JCS026104 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. EM images of WI-38 nuclei contain only one large heterochromatin body per nucleus similar in size to the Barr body identified by DAPI staining. Here we show the complete set of serial sections (200-nm thick) from the nucleus shown in Fig. 3. Top section (section 1) shows grazing sections of nucleoli which bulge out from top of the nucleus. Sections 7-11 show the Barr body (arrows) consists of heterochromatin fibers which are distinct from surrounding chromatin. Asterisks in other sections show the approximate location of the Barr body seen in sections 7-11 and possible remnants of Xi chromatin. Nu indicates nucleolus. Arrowheads point to non-Xi heterochromatin associated with the nuclear envelope or nuclear envelope invaginations (Inv). These other heterochromatin regions are noticeably smaller and easily distinguishable from the Xi. Scale bar: 2 µm.

• Supplemental Figure S2 -

  Fig. S2. Intensity projection of a serial section stack shows comparable appearance of the Barr body and surrounding chromatin to a single optical section from a live confluent WI-38 cell. (A) Electron micrograph of a single 60-nm nuclear cross-section. (B) Intensity projection of a nuclear region aligned on the above section. (C) Higher-magnification view of projection. (D) Live cell expressing histone H2B-GFP. Nu indicates nucleolus; arrows point to the Xi.

• Supplemental Figure S3 -

  Fig. S3. Nuclear pore staining shows attachment of the Xi to the nuclear envelope in a high percentage of the WI-38 cells with an ‘intranuclear’ Xi. Rabbit anti-H3-3mK27 antibody staining (green), mouse anti-nuclear pore antibody staining (red). (A) Xi is located over a grazing optical section through the nuclear envelope. (B) The envelope touches the Xi via an invagination. (C) The Xi is anchored to the nuclear envelope by a chromatin fiber. Arrowheads indicate connections of the Xi to the nuclear envelope. Scale bar: 2 µm.

• Supplemental Figure S4 -

  Fig. S4. Attachment of MEF chromocenters to the nuclear envelope. (A,B) Regions from two different MEF nuclei demonstrating attachment of chromocenters to the nuclear envelope. Representative 200-nm serial sections are shown. See supplementary material Movie 3 for the complete set of serial sections for the nucleus shown in B. Chromocenters (arrowheads) display slightly less-dense heavy-metal staining than nucleoli (Nu). Xi heterochromatin is marked by arrows.

• Supplemental Figure S5 -

  Fig. S5. Nucleolar associations of the Xi in confluent WI-38 cells. (A) LM shows a connection (arrowhead) of the Xi (anti-H3-3mK27 antibody, red) and nucleoli (anti-fibrillarin antibody; green) in 40% of nuclei. (B-D) TEM images of 200-nm serial sections show examples of Xi (arrows)-nucleolar (Nu) attachment observed in 38% of nuclei. (B) Xi attached simultaneously to both the nuclear envelope and nucleolus. (C) Xi sandwiched between nucleolus and nuclear envelope invagination (Inv). (D) Protrusion of nucleolus in direct contact with the Xi (identified by H3-3mK27 immunogold staining). This cell has a large nucleus and may be tetraploid, with the Xi appearing as a bi-lobed, double chromatin mass. Adjacent sections show clear connection of this bi-lobed Xi to the nuclear envelope (data not shown).

• Supplemental Figure S6 -

  Fig. S6. Fig. 5D′′′ shown at appropriate magnification for optimal stereopair viewing.

• Movie 1 -

  Movie 1. Porous structure of the Barr body. Xi heterochromatin has an open structure with intervening tunnels of continuous interchromosomal spaces. All 21 aligned 60-nm serial sections of a confluent WI-38 nucleus are shown. Display time is 1 frame per second (f/s).

• Movie 2 -

  Movie 2. Rotating solid model of a Barr body. Reconstructed from stack of interpolated and filtered images of 60 nm serial sections through a confluent WI-38 nucleus. Display time is 7 f/s.

• Movie 3 -

  Movie 3. Chromocenters of mouse embryonic fibroblasts are attached to the nuclear envelope. Complete set of 200-nm sections (17 images) through a nucleus demonstrates attachment of all chromocenters (arrowheads) to the nuclear envelope. Chromocenters are distinguished from darkly stained nucleoli (Nu) by their slightly lower, but still dense staining. Xi heterochromatin is marked by an arrow. Display time is 1 f/s.