1. The Nucleus

2. Nuclear Morphology

3. Chromosome Territories • Chromosomes occupy discrete regions of interphase nucleus • Chromatin – both heterochromatin and euchromatin • Interchromasomal domain

4. Sites of RNA Synthesis and Processing • Perichromatin fibrils – >10000 sites of Pol II transcription & RNA processing • Speckles & Cajal bodies • sites of storage for RNA splicing components

5. Nucleolar Subdomains • rRNA transcription at FC/DFC boundary • Ribosomal assembly in GCs • RNA Pol I & factors in FC

6. Nucleolar Organizing Regions (NORs) • rDNA on chromosomes 13, 14, 15, 21 & 22 • Remains associated with Pol I & transcription factors throughout mitosis

7. Nuclear Envelope

8. Nuclear Lamina • Lamin B – most prevalent • Lamin A & C – differentiation specific

9. Nuclear Envelope Disassembly & Reassembly During Cell Division • Lamins A & C • dephosphorylated • Lamin B • phosphorylated and demethylated • Remain attached to NE remnants

10. Nuclear Pore Complexes • ~ 3000-5000 pores/nucleus • 130 nm in diameter • 55 nm central channel • Mammalian pores composed of >100 proteins • Nucleoporins major class of pore proteins

11. Nuclear/Cytoplasmic Trafficking • Nuclear Localization Signal – NLS • Many defined a. a. sequences • Basic NLS – very common • Stretch of basic a.a. residues • SV-40 T Ag – PKKKRKV • Direct import of protein into nucleus • Nuclear Export Signal – NES • Direct export of nuclear proteins • Critical for recycling of import factors • Nuclear Retention Signal – NRS • Critical for retention of various RNA processing factors

12. Demonstration of NLS C E D F Figure 16-13. A NLS ICAD causes specific import into the nucleus of an ICAD-GFP fusion protein. Panels A, C & E show cells transfected with ICAD-GFP without a NLS, panels B, C & F are cells transfected with ICAD-GFP with a NLS. A & B- Composite fluorescence image of GFP and DAPI. C & D- GFP fluorescence showing cytoplasmic distribution of ICAD-GFP lacking a NLS (C). E & F- Nuclei stained with DAPI.

13. Demonstration of NES Site of injection, nuclear vs cytoplasmic, is indicated by position of arrow

14. Nuclear Import/Export Cycles • Adapter = importin  • Receptor = importin  • Export receptor = CAS • Ran – small GTPase • RanGAP – activates GTPase activity • RanGEF – exchanges GDP for GTP

15. Some Structures

16. Regulated Nuclear Localization