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Nuclear structure and transport Part I: Organization of the nucleus Stine F. Pedersen Dept. of Biology Cell Biology 2008 Dias 1 To be or not to be eukaryotic… Pro = “before”, karyon = “nucleus” Prokaryotes (Eubacteria and Archaea) lack a nucleus (and other real organelles) Their genetic material is found in a mass - the nucleoid which is freely exposed in the cytoplasm (but nonetheless highly ordered) Dias 2 To be or not to be eukaryotic… Eu = “true”, karyon = “nucleus” Eukaryotic cells contain a double membrane-bound nucleus, as well as several other organelles The presence of a nucleus separates (most of) the genetic material from the rest of the cell Dias 3 Dias 4 To be or not to be eukaryotic… • Eukaryotes contain much more chromatin than prokaryotes • The nucleus protects the fragile chromatin from breaking during interphase, when active chromatin is relatively loosely packed • During mitosis, when the nuclear envelope breaks down, chromatin is compacted into chromosomes, which are more resistant to breakage • Transport to and from the nucleus is regulated, allowing highly controlled gene regulation Dias 5 To be or not to be eukaryotic… • Eukaryotes contain much more chromatin than prokaryotes • The nucleus protects the fragile chromatin from breaking during interphase, when active chromatin is relatively loosely packed • During mitosis, when the nuclear envelope breaks down, chromatin is compacted into chromosomes, which are more resistant to breakage • Transport to and from the nucleus is regulated, allowing highly controlled gene regulation Dias 6 The evolution of the nucleus allowed transcription-translation uncoupling In prokaryotes, translation of mRNA begins before its synthesis is completed In eukaryotes, the separate nuclear compartment allows for much more complex regulation, and transcription and translation are separated. Dias 7 Most, but not all, cells have a single nucleus Nuclei range in size from about 1 µm (S. cerevisiae) to 400 µm (X. laevis oocytes) Most have just one, but some cells contain multiple nuclei, and a few lack a nucleus Dias 8 Most, but not all, cells have a single nucleus Nuclei range in size from about 1 µm (S. cerevisiae) to 400 µm (X. laevis oocytes) Most have just one, but some cells contain multiple nuclei, and a few lack a nucleus Immature erythroblasts Dias 9 Mature mammalian red blood cells Mature mammalian erythrocytes lack a nucleus As erythroblasts mature to become erythrocytes, most of their DNA is permanently silenced, seen as an increase in the fraction of heterochromatin Eventually, in mammalian erythrocytes, the nucleus is lost entirely Polychromatophilic: showing a bluegray tint on Wright-stained smears, indicating the presence of cytoplasmic RNA – i.e., immature Dias 10 Overview of nuclear organization The nucleus is separated from the cytoplasm by the a double membrane, the nuclear envelope The lumen of the nuclear envelope is continuous with the endoplasmic reticulum Transport between the cytoplasm and nucleus occurs through nuclear pore complexes (NPCs) Dias 11 Overview of nuclear organization Some important nuclear subcompartments (not membrane bound): 1. Nucleolus: site of synthesis of ribosomal subunits and ribosome assembly 2. Speckles: storage of RNA splicing factors 3. Cajal bodies: sn- & snoRNA synthesis and posttranscriptional processing? 4. Replication factories: sites of DNA synthesis The non-nucleolar, nonchromosomal regions is called the nucleoplasm Dias 12 Overview of nuclear organization The DNA (chromatin) that appears dark in electron micrographs is heterochromatin: highly folded and not actively transcribed; usually found near the nuclear envelope The chromatin containing the active genes is more dispersed, and is called euchromatin Dias 13 Organization of the chromosomes The images of individual chromosomes were produced by fluorescent in situ hybridisation (FISH) using probes specific for different chromosomes Dias 14 Organization of the chromosomes Each chromosome has its own chromosome territory, and is anchored to the nuclear membrane through its telomers Interchromosomal domains NPC contain poly(A)+ RNA undergoing processing and diffusing to the nearby NPCs Highly/actively transcribed genes are generally closest to the interchromosomal regions, facilitating export of the mRNAs to the cytoplasm Dias 15 Organization of the chromosomes Dias 16 The nucleus contains multiple subdomains Kumaran et al. 2008, Cell Dias 17 But first…RNA classes – a quick reminder • snRNA: Small nuclear RNA - a class of small RNA found in the nucleus and involved in several important processes including RNA splicing. snRNA is always associated with specific proteins; the complexes are called small nuclear ribonucleoproteins (snRNP) or snurps. The major spliceosome is composed of the U1, U2, U4, U5, and U6 snRNPs. • snoRNA: Small nucleolar RNAs - small RNAs involved in RNA biogenesis and in chemical modification of rRNA, tRNA, and snRNA. Found e.g. in cajal bodies. • rRNA: ribosomal RNAs - the large ribosomal subunit (60S) in eukaryotes is composed of 3 rRNAs and about 50 proteins, the small subunit (40S) of one (18S) rRNA and about 30 proteins. • tRNA: transfer RNA - small RNAs (usually ~ 75-100 nucleotides) that transfer specific amino acids to the polypeptide chain growing at the ribosomal site of protein synthesis during translation. • hnRNP: heterogenous nuclear ribonucleoprotein particles – complexes formed between pre-mRNA and multiple proteins – some proteins are shed in the nucleus, but some are exported to the cytosol with the mature mRNA – the complex is called the messenger ribonucleoprotein particle (mRNP) when the RNA is mature. Dias 18 Structure and functions of the nucleolus The nucleolus is the site of rRNA transcription and processing, and ribosome subunit assembly. The spatial separation of these processes is seen as nucleolus subcompartments tRNA genes are also transcribed and processed in the nucleolus The nucleolus has no membrane, is disassembled during mitosis, and is only detectable during production of ribosomal subunits Dias 19 Some other nuclear subcompartments Not visible by light microscopy – but by immunocytochemistry Speckles: storage of RNA splicing factors (but not sites of splicing) Cajal and Gemini bodies: probably synthesis / posttranscriptional processing of snRNAs and snoRNAs, and assembly of these into ribonucleoprotein complexes Promyelocytic leukemia (PML) bodies: function unknown and likely promiscuous Dias 20 Other nuclear subcompartments Replication factories are large nuclear regions, in which DNA replication becomes concentrated in S-phase – likely organized by the nuclear matrix, a form of nuclear ”skeleton” Similar sites may exist for transcription, termed transcription factories Bromodeoxyuridine labeling of newly incorporated DNA Dias 21 Overall organization of the nuclear envelope Outer membrane and lumen continuous with ER, and covered with ribosomes Dias 22 Nuclear pore complexes for transport between nucleus and cytosol Inner nuclear membrane, reinforced by the nuclear lamina The nuclear envelope and the endosymbiont hypothesis The double nuclear membrane may, similar to those of chloroplasts and mitochondria, reflect that the nucleus arose by endosymbiosis Dias 23 Structure of the nuclear lamina A meshwork consisting of the intermediate filament proteins, lamin A and B, and lining the inner nuclear membrane of most interphase eukaryotic cells Lamin B Lamin DiasA24 Structure of the nuclear lamina The nuclear lamina is connected to the inner nuclear membrane via: 1. interactions with lamina-associated proteins (LAPs) and NPCs 2. farnesylation (lipid anchor) Dias 25 Regulation and functions of the nuclear lamina 1. Mechanical reinforcement of the nucleus (many small unicellular eukaryotes lack a nuclear lamina) 2. Disassembly and rebuilding of the nuclear envelope prior to and after mitosis: the nuclear lamins are phosphorylated by cyclindependent kinase (Cdk1) early in mitosis, resulting in dissolution of the lamina and the nuclear envelope; lamins may also play a role in rebuilding of the nucleus after mitosis 3. The nuclear lamins interacts with the chromatin (both DNA and histones) and are thought to be involved in chromatin decondensation, DNA replication, and control of gene expression and cell proliferation 4. During apoptosis, the nuclear lamina is disassembled in the early stages by caspase-mediated proteolysis Dias 26 Laminopathies are genetic diseases resulting from mutations in lamins and LAPs Laminopathies are thought to reflect involvement of the lamins in: 1. Mechanical strength of the nuclear envelope 2. Control of cell cycle progression 3. Control of gene expression 4. DNA damage (prelamin A) Laminopathies are serious: • Premature ageing • Cardiac disease • Lipid synthesis disorders and diabetes • Muscle- and neuro-degeneration • Stem cell differentiation defects Dias 27 Nuclear pore complexes (NPCs) NPCs are huge (1,2 x 105 kDa), symmetrical macromolecular complexes, which mediate transport of ions and most molecules between nucleus and cytosol (how is the subject of the next lecture!) Den endelige NPC model, baseret på de to Nature 2007 papers Nature 450(7170): 683–694 and 695–701 Dias 28 NPC structure: cytoplasmic and nuclear views • • NPCs generally have 8-fold symmetry, and are ~120 nm in diameter The nuclear and cytoplasmic faces have ”basket” and ”fibrillar” structures, respectively Dias 29 NPC structure model: side view • • • NPCs are anchored to the nuclear envelope via integral membrane proteins The nuclear envelope is fused at NPC sites NPCs penetrate, and are attached to, the nuclear lamina Fibrils Terminal structures Nuclear basket Dias 30 The NPC is composed of multiple modules The NPC consists of multiples of 8 copies of ~30 proteins, the nucleoporins, assembled into modules In interphase, some nucleoporins are stably, and some dynamically, associated with the NPC Early in mitosis, the NPC is disassembled into submodules, which rapidly reassemble late in mitosis Dias 31 NPCs are highly conserved from yeast to vertebrates Dias 32 How was all this discovered? Atomic force microscopy (AFM) can provide information on NPC dynamics in living cells Cryo-EM has greater resolution, but is not dynamic Dias 33 How was all this discovered? In this manner, many nucleoporins were found to exhibit socalled PheGly (FG) repeats that may serve as docking sites for cargo, and coiled coil domains, thought to be important for overall NPC structure Nucleoporins can be purified, separated and quantified by SDSPAGE, and their identity determined by mass spectrometry Dias 34 And… How was all this discovered? Relative nucleoporin abundance obtained from SDS-PAGE gel quantification, combined with immuno-EM, can be used to determine NPS organization Dias 35 Evidence from multiple sources Biochemical Genetic Immunocytochemistry, Cryo-EM, AFM FRAP, etc Molecular identity, Molecular identity, (Co)-localization, protein motifs, function, evolution dynamics Structure, est. overall size, interactions some functions Current working model(s) Dias 36 Essential terms: Nucleoplasm Nuclear envelope Nuclear lamina Nuclear pore complex Nucleoporin Nucleolus Speckles Replication factories Cajal-, Gemini-, and PML bodies Heterochromatin Euchromatin Chromosome domains Interchromosome domains Dias 37