Lecture 2

1. Lecture 2 Nuclear Envelope, NPC, Nucleolus & Nuclear Matrix

2. Endoplasmic Reticulum (ER) Properties of the Nuclear Envelope (NE) • Nuclear envelope appears as flattenedER cisternaesurrounding the nucleus. • It is composed of two layers: outer and inner nuclear membrane which areconnected together by thenuclear pore complex (NPC). • ER properties of the NE: (a)Overall Morphology;(b)Presence of ribosomes on outer nuclear membrane;(c)Connections with rough ER;(d)Similar composition of phospholipids and other membrane bound enzymes • NE is specialized in terms of thenuclear pore complexandnuclear lamina.

3. Biochemistry of Isolated Nuclear Membranes: Resembles ER > (PE)

4. Assembly and Disassembly of Nuclear Envelope • Nuclear envelope (NE)is a cell cycle dependent structure that disperses at the onset of mitosis (late prophase) and reassembles around the reforming nucleus in the late telophase. • Inhibition of protein synthesis bycycloheximidein late G2 phase has no apparent affect on nuclear assembly in telophase indicating that no new protein synthesis is required for reassembly of the nuclear envelope. • This reassembly involves ~ 10,000 nuclear pores in a matter of minutes. • The correlations of breakdown of the nuclear envelope, chromosome formation mitosis & NE reassembly after mitosis are essential for cell division and the ability of cells to divide in an orderly manner.

5. Assembly and Disassembly of Nuclear Envelope contd… (Role of the Nuclear Lamina) • The proteins that compose the nuclear lamina(lamins A, B,C)are involved in thedisassembly/reassemblyof the nuclear envelope during cell cycle viaphosphorylation (P)/dephosphorylation (deP). • Yeast genetic studies have identifiedcdc2as an essential gene for cell division in yeast. This is a cyclin dependant protein kinase calledcyclin B-cdc2 (cdk1) kinase.Cyclins are regulatory proteins that mediate the enzymatic activity of protein kinases that plays a major role in the regulation of the cell cycle. • Lamin phosphorylation/ dephosphorylation during cellcycle by cdk1 kinase/cdc14 . late prophase early telophase (cdc14)

6. Phosphorylation (P)/De(P) of the Nuclear Lamins Correlates with Nuclear Envelope Assembly/Disassembly 2-D Gel Shift –Phosphorylation of the nuclear lamin proteins in late prophase correlates with the disassembly of the nuclear envelope and dephosphorylation of the lamins correlates with nuclear envelope reassembly. This is indicated by the increased phosphorylation during prophase and the dephosphorylation during telophase of the nuclear lamins in a 2 D-gel shift AP experiment(AP = alkaline phosphatase, acidic is left; basic is right).

7. Experimental Basis for the Role of Nuclear Lamin Phosphorylation in Nuclear Envelope Disassembly[Heald & McKeon, Cell 61 (1990) 579-589] DNA transfection experiments – in which human lamin A gene mutated at two sites ( S-22 and S-392 which are the phosphorylation sites for cdc2 kinase) to alanine or isoleucine (cannot be phosphorylated) are then transfected into mammalian cells. Results show that mitosis proceeds up to a point with no breakdown of nuclear envelope. Therefore phosphorylation of S-22 and S-392 by cdc2 kinase is essential for nuclear envelope breakdown. Normal lamin A gene Mutant lamin A gene Interphase Prophase Metaphase Anti-lamin A DNA (DAPI) Anti-lamin A DNA (DAPI)

8. Experimental Basis for the Role of Nuclear Lamin Dephosphorylation in Nuclear Envelope Assembly [Burke & Gerace, Cell 44 (1986) 639-652] Incubate at 330C and measure nuclear envelope assembly around the chromosomes and dephosphorylation of lamins by 2-D gel shift Mitotic CHO cells Disrupt mitotic extract Assembly of nuclear envelope in mitotic extracts- In mitotic cells incubated in vitro can follow the assembly of nuclear envelope around chromosomes in association with dephosphorylation of nuclear lamins as observed by shifts in the PI of the lamin proteins on 2-D gels. If dephosphorylation of nuclear lamins is inhibited there is a corresponding inhibition of nuclear envelope assembly. 2-D gel shift

9. Nuclear Pore Complex (NPC) Field emission scanning EM micrographs of NPC • Nuclear pore complex connects the outer nuclear membrane to the inner membrane which allows for the nucleocytoplasmic transport of materials (mRNA’s, tRNA’s, proteins etc) . • 3-D microscopy indicates an asymmetrical organization of the pore complex: “nuclear basket” • ~100 different nuclear pore proteins (NUP’s/ (nucleoporins); total mass >10,000 kDa. • Positioning on NPC: (a) Cytoplasmic face : NUP’s 180 & 124; (b) Nuclear face : NUP 153; (c) Both faces: NUP’s 62 & 155 • Sequence of the NUP’s (a) pentameric degenerative repeats: (XFXFG, docking sequence for importin beta) : NUP’s 62, 78, 113 & 153; (b) Tetrameric degenerative repeats: (GLFG , docking sequence for importin beta) : NUP’s 49, 57, 100, 145, 166; (c) No degenrative repeats: NUP 155 • Many NUP’s are glycoproteins with single 0-linked N-acetylglucosamine residues. After detergent Nuclear face Cytoplamic face NUP 159 NUP 96 Asymmetric (cytoplasmic) Symmetric

10. Nucleolus • The Nucleolus (‘tiny nucleus”) is a reticular fibrogranular structure in the nucleus that is specialized for transcription of ribosomal RNA and its packaging into pre-ribosomal subunits. • The genes for ribosomal RNA are highly amplified and located on five different chromosomes (13, 14, 15, 21, 22) called the nucleolar organizer regions (NOR). • rDNA genes are concentrated within the numerous fibrillar centers (fc) that compose the nucleolus. • rRNA is believed to be transcribed at the borders of fc and the dense fibrillar component (dfc) where ribosomal proteins associate to form pre-ribosomal RNP particles. • Progressive processing of the pre–rRNP particles occurs within the granular component (gc) where mature ribosomal subunits are released for transport into the cytoplasm. • The massive rRNA transcription is illustrated by the “Christmas tree” structures composed of repeating nascent rRNP strands that grow in assembly line fashion along the rDNA transcription units.

11. Nuclear Matrix: Structural Architecture of the Cell Nucleus A fundamental question is whether there is an overall framework structure that can serve to position and order components and macromolecular complexes in the cell nucleus ??? Electron microscopy using EDTA regressive staining have revealed a non chromatin structure in the cell nucleus that resembles a network of fibrogranular structure. This fibrogranular structure nuclear network is called the in situ nuclear matrix. (standard) Whole cell (EDTA) Isolated nuclear matrix Whole cell EDTA regressive staining

12. Procedure of Nuclear Matrix Isolation

13. Components of Isolated Nuclear Matrix Isolated nuclear matrix is composed of: nuclear lamina, residual nucleoli and a fibrogranular internal nuclear matrix

14. High magnification electron microscopy of nuclear matrix

15. Whole Mount Electron Microscopy Demonstrating Fibrogranular Structure of the Internal Nuclear Matrix

16. 2-D PAGE of Nuclear Matrix Proteins

17. Functional Properties Associated with Nuclear Matrix

18. Functional Properties Associated with Nuclear Matrix contd..