Eukaryotic Cell Physical Compartments Cell Nucleus From CellBiology Contents 1 Introduction 1.1 Lecture Audio 2 Objectives 3 Eukaryotes 4 Eukaryote Gene Expression 4.1 DNA -> mRNA -> Protein 4.2 Nucleus 4.3 Cytoplasm 5 Membrane Evolution 6 Nuclear Compartment 6.1 Nucleus Size 7 Nuclear Envelope 8 Nuclear Cytoskeleton 8.1 Nuclear Lamins 9 Nuclear Transport History 10 Nuclear Pores 11 Nuclear Bodies 11.1 Cajal Bodies 11.2 PML Bodies 12 Chromosomes 12.1 Chromosome Territories 13 Nucleolus 13.1 Appearance 13.2 Function 14 Chromosome Features 14.1 Telomere 14.2 Centromere 14.3 Replication Origins 15 Chromosome DNA Packing 15.1 Nucleosomes 15.2 Histones 16 Abnormalities 16.1 Hutchinson-Gilford Progeria Syndrome 16.2 Emery-Dreifuss Muscular Dystrophy 17 Eukaryote Gene Expression 17.1 Protein Modification 17.2 Exocytosis 18 History 19 References 19.1 Textbooks 19.2 Books 19.3 Reviews 19.4 Articles 20 Acronyms 21 Movies 22 2016 Course Content Introduction This lecture introduces the nucleus and how information is transferred from stable stored information (DNA) converted to an intermediate (mRNA, rRNA, tRNA) of variable stability, exported from the nucleus to the cytoplasm where mRNA is then translated into Protein. This is gene expression, the products of this process are used either within the cell, exported (exocytosis) or used to replace worn out components. We will study this topic looking at the key organelle in this process, the nucleus. Nucleolar Assembly Movie | Movie - nuclear pore complexes (http://jcb.rupress.org/cgi/content/full/154/6/1147/FIG6/DC1)
17 Eukaryote Gene Expression17.1 Protein Modification17.2 Exocytosis
18 History19 References
19.1 Textbooks19.2 Books19.3 Reviews19.4 Articles
20 Acronyms21 Movies22 2016 Course Content
IntroductionThis lecture introduces the nucleus and how information is transferred from stable stored information (DNA) converted to an intermediate(mRNA, rRNA, tRNA) of variable stability, exported from the nucleus to the cytoplasm where mRNA is then translated into Protein. This isgene expression, the products of this process are used either within the cell, exported (exocytosis) or used to replace worn out components.
We will study this topic looking at the key organelle in this process, the nucleus.
Nucleolar Assembly Movie | Movie - nuclear pore complexes (http://jcb.rupress.org/cgi/content/full/154/6/1147/FIG6/DC1)
MH - note that content listed below will not match exactly current lecture structure but has been selected as having similar content.
ObjectivesUnderstand the concept of the cell nucleusUnderstand the overall structure and components within the nucleusUnderstand the functions of the nucleusBrief understanding of chromosomal structure
EukaryotesDifference between Prokaryotes and Eukaryotes
DNA (transcription) -> mRNA (translation) -> Protein (function)
Nucleus
DNA (transcription) -> mRNA Nuclear processing (export)
DNA -> mRNA splicing (introns removed, exons joined) -> mRNA
Cytoplasm
mRNA (translation by ribosomes) -> Protein (processing)
Protein Processing cytoplasm (free ribosomes), rough endoplasmic reticulum(bound ribosomes)
Some proteins are returned to the nucleus by a Nuclear Localization Sequence (NLS)
SV40 Large T-antigen (http://www.plosone.org/article/fetchObject.action?uri=info:doi/10.1371/journal.pone.0010475.g001&representation=PNG_M) - PKKKRKV (single part)nucleoplasmin - KR[PAATKKAGQA]KKKK (two part) two clusters of basic amino acids separated by 10 amino acids.
Membrane EvolutionPostulated that an early "coating" structure lead to the infolding of the primitive plasma membrane to form the many membrane coveredorganelles in the cytoplasm.
These modules may also be the evolutionary precursor to the nuclear pore structures and account for the double membrane that coats thenucleus.
Splicing speckles or SC 35 domainsthought to be sites of storage of mRNA splicing factors
nuclear bodies - Cajal and PML
Links: MBOC - A cross-sectional view of a typical cell nucleus (http://www.ncbi.nlm.nih.gov/books/NBK26821/?rendertype=figure&id=A606)
Nucleus Size
cell "karyoplasmic ratio" relatively constant (ratio of nuclear volume to cell volume)most other cellular organelles (ER and mitochondria) can vary greatly inamounts
multinucleated fission yeast cellsrelative amount of cytoplasm surrounding each nucleus controls the size ofindividual nuclei
Links: Nuclear size control in fission yeast. Neumann FR, Nurse P. J Cell Biol. 2007 Nov19;179(4):593-600. PMID: 17998401 (http://www.ncbi.nlm.nih.gov/pubmed/17998401)
Nuclear EnvelopeForms structural compartmentNuclear envelope two concentric membranesBreaks down each mitosis (recycled)Outer membrane continuous with Endoplasmic Reticulum (Endoplasmic Reticulumis covered in Lecture 5)Contains holes “nuclear pores”
The Cell - Figure 8.1. The nuclear envelope (http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=nuclear%20envelope&rid=cooper.figgrp.1325)
Nuclear Cytoskeleton
The nuclear cytoskeleton has 2 layers
outer - less organised surrounds nuclear envelopeinner - nuclear lamina - thin shell (20 nm) underlying the nuclear envelope
associates with both the inner nuclear membrane and underlying chromatincan regulate gene expressionprovides anchor sites for nuclear pore complexesbroken down each cell division
Nuclear Envelope showing underlying nuclearmatrix (green) (EM Image Martin Goldberg)
Muscle satellite celllamin A/C expression
heterochromatin (green)LaminA/C (red)[1]
Links: MBOC - A cross-sectional view of a typical cell nucleus (http://www.ncbi.nlm.nih.gov/books/NBK26821/?rendertype=figure&id=A606)
Nuclear Lamins
Intermediate filaments covered in Cytoskeleton Lecture – Intermediate Filaments
Lamins - Class V intermediate filamentsVertebrate lamins are classified into 2 types - A and B10 nm in diameter, forms rope-like networkspolypeptide form dimers - central alpha-helical regions of two polypeptide chainsare wound around each otherassembly - head-to-tail association of dimers form linear polymers, side-by-sideassociation of polymers form filamentsB-type - B1 and B2 (586 aa protein, Mr 66,334 Da) lamins are ubiquitouslyexpressed throughout developmentA-type - lamins A and C (Mr 74 kD and 65 kD) lamins in many organismsexpression does not appear until midway through embryogenesis (possible role indifferentiation)lamins phosphorylation state affects nuclear envelope assembly state(dephosphorylation nuclear envelope assembly, phosphorylation nuclear envelopedisassembly)Lamins also link DNA to nuclear envelope (Lamin B1 interacts with chromatin)
Nuclear lamina and lamina-interactingproteins
Nucleoskeleton to the cytoskeletoncomplexes
Lamin Abnormalities - (laminopathies) mutations in lamins can lead to human disease (Hutchinson-Gilford Progeria Syndrome)
Links: MBOC - A cross-sectional view of a typical cell nucleus (http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=mboc4.figgrp.606) |Nature Medicine - Image - The nuclear membranes include the interconnected but distinct inner and outer nuclear membranes and the nuclearpore membrane (http://www.nature.com/nm/journal/v6/n2/fig_tab/nm0200_136_F1.html) MBOC - The breakdown and re-formation of thenuclear envelope during mitosis (http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=mboc4.figgrp.2174) | The Cell - Intermediate FilamentProteins (http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=cooper.table.1810) | The Cell - Model of lamin assembly(http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=cooper.figgrp.1328) | Abcam - Lamin B1 antibody - Nuclear Envelope Marker
1999 Nobel Prize Medicine - Günter Blobel for the discovery that "proteins have intrinsic signals that govern their transport and localizationin the cell"
Links: 1999 Nobel Prize Medicine - Günter Blobel (http://nobelprize.org/nobel_prizes/medicine/laureates/1999/press.html)
Protein complexExternal diameter of about 120 nm (30 times the size of a ribosome)Channel diameter 25 nmchannels between nucleus and cytoplasm (import/export)passive passage of small polar molecules, ions, (<40-60 kDa)active (selective/ regulated) passage of larger macromolecules, proteins and RNAsimportin - cytosolic protein
Links: JCB Movie - Nuclear Pore Complex movement in interphase(http://jcb.rupress.org/content/suppl/2001/07/11/200101089.F4.DC1)
Nuclear BodiesFunctional compartments
Nucleus Movie 1
Cajal Bodies
also called - nucleolar accessory bodies, coiled body, gems0.1 - 2.0 microns, 1-10/ nucleusGems and Cajal bodies two forms of same structureGEMS (Gemini of coiled bodies)
proposed sites where small nuclear ribonucleoproteins (snRNPs) and small nucleolar RNAs (snoRNPs) are modified.snRNPs are particles that combine with pre-mRNA and various proteins to form spliceosomessnoRNAs are a class of small RNA molecules that are involved with modifications of ribosomal RNAs (rRNAs) and other RNAgenes
Cajal bodies were first reported in 1903 by the Spanish cytologist/histologist Ramón y Cajal, who christened them "nucleolar accessorybodies".
Cajal, S.R. 1903. Un sencillo método de coloración selectiva del retículo protoplásmico y sus efectos en los diversos órganos nerviosos devertebrados e invertebrados. Tra. Lab. Invest. Biol. 2:129–221.
See also Nature Reviews - The centennial of the Cajal body
PML Bodies
promyelocytic leukaemia nuclear bodiesalso called PODs, ND10 or Kremer bodiesFunction unknownregulation of diverse cellular functions?viral infection, cellular transformation, innate immunity, growth control, apoptosis
Chromosomesnot “visible” at interphase, condense for mitosis (1,000 fold)
condensation allows chromosomes to move along mitotic spindle without breaking or tanglingeukaryotes have separate chromosomes
Human 23 pairs, 22 autosome pairs, 2 sex chromosomesdiploid 2 copies of each chromosome (inherited one male/one female)
except male sex chromosomes X from mother Y from fatherDNA and proteinpacking of DNADNA structureencodes genome (humans 30,000 genes, draft sequence published in 2001)DNA genes encode RNA and proteinsDNA can also encodes nothing
Space within the nucleus occupied by individual chromosomesSeveral different models as to how these territories interact
Intrachromosomal domains possibly RNA processing and transport
Links: MBOC - Selective painting of two interphase chromosomes in a human peripheral lymphocyte(http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=mboc4.figgrp.686) | The Cell - Chromosome Territories(http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=cooper.figgrp.1352)
Nucleolus
Appearance
Fibrillar center, dense fibrillar component, and granular componentNucleolus changes during the cell cycle:
during mitosis - nucleolus breaks up as chromosomes condenseafter mitosis - nucleolus reforms from coalesce of tips of 10 chromosomes
Function
Sites of ribosomal (rRNA) gene transcription, processing, and ribosome assemblyNucleolus size depends on cell metabolic activitySites of ribosomal (rRNA) gene transcription, processing, and ribosome assemblyAll cells contain multiple copies of rRNA genes
Links: Movie - Dynamics of nucleolus-derived foci throughout the cytoplasm and the disappearance of NDF during telophase(http://jcb.rupress.org/content/suppl/2000/08/03/150.3.433.F2.DC1) | Movie - Dynamics of nucleolar reassembly in telophase wereanalyzed by the visualization of fibrillarin-GFP (http://jcb.rupress.org/content/suppl/2000/08/03/150.3.433.F3.DC1)
Chromosome Features
2 telomeres, centromere, replication originsTelomere- at ends of chromosome (bacterial DNA circular)Centromere- holds duplicated DNA togetherKinetochore - protein complex forms around the centromere forms during mitosisChromatin - DNA packed by DNA binding proteins (histones and non-histones) form 30nm DNA fibre2 types of chromatin in interphase nuclei (based on cytology)
at ends of all chromosomes (not bacterial DNA circular)roles in chromosome replication and maintenancereplication
for replicating the ends of linear chromosomesmaintenance
proposed to provide each cell with a replication counting mechanism that helps prevent unlimited proliferationeach cell division shortens telomere 50–100 nucleotidesDNA 100s to 1,000s repeats of a simple-sequence containing clusters of G residues (humans AGGGTT)Telomerase enzyme maintains length
Centromere
directs movement of each chromosome into daughter cells every time a cell dividescentromere embedded in heterochromatinsatellite DNA sequences (AT-rich) repeated many thousands of timesproteins assemble on this to form Kinetochore
DNA replication initiates at multiple origins (ori)in both prokaryotic and eukaryotic DNAmultiple origins in eukaryotes (human genome about 30,000 origins)each origin produces two replication forks (moving in opposite directions)
Chromosome DNA Packing
Euchromatin ("good chromatin") - light, transcriptionally active, about 10% of allchromatin.
Heterochromatin - condensed, transcriptionally inactive, about 90% of all chromatin.
Nucleosomes
formed by DNA wrapped around histonesunit particle of chromatin (nucleosomal histones) (discovered 1974)EM unfolded DNA has "beads on a string" appearancesecond order folding forms 300 nm fibrecondensed DNA for mitosis 700 nm fibre
Histones
only in eukaryotessmall proteins positively charged (binds negatively charged DNA)not sequence specific binding (as in transcription factors)4 core histones (H2A, H2B, H3, and H4)2 linker histones (H1/H5)
Abnormalities
Hutchinson-Gilford Progeria Syndrome
In more than 80% of cases the gene defect responsible for HGPS is a single spontaneous mutation in codon 608 of the LMNA gene,which encodes both lamin A and lamin C
single-base substitution in exon 11 that reveals a cryptic splice site in the LMNAgene thus producing a truncated proteinprogerin is a mutant form of the nuclear architectural protein lamin A
Loss of a-Type Lamin Expression Compromises Nuclear Envelope Integrity Leading toMuscular Dystrophy (http://jcb.rupress.org/cgi/content/abstract/147/5/913)
Eukaryote Gene ExpressionDNA -> mRNA -> Protein
DNA (transcription) -> mRNA (translation) -> Protein (function)
DNA -> mRNA splicing (introns removed, exons joined) -> mRNA
DNA -> rRNA, tRNA, siRNA (RNA interference (RNAi) pathway
Nucleus DNA (transcription) -> mRNA Nuclear processing (export) Cytoplasm mRNA(translation) -> Protein (cytoplasm, rough endoplasmic reticulum)
Protein Modification
Protein - cytoplasmic (free ribosomes), rough endoplasmic reticulum (bound ribosomes)
HistoryBelow are some example historical research finding related to cell junctions from the JCBArchive (http://jcb.rupress.org/misc/fromthearchive.shtml) and other sources.
1961 The nucleolar origin of rRNA (http://jcb.rupress.org/cgi/content/full/168/4/524-a)Base compositions and half-lives suggest to Jan-Erik Edström that the nucleolus is thesource of rRNA.
References1. Neveen A Hosny, Mingying Song, John T Connelly, Simon Ameer-Beg, Martin M
Knight, Ann P Wheeler Super-resolution imaging strategies for cell biologistsusing a spinning disk microscope. PLoS ONE: 2013, 8(10);e74604 PubMed24130668 | PLoS One.(http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074604)
2. Alexander von Appen, Martin Beck Structure Determination of the Nuclear PoreComplex with Three-Dimensional Cryo electron Microscopy. J. Mol. Biol.:2016; PubMed 26791760
Textbooks
Molecular Biology of the Cell (4th ed.) - Part 1 Chapter 8 - The Cell Nucleus -Three views of a cell membrane (http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=mboc4.figgrp.1862)Molecular Cell Biology - The Dynamic Cell(http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=mcb.chapter.145)The Cell- A Molecular Approach - An Overview of Cells and Cell Research (http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=cooper.chapter.89)
"cell nucleus" Molecular Biology of the Cell (http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=Books&cmd=search&doptcmdl=DocSum&term=cell+nucleus+AND+mboc4%5Bbook%5D) | Molecular Cell Biology(http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=Books&cmd=search&doptcmdl=DocSum&term=cell+nucleus+AND+mcb%5Bbook%5D) | The Cell- A molecular Approach(http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=Books&cmd=search&doptcmdl=DocSum&term=cell+nucleus+AND+cooper%5Bbook%5D)"nuclear envelope" Molecular Biology of the Cell (http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=Books&cmd=search&doptcmdl=DocSum&term=nuclear+envelope+AND+mboc4%5Bbook%5D) | Molecular Cell Biology(http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=Books&cmd=search&doptcmdl=DocSum&term=nuclear+envelope+AND+mcb%5Bbook%5D) | The Cell- A molecular Approach(http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=Books&cmd=search&doptcmdl=DocSum&term=nuclear+envelope+AND+cooper%5Bbook%5D)"nuclear pore" Molecular Biology of the Cell (http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=Books&cmd=search&doptcmdl=DocSum&term=nuclear+pore+AND+mboc4%5Bbook%5D) | Molecular Cell Biology(http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=Books&cmd=search&doptcmdl=DocSum&term=nuclear+pore+AND+mcb%5Bbook%5D) | The Cell- A molecular Approach(http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=Books&cmd=search&doptcmdl=DocSum&term=nuclear+pore+AND+cooper%5Bbook%5D)
Books
The Cytoskeleton - cellular architecture and choreography (http://books.google.com/books?id=JNHuxHzTm7IC)
Reviews
Selma Osmanagic-Myers, Thomas Dechat, Roland Foisner Lamins at the crossroads of mechanosignaling. Genes Dev.: 2015, 29(3);225-37PubMed 25644599
Katherine L Wilson, Scott C Dawson Evolution: functional evolution of nuclear structure. J. Cell Biol.: 2011, 195(2);171-81 PubMed22006947
Michael D Huber, Larry Gerace The size-wise nucleus: nuclear volume control in eukaryotes. J. Cell Biol.: 2007, 179(4);583-4 PubMed17998404
Roderick Y H Lim, Ueli Aebi, Birthe Fahrenkrog Towards reconciling structure and function in the nuclear pore complex. Histochem.Cell Biol.: 2008, 129(2);105-16 PubMed 18228033
Articles
Wenjing Li, Brian J Ferguson, Walid T Khaled, Maxine Tevendale, John Stingl, Valeria Poli, Tina Rich, Paolo Salomoni, Christine J WatsonPML depletion disrupts normal mammary gland development and skews the composition of the mammary luminal cell progenitorpool. Proc. Natl. Acad. Sci. U.S.A.: 2009, 106(12);4725-30 PubMed 19261859
J Ellenberg, E D Siggia, J E Moreira, C L Smith, J F Presley, H J Worman, J Lippincott-Schwartz Nuclear membrane dynamics andreassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. J. Cell Biol.: 1997, 138(6);1193-206 PubMed 9298976
L Yang, T Guan, L Gerace Integral membrane proteins of the nuclear envelope are dispersed throughout the endoplasmic reticulumduring mitosis. J. Cell Biol.: 1997, 137(6);1199-210 PubMed 9182656
AcronymsAA - amino acidDNA - deoxyribonucleic acidEM - electron microscopyFL - flourescentGEMS - Gemini of coiled bodiesINM - inner nuclear membraneKASH - Klarsicht/ANC-1/Syne-1 homology domain–containing proteinLEM domain - fold identified in LAP2, emerin, and MAN1 confers direct binding to dsDNALINC - nucleoskeleton to the cytoskeleton complexesmRNA - messenger RNANE - nuclear envelope
MoviesNuclear pore complexes are fixed in place Daigle et al. (http://jcb.rupress.org/cgi/content/abstract/154/1/71) report that nuclear porecomplexes (NPCs) undergo limited movements (http://jcb.rupress.org/cgi/content/full/200101089/F4/DC1) that match the deformationsof the nuclear envelope as tracked using a grid (http://jcb.rupress.org/cgi/content/full/200101089/F4/DC2) of bleached nuclear lamins.NPCs are therefore remarkably stable complexes, and are probably anchored to a protein network in the nuclear envelope.
Nucleoporins reassemble around post-mitotic chromatin A conserved nuclear pore subcomplex was characterized and tracked byBelgareh et al. (http://jcb.rupress.org/cgi/content/abstract/154/6/1147), who found that the proteins were recruited(http://jcb.rupress.org/cgi/content/full/154/6/1147/FIG6/DC1) during telophase in a rim pattern surrounding the chromosomes. A lowlevel of staining was also apparent on the kinetochores throughout mitosis.
Nucleolar re-formation after mitosis Savino et al. (http://jcb.rupress.org/cgi/content/abstract/153/5/1097) follow the re-formation(http://jcb.rupress.org/cgi/content/full/153/5/1097/F3/DC1) of nucleoli after mitosis. Prenucleolar bodies (PNB) form on thechromosome surface and nucleolar material flows along links between PNBs(http://jcb.rupress.org/cgi/content/full/153/5/1097/F4/DC2) and towards (http://jcb.rupress.org/cgi/content/full/153/5/1097/F6/DC1) adeveloping nucleolar organizer region (http://jcb.rupress.org/cgi/content/full/153/5/1097/F4/DC1) (NOR). Eventually this leads to thefusion (http://jcb.rupress.org/cgi/content/full/153/5/1097/F9/DC1) of nucleoli to form a single entity.
Processing complexes may help reassemble nucleoli Nucleolar reassembly (http://jcb.rupress.org/cgi/content/full/150/3/433/F3/DC2)during telophase is shown by Dundr et al. (http://jcb.rupress.org/cgi/content/abstract/150/3/433) to require mitotically preservedprocessing complexes.
Speckles - A splicing factor has limited mobility Based on the limited mobility(http://jcb.rupress.org/cgi/content/full/150/1/41/F1/DC1) of a splicing factor, Kruhlak et al.(http://jcb.rupress.org/cgi/content/abstract/150/1/41) determine that the factor undergoes frequent but transient interactions withrelatively immobile nuclear binding sites, both when associated with speckles and when dispersed in the nucleoplasm. This a 3-D videothat should be viewed using red/green 3-D glasses.
