CELB30090 Advanced Cell Biology. Prof. Jeremy C. Simpson. Lecture 12 The nucleus form and function

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1 CELB30090 Advanced Cell Biology Prof. Jeremy C. Simpson Lecture 12 The nucleus form and function

2 Today s lecture... Functions of the nucleus Composition and organisation of the nucleus Subnuclear structures The nuclear envelope and nuclear pores Transport into and out of the nucleus

3 The subcellular complexity of cells

4 Primary functions of the nucleus storage and maintenance of the cell s genetic information control of gene expression through transcription synthesis of rrna and assembly of ribosomes transmission light image fluorescence image side view typical diameter : 10 20µm typical volume : 10% of cell 10µm

5 Organisation of the nucleus shows a high level of organisation (eg: subnuclear structures and chromosome territories) no internal membranes, but is compartmentalised the typical interphase (non mitotic) cell nucleus is composed of: DNA / chromosomes present as highly extended nucleoprotein fibers called chromatin nuclear matrix protein containing fibrillar network nucleolus one or more; irregularly shaped, electron dense, amorphous structures nucleoplasm the fluid substance in which the solutes of the nucleus are dissolved subnuclear particles various discrete specialised structures within the nucleoplasm nuclear envelope a double membrane surrounding the nucleus, contains channels (nuclear pores)

6 Spector DL (2001) J Cell Sci 114: Organisation of the nucleus

7 Visualisation of nuclear structures by light and electron microscopy Conventional wide field fluorescence image of a paraformaldehydefixed HeLa cell stained with markers for : the nuclear pore complex (red) the nucleolus (green) chromatin (blue) The resolution is ca. 200 nm. Field emission scanning electron micrograph of a dry fractured HeLa cell. The fracture exposes : NCL the nuclear pore complex (NPC), clearly visible on the surface of the nucleus CHR the chromatin (CHR) within the nucleus NPC the dense nucleolar (NCL) region The spatial resolution of structures is within 3 to 5 nm.

8 Organisation of the nucleus DNA / chromosomes the total length of DNA is ca. 2m per cell DNA is combined with histone proteins and organised into a precise, compact structure a dense string like fibre called chromatin DNA histone units are termed nucleosomes, and in the electron microscope, uncondensed chromatin has a beads on a string appearance nucleosomes are further coiled, compacting the DNA by a factor of 40 the overall negative charge of the DNA is neutralised by the positive charge of the histones there are two types of chromatin. Euchromatin is the genetically active portion (being transcribed), and heterochromatin contains inactive DNA and is the portion of chromatin that is most condensed, since it is not being used during interphase the chromatin is dispersed throughout the nucleus. This exposes the euchromatin and makes it available for transcription

9 Organisation of the nucleus nuclear matrix a highly proteinaceous fibrillar network can be isolated after treatment of nuclei with non ionic detergents and high salt, thereby removing lipids and chromatin proteins acts as both a skeleton to maintain the shape of the nucleus, and as a scaffold on which to organise the chromatin anchors much of the machinery involved in various nuclear activities, including transcription, RNA processing and replication Isolated nucleus treated with detergent and salt Cell extracted with detergent and then treated with nuclease and salt DNA loops

10 Organisation of the nucleus nucleolus one or more per nucleus; irregularly shaped, electron dense, amorphous structures site of rrna synthesis, rrna processing, and assembly of ribosomal subunits most mammalian cells contain 1 5 nucleoli, each µm in diameter the nucleolus is differentiated into three clearly identifiable regions: 1. The fibrillar centres (probably contains the DNA encoding the rrna). 2. The dense fibrillar component (transcription and processing of rrna). 3. The granular component (pre ribosomal particles, plus large and small ribosomal subunits).

11 Organisation of the nucleus subnuclear particles Cajal bodies (coiled bodies) typically µm in diameter, 1 10 per nucleus, number varies over the cell cycle likely sites of assembly or modification of the transcription machinery role in biogenesis and trafficking of small nuclear ribonucleoproteins (snrnps) en route to nucleoli or splicing speckles bound to the nucleolus by coilin proteins Nuclear speckles localised in a pattern of speckles per nucleus, highly dynamic, sometimes diffusely distributed throughout the nucleoplasm possible function as dynamic storage depots that supply splicing factors for use at nearby transcription sites PML bodies typically µm in diameter, per nucleus possible functions include transcription, DNA repair, viral defence, stress, cell cycle regulation, proteolysis and apoptosis defined by the presence of the PML protein

12 Organisation of the nucleus nuclear envelope double membrane surrounding the nucleus containing protein channels (nuclear pores) the outer membrane is contiguous with the rough endoplasmic reticulum and is often studded with ribosomes the inner and outer nuclear membranes (separated by nm) are fused together in places, forming nuclear pores that serve in the transit of materials between the nucleus and cytoplasm the average mammalian cell contains ca nuclear pores the nuclear pore complex (NPC) has an extensive substructure, in which a basket extends into the nucleoplasm the peripheral nuclear lamina lies inside the nuclear envelope and is composed of lamins A/C and B and is thought to regulate nuclear envelope structure and anchor interphase chromatin at the nuclear periphery

13 Nuclear pores protein pores allow molecules to be transported across nuclear envelope for example, RNAs and proteins move in both directions between the nucleus and cytoplasm 1. Gene replication and transcription machinery in the nucleus require a large number of proteins that are made in cytoplasm and transported into nucleus across the nuclear envelope 2. mrnas, trnas, and ribosomal subunits are made in the nucleus and are transported to the cytoplasm 3. HeLa cells contain ca. 10 million ribosomes, therefore to support its growth, one HeLa cell nucleus must import ca. 560,000 ribosomal proteins and export ca. 14,000 ribosomal subunits per minute gold particles and EM have been used to visualise transfer through the NPC

14 Nuclear pores nuclear pores contain a complex, basketlike apparatus, the nuclear pore complex (NPC), which appears to fill the pore like a stopper, projecting into both the cytoplasm and nucleoplasm the NPC is a large, supramolecular complex (molecular mass of MDa in mammals) NPCs exhibit octagonal symmetry due to the 8 fold repetition of a number of structures NPCs contain ca. 30 different proteins (termed nucleoporins or nups) each nucleoporin is present in at least 8 copies, in total each NPC contains ca nucleoporin proteins EM showing cytoplasmic face of nuclear envelope EM showing nuclear face of nuclear envelope

15 The nuclear pore complex cytoplasm nucleus transmembrane nucleoporins scaffold subcomplexes D Angelo MA & Hetzer MW (2008) Trends Cell Biol 18:

16 Transport through the nuclear pore complex ions and small metabolites can diffuse through NPCs molecules with a mass > 40 kda need to be actively transported (nucleocytoplasmic transport) transport is carried out by a large family of transport receptors known as karyopherins in yeast, and importins and exportins in mammals (named depending on their direction of transport) NPCs can perform 1000 translocations events per second, shuttling a mass of 100 MDa per second every nuclear pore gets renewed during each cell division cycle, however how NPCs are maintained during interphase is unknown specific amino acid sequences within proteins act as signals for nuclear import or export. These are termed nuclear localisation signals (NLS) and nuclear export signals (NES) Typical NLS : Pro Pro Lys Lys Lys Arg Lys Val (positive charge) Typical NES : Leu Ala Leu Lys Leu Ala Gly Leu Asp Ile (hydrophobic)

17 Transport through the nuclear pore complex Nuclear entry 1. Cargo with an NLS binds to a receptor (importin) 2. Docking at NPC 3. Translocation through pore 4. Disassembly of complex, aided by the GTPase Ran Nuclear export 1. Cargo with an NES binds to a receptor (exportin), aided by the GTPase Ran 2. Docking at NPC 3. Translocation through pore 4. Disassembly of complex, aided by the GTPase activating protein RanGAP Terry LJ et al. (2007) Science 318:

18 The small GTPase Ran in the nuclear import / export cycle The small GTPase Ran act as a molecular switch controlling nuclear import and export (1) (6) (1) Ran is concentrated in the nucleus because of nuclear transport factor 2 (NTF2) mediated active import (2) Ran GTP concentration is high in the nucleus (3) (2) (3) Binding / loading of GTP occurs by guanine exchange factors (GEFs) (eg: RCC1), causing a conformational change in Ran, allowing it to bind other molecules (eg: importins) (4) (5) (4) Ran GTP causes dissociation of imported complexes Clarke PR and Zhang C (2008). Nat Rev Mol Cell Biol 9: Binding of GTP = active Hydrolysis of GTP = inactive (5) Ran GTP binds to chromosome region maintenance protein 1 (CRM1) and promotes the assembly of export complexes containing proteins with an NES (6) In the cytoplasm Ran GTP is hydrolysed by GTPase activating proteins (GAPs) (eg: RanGAP1)

19 Key take home point The nucleus maintains a complex and specific architecture, with the transport of materials into and out the nucleus being a highly regulated process governed through the activity of the small GTPase Ran