2009 Lecture 4

From CellBiology
Eukaryotic Cell Physical Compartments

Cell Nucleus

Introduction

This lecture introduces 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 at the level of the cellular components and organelles involved in the process: ribosomes, endoplasmic reticulum, Golgi apparatus, vesicles (transport and secretory).

Dr Mark Hill (2009)

Lecture Audio

The University has a system for automated recording of lectures called Lectopia. Lectopia requires login using your student number and unipass. I will be adding the link to each iLecture Audio following the Lecture. Due to the automated recording method, most lectures begin 4-5 minutes into MP3 recordings and occasionally stop before the end of the lecture. 2009 All Audio Files

Lecture 4: Cell Nucleus Lecture Date: 18-03-2009 Lecture Time: 10:00 Venue: BioMed E Speaker: Mark Hill

Archive

MH - note that content listed below will not match exactly current lecture structure but has been selected as having similar content.

  • 2008 Online Lecture 2008 Online content
  • 2007 This lecture was previously was part of a Laboratory.

Objectives

  • Understand the concept of the cell nucleus
  • Understand the overall structure and components within the nucleus
  • Understand the functions of the nucleus
  • Brief understanding of chromosomal structure


Eukaryotes

Difference between Prokaryotes and Eukaryotes

  • Cytoskeleton
  • DNA structure
    • circular, linear
    • Packing (histones)
    • RNA processing (splicing)

Eukaryote Gene Expression

rough endoplasmic reticulum (tem)

DNA -> mRNA -> Protein

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)

Membrane Evolution

Nucleus Membrane Evolution

Postulated that an early "coating" structure lead to the infolding of the primitive plasma membrane to form the many membrane covered organelles in the cytoplasm.

These modules may also be the evolutionary precursor to the nuclear pore structures and account for the double membrane that coats the nucleus.

Nuclear Compartment

  • Nuclear envelope
  • Nuclear cytoskeleton
  • Nucleolus
  • Chromosome territories
  • Interchromatin compartment
  • “speckles” interchromatin granule clusters
    • Splicing speckles or SC 35 domains
    • thought to be sites of storage of mRNA splicing factors
  • nuclear bodies - Cajal and PML

Nucleus Size

  • cell "karyoplasmic ratio" relatively constant (ratio of nuclear volume to cell volume)
    • most other cellular organelles (ER and mitochondria) can vary greatly in amounts
  • multinucleated fission yeast cells
    • relative amount of cytoplasm surrounding each nucleus controls the size of individual nuclei
Links: Nuclear size control in fission yeast. Neumann FR, Nurse P. J Cell Biol. 2007 Nov 19;179(4):593-600. PMID: 17998401

Nuclear Envelope

Nuclear architecture
  • Forms structural compartment
  • Nuclear envelope two concentric membranes
  • Breaks down each mitosis (recycled)
  • Outer membrane continuous with Endoplasmic Reticulum (Endoplasmic Reticulum is covered in Lecture 5)
  • Contains holes “nuclear pores”

Em nuclear envelope2.jpg

The Cell - Figure 8.1. The nuclear envelope

Nuclear Cytoskeleton

  • The nuclear cytoskeleton has 2 layers MBOC - A cross-sectional view of a typical cell nucleus
    • outer - less organised surrounds membrane
    • inner - nuclear lamina - thin shell (20 nm) underlying the membrane (nuclear envelope)
    • associates with both the inner nuclear membrane and underlying chromatin
    • provides anchor sites for nuclear pore complexes
  • broken down each cell division

Nuclear Lamins

  • intermediate filaments
  • large family of different filament types (covered in Cytoskeleton Lecture – intermediate filaments)
  • 10 nm in diameter, forms rope-like networks
  • lamins, Class V intermediate filaments
    • polypeptide form dimers
    • central alpha-helical regions of two polypeptide chains are wound around each other
  • assembly
    • head-to-tail association of dimers form linear polymers
    • side-by-side association of polymers form filaments

Nuclear Envelope and Nuclear Cytoskeleton

Em nuclear envelope2.jpg

Nuclear Envelope showing underlying nuclear matrix (green)

(EM Image Martin Goldberg)

  • Vertebrate lamins are classified into 2 types - A and B
  • Mammalian somatic cells show 2 species of each type
    • A type - lamins A and C (Mr 74 kD and 65 kD)
    • B type - B1 and B2 (586 aa protein, Mr 66,334 Da)
  • lamins phosphorylation state affects nuclear envelope assembly state
    • phosphorylation nuclear envelope disassembly
    • dephosphorylation nuclear envelope assembly
  • Mutations in lamins can lead to human disease
    • More? Hutchinson-Gilford Progeria Syndrome
  • Lamins also link DNA to nuclear envelope
    • Outer laminar surface is connected to inner nuclear membrane
    • inner laminar surface attached to the chromatin

Links: MBOC - A cross-sectional view of a typical cell nucleus | Nature Medicine - Image - The nuclear membranes include the interconnected but distinct inner and outer nuclear membranes and the nuclear pore membrane MBOC - The breakdown and re-formation of the nuclear envelope during mitosis | The Cell - Intermediate Filament Proteins | The Cell - Model of lamin assembly | Abcam - Lamin B1 antibody - Nuclear Envelope Marker | OMIM - Lamin A/C | OMIM - Lamin B1 |

Nuclear Transport History

1999 Nobel Prize Medicine - Günter Blobel for the discovery that "proteins have intrinsic signals that govern their transport and localization in the cell"

Proteintransport.gif



Links: 1999 Nobel Prize Medicine - Günter Blobel

Nuclear Pores

Em nuclear envelope4.jpg


Nuclear pore cytoplasmic2.jpg NuclearPore1.jpg

  • Protein complex
  • External diameter of about 120 nm (30 times the size of a ribosome)
  • Channel diameter 25 nm
  • channels between nucleus and cytoplasm (import/export)
  • passive passage of small polar molecules, ions,
  • active (selective/ regulated) passage of macromolecules, proteins and RNAs

JCB Movie: Nuclear Pore Complex movement in interphase (9.6 MB)

Nuclear Bodies

  • functional compartments

Cajal Bodies

Cajal bodies.gif

  • also called - nucleolar accessory bodies, coiled body, gems
  • 0.1 - 2.0 microns, 1-10/ nucleus
  • Gems and Cajal bodies two forms of same structure
  • GEMS (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 spliceosomes
    • snoRNAs are a class of small RNA molecules that are involved with modifications of ribosomal RNAs (rRNAs) and other RNA genes


Cajal bodies were first reported in 1903 by the Spanish cytologist/histologist Ramón y Cajal, who christened them "nucleolar accessory bodies".

Cajal, S.R. 1903. Un sencillo método de coloración selectiva del retículo protoplásmico y sus efectos en los diversos órganos nerviosos de vertebrados e invertebrados. Tra. Lab. Invest. Biol. 2:129–221.

See also Nature Reviews - The centennial of the Cajal body

PML Bodies

PMLbodies.jpg

  • promyelocytic leukaemia nuclear bodies
  • also called PODs, ND10 or Kremer bodies
  • Function unknown
  • regulation of diverse cellular functions?
  • viral infection, cellular transformation, innate immunity, growth control, apoptosis
  • dynamic hubs sensing stress and DNA damage

Chromosomes

  • not “visible” at interphase, condense for mitosis (1,000 fold)
    • condensation allows chromosomes to move along mitotic spindle without breaking or tangling
  • eukaryotes have separate chromosomes
    • Human 23 pairs, 22 autosome pairs, 2 sex chromosomes
  • diploid 2 copies of each chromosome (inherited one male/one female)
    • except male sex chromosomes X from mother Y from father
  • DNA and protein
  • packing of DNA
  • DNA structure
  • encodes genome (humans 30,000 genes, draft sequence published in 2001)
  • DNA genes encode RNA and proteins
  • DNA can also encodes nothing

Chromosome Territories

Nucleus cartoon3.jpg Human Chromosome Territories G0 Fibroblast Nucleus Nucleus territory1.jpg

  • Space within the nucleus occupied by individual chromosomes
    • Several 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 | The Cell - Chromosome Territories

Nucleolus

  • Appearance
    • Fibrillar center, dense fibrillar component, and granular component
    • Nucleolus changes during the cell cycle:
      • during mitosis - nucleolus breaks up as chromosomes condense
      • after mitosis - nucleolus reforms from coalesce of tips of 10 chromosomes
  • Function
    • Sites of ribosomal (rRNA) gene transcription, processing, and ribosome assembly
    • Nucleolus size depends on cell metabolic activity
    • Sites of ribosomal (rRNA) gene transcription, processing, and ribosome assembly
    • All cells contain multiple copies of rRNA genes

Chromosome Features

HumanchromosomeXX1.jpg

  • 2 telomeres, centromere, replication origins
  • Telomere- at ends of chromosome (bacterial DNA circular)
  • Centromere- holds duplicated DNA together
  • Kinetochore - protein complex forms around the centromere forms during mitosis
  • Chromatin - DNA packed by DNA binding proteins (histones and non-histones) form 30nm DNA fibre
  • 2 types of chromatin in interphase nuclei (based on cytology)
    • heterochromatin - highly condensed (restricted gene transcription)
    • euchromatin - less condensed (gene transcription)


Telomere

Chromosome telomeres.gif

  • at ends of all chromosomes (not bacterial DNA circular)
  • roles in chromosome replication and maintenance
  • replication
    • for replicating the ends of linear chromosomes
  • maintenance
    • proposed to provide each cell with a replication counting mechanism that helps prevent unlimited proliferation
  • each cell division shortens telomere 50–100 nucleotides
  • DNA 100s to 1,000s repeats of a simple-sequence containing clusters of G residues (humans AGGGTT)
  • Telomerase enzyme maintains length

Centromere

Centromeres2.jpg

  • directs movement of each chromosome into daughter cells every time a cell divides
  • centromere embedded in heterochromatin
  • satellite DNA sequences (AT-rich) repeated many thousands of times
  • proteins assemble on this to form Kinetochore
    • attachment site for spindle microtubules

Links: MBOC - Centromere

Replication Origins

DNA replication fork.png

  • DNA replication initiates at multiple origins (ori)
  • in both prokaryotic and eukaryotic DNA
  • multiple origins in eukaryotes (human genome about 30,000 origins)
  • each origin produces two replication forks (moving in opposite directions)

Chromosome DNA Packing

Chromatin Structures


Nucleosomes

Nucleosome
  • formed by DNA wrapped around histones
  • unit particle of chromatin (nucleosomal histones) (discovered 1974)
  • EM unfolded DNA has "beads on a string" appearance
  • second order folding forms 300 nm fibre
  • condensed DNA for mitosis 700 nm fibre

Histones

  • only in eukaryotes
  • small 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.jpg

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
  • progerin is a mutant form of the nuclear architectural protein lamin A


Eukaryote Gene Expression

DNA -> 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)

Exocytosis

Rough Endoplasmic Reticulum -> transport vesicle -> Golgi apparatus -> secretory vesicle

History

Below are some example historical research finding related to cell junctions from the JCB Archive and other sources.

1961 The nucleolar origin of rRNA Base compositions and half-lives suggest to Jan-Erik Edström that the nucleolus is the source of rRNA.

References

Textbooks


Search Online Textbooks

Books

Reviews

  • Scaffidi P, Misteli T. Lamin A-dependent misregulation of adult stem cells associated with accelerated ageing. Nat Cell Biol. 2008 Mar 2; [Epub ahead of print]
  • The size-wise nucleus: nuclear volume control in eukaryotes. Huber MD, Gerace L. J Cell Biol. 2007 Nov 19;179(4):583-4. Epub 2007 Nov 12. PMID: 17998404
  • Lanctôt C, Cheutin T, Cremer M, Cavalli G, Cremer T. Lanctôt C, Cheutin T, Cremer M, Cavalli G, Cremer T. Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions. Nat Rev Genet. 2007 Feb;8(2):104-15. Review.
  • Gorski SA, Dundr M, Misteli T. The road much traveled: trafficking in the cell nucleus. Curr Opin Cell Biol. 2006 Jun;18(3):284-90.
  • Towards reconciling structure and function in the nuclear pore complex. Lim RY, Aebi U, Fahrenkrog B. Histochem Cell Biol. 2008 Feb;129(2):105-16. Epub 2008 Jan 29. Review. PMID: 18228033

Articles

  • Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. Ellenberg J, Siggia ED, Moreira JE, Smith CL, Presley JF, Worman HJ, Lippincott-Schwartz J. J Cell Biol. 1997 Sep 22;138(6):1193-206. PMID: 9298976
  • Integral membrane proteins of the nuclear envelope are dispersed throughout the endoplasmic reticulum during mitosis. Yang L, Guan T, Gerace L. J Cell Biol. 1997 Jun 16;137(6):1199-210. PMID: 9182656
  • PML depletion disrupts normal mammary gland development and skews the composition of the mammary luminal cell progenitor pool. Li W, Ferguson BJ, Khaled WT, Tevendale M, Stingl J, Poli V, Rich T, Salomoni P, Watson CJ. Proc Natl Acad Sci U S A. 2009 Mar 4. Epub ahead of print PMID: 19261859

Acronyms

  • AA - amino acid
  • DNA - deoxyribonucleic acid
  • EM - electron microscopy
  • FL - flourescent
  • GEMS - Gemini of coiled bodies
  • mRNA - messenger RNA
  • OMIM - Online Mendelian Inheritance in Man Database
  • PML Bodies - promyelocytic leukaemia nuclear bodies
  • RER - rough endoplasmic reticulum
  • RNA - ribonucleic acid
  • rRNA - ribosomal RNA
  • SER - smooth endoplasmic reticulum
  • SUMO - small ubiquitin-related modifier
  • tRNA - transfer RNA

2009 Course Content

Lectures

Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton 1 Intermediate Filaments | Cytoskeleton 2 Microtubules | Cytoskeleton 3 Microfilaments | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells | Stem Cells | Development | Revision

Laboratories

Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Tissue Culture 1 | Tissue Culture 2 | Microarray Lab visit

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G