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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
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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.
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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)
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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
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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
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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
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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
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NPCs are highly conserved
from yeast to vertebrates
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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