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 D E 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