained with mCling-atto647 (grey) and proteins were stained with Alexa Fluor 488 NHS ester (blue). Scale bar: 3 µm. ii: Magnified view of nuclear tunnel-nucleolus contact in the boxed area of the image (i). The arrow points to an FC/DFC region of the nucleolus. Scale bar: 500 nm. Length expansion factor: 3.9.

(B) i: Image of an active nucleolus contacted by a nuclear tunnel in a UCI082014 cell. The cell was stained with anti-RPA194 antibodies (yellow), which mark Pol I, anti-Nup153 antibodies (green), which mark the nuclear tunnel, and NHS ester (grey). Scale bar: 5 µm. ii: Magnified view of NE-nucleolus contact in the boxed area of the image (i). Scale bar: 1 µm. Length expansion factor: 3.9.

(C) i: Image of an inactive nucleolus without contact with NE in a UCI082014 cell. The cell was stained with anti-RPA194 antibodies (yellow), anti-Nup153 antibodies (green), and NHS ester (grey). Scale bar: 5 µm. ii: Magnified view of NE-nucleolus contact in the boxed area of the image (i). Scale bar: 1 µm. Length expansion factor: 3.9.

(D) Barchart of RPA194 cluster density in the nucleoli contacted by tunnels or suspended in UCI082014 cells. Each bar represents the mean ± standard error of more than 15 nucleoli from 3 independent experiments. ** indicates p<0.01 by unpaired t test.

(E) i: Image of an MCF-10A cell stained with anti-H3K9me3 antibodies (pink), anti-Nup153 antibodies (green), and NHS ester (grey). Scale bar: 3 µm. ii: Magnified view of tunnel-nucleolus contact in boxed area of image (i). Scale bar: 1 µm. Length expansion factor: 3.9.

(F) i: Image of a UCI082014 cell stained with anti-eIF6 (orange) antibodies, anti-Nup153 (green), and NHS ester (grey). Scale bar: 3 µm. ii: Magnified view of tunnel-nucleolus contact in boxed area of image (i). Scale bar: 500 nm. Length expansion factor: 3.7.

(G) i: Image of a UCI082014 cell stained with anti-Nup153 antibodies (green) and anti-exportin1 (magenta) antibodies. Scale bar: 5 µm. ii: Magnified view of a nuclear tunnel in boxed area of image (i). Scale bar: 500 nm. iii: Magnified view of Nup153 channel of image (ii). iv: Magnified view of exportin1 channel of image (ii). Length expansion factor: 4.0.

(H) i: Image of a UCI082014 cell stained with anti-Sec61b antibodies (yellow) and anti-RPS3 (pink) antibodies, and NHS ester (blue). Scale bar: 5 µm. ii: Magnified view of tunnel-nucleolus contact in boxed area of image (i). Scale bar: 500 nm. Length expansion factor: 3.9.

(I) i: Image of a UCI082014 cell stained with anti-Tom20 antibodies (purple) and NHS ester (blue) and. Scale bar: 5 µm. ii: Magnified view of tunnel-nucleolus contact in boxed area of image (i). Scale bar: 500 nm. Length expansion factor: 3.7.

(J) i: Image of a UCI082014 cell stained with anti-alpha-tubulin antibodies (magenta) and NHS ester (blue). Scale bar: 3 µm. ii: Magnified view of tunnel-nucleolus contact in boxed area of image (i). Scale bar: 500 nm. Length expansion factor: 4.0.

(K) Model of the organization of ribosome biogenesis-associated components near the nuclear tunnel