Transcript Lecture 2 Date: 11/17/03 - Department of Biological Sciences

Lecture 2
Nuclear Envelope, NPC,
Nucleolus & Nuclear Matrix
Endoplasmic Reticulum (ER) Properties of
the Nuclear Envelope (NE)
• Nuclear envelope appears as flattened ER cisternae surrounding the
nucleus.
• It is composed of two layers: outer and inner nuclear membrane which are
connected together by the nuclear pore complex (NPC).
• ER properties of the NE: (a) Overall Morphology; (b) Presence of ribosomes
on outer nuclear membrane; (c) Connections with rough ER; (d) Similar
composition of phospholipids and other membrane bound enzymes
• NE is specialized in terms of the nuclear pore complex and nuclear lamina.
Biochemistry of Isolated Nuclear
Membranes: Resembles ER
Phospholipid composition
phosphatidyl choline > phosphatidyl ethanolamine
(PC) > (PE)
Membrane bound enzymes
Glucose-6-phosphatase
Electron transport systems
Drug detoxification ---------
NADPH- cytochrome P- 450 system
Fatty acid metabolism ------ NADH cytochrome b5 reductase / fatty acid
desaturase
Assembly and Disassembly of Nuclear Envelope
• Nuclear envelope (NE) is a cell cycle
dependent structure that disperses at the
onset of mitosis (late prophase) and
reassembles around the reforming nucleus
in the late telophase.
• Inhibition of protein synthesis by
cycloheximide in late G2 phase has no
apparent affect on nuclear assembly in
telophase indicating that no new protein
synthesis is required for reassembly of the
nuclear envelope.
• This reassembly involves ~ 10,000 nuclear
pores in a matter of minutes.
• The correlations of breakdown of the
nuclear envelope, chromosome formation
mitosis & NE reassembly after mitosis are
essential for cell division and the ability of
cells to divide in an orderly manner.
Assembly and Disassembly of Nuclear Envelope
contd… (Role of the Nuclear Lamina)
• The proteins that compose the
nuclear lamina (lamins A, B,C)
are involved in the
disassembly/reassembly of the
nuclear envelope during cell
cycle via phosphorylation
(P)/dephosphorylation (deP).
• Yeast genetic studies have
identified cdc2 as an essential
gene for cell division in yeast.
This is a cyclin dependant
protein kinase called cyclin Bcdc2 (cdk1) kinase. Cyclins are
regulatory proteins that mediate
the enzymatic activity of protein
kinases that plays a major role in
the regulation of the cell cycle.
• Lamin phosphorylation/
dephosphorylation during cell
cycle by cdk1 kinase/cdc14 .
late prophase
early telophase
(cdc14)
Phosphorylation (P)/De(P) of the Nuclear Lamins
Correlates with Nuclear Envelope
Assembly/Disassembly
2-D Gel Shift – Phosphorylation
of the nuclear lamin proteins in
late prophase correlates with the
disassembly of the nuclear
envelope and dephosphorylation
of the lamins correlates with
nuclear envelope reassembly.
This is indicated by the increased
phosphorylation during prophase
and the dephosphorylation
during telophase of the nuclear
lamins in a 2 D-gel shift AP
experiment (AP = alkaline
phosphatase, acidic is left; basic
is right).
Experimental Basis for the Role of Nuclear Lamin
Phosphorylation in Nuclear Envelope Disassembly
[Heald & McKeon, Cell 61 (1990) 579-589]
DNA transfection experiments – in which human lamin A gene
mutated at two sites ( S-22 and S-392 which are the phosphorylation sites
for cdc2 kinase) to alanine or isoleucine (cannot be phosphorylated) are
then transfected into mammalian cells. Results show that mitosis proceeds
up to a point with no breakdown of nuclear envelope. Therefore
phosphorylation of S-22 and S-392 by cdc2 kinase is essential for nuclear
envelope breakdown.
Normal lamin A gene
Mutant lamin A gene
Interphase
Prophase
Metaphase
Anti-lamin A DNA (DAPI)
Anti-lamin A DNA (DAPI)
Experimental Basis for the Role of Nuclear Lamin
Dephosphorylation in Nuclear Envelope Assembly
[Burke & Gerace, Cell 44 (1986) 639-652]
Mitotic CHO cells
Disrupt mitotic extract
Assembly of nuclear
envelope in mitotic
extracts- In mitotic cells
incubated in vitro can follow
the assembly of nuclear
envelope around
chromosomes in association
with dephosphorylation of
nuclear lamins as observed
by shifts in the PI of the
lamin proteins on 2-D gels.
If dephosphorylation of
nuclear lamins is inhibited
there is a corresponding
inhibition of nuclear
envelope assembly.
Incubate at 330C and measure nuclear envelope
assembly around the chromosomes and
dephosphorylation of lamins by 2-D gel shift
2-D gel shift
Nuclear Pore Complex (NPC)
• Nuclear pore complex connects the outer nuclear
membrane to the inner membrane which allows for
the nucleocytoplasmic transport of materials
(mRNA’s, tRNA’s, proteins etc) .
• 3-D microscopy indicates an asymmetrical
organization of the pore complex: “nuclear basket”
Field emission scanning EM micrographs of NPC
Cytoplamic
face
Nuclear face
• ~100 different nuclear pore proteins (NUP’s/
(nucleoporins); total mass >10,000 kDa.
• Positioning on NPC: (a) Cytoplasmic face : NUP’s
180 & 124; (b) Nuclear face : NUP 153; (c) Both
faces: NUP’s 62 & 155
• Sequence of the NUP’s (a) pentameric degenerative
repeats: (XFXFG, docking sequence for importin
beta) : NUP’s 62, 78, 113 & 153; (b) Tetrameric
degenerative repeats: (GLFG , docking sequence for
importin beta) : NUP’s 49, 57, 100, 145, 166; (c) No
degenrative repeats: NUP 155
• Many NUP’s are glycoproteins with single 0-linked
N-acetylglucosamine residues.
NUP 96
Symmetric
NUP 159
Asymmetric
(cytoplasmic)
After
detergent
•
The Nucleolus (‘tiny nucleus”) is a reticular
fibrogranular structure in the nucleus that is
specialized for transcription of ribosomal RNA and its
packaging into pre-ribosomal subunits.
• The genes for ribosomal RNA are highly amplified and
located on five different chromosomes (13, 14, 15, 21,
22) called the nucleolar organizer regions (NOR).
• rDNA genes are concentrated within the numerous
fibrillar centers (fc) that compose the nucleolus.
• rRNA is believed to be transcribed at the borders of
fc and the dense fibrillar component (dfc) where
ribosomal proteins associate to form pre-ribosomal
RNP particles.
• Progressive processing of the pre–rRNP particles
occurs within the granular component (gc) where
mature ribosomal subunits are released for transport
into the cytoplasm.
• The massive rRNA transcription is illustrated by the
“Christmas tree” structures composed of repeating
nascent rRNP strands that grow in assembly line
fashion along the rDNA transcription units.
Nucleolus
Nuclear Matrix: Structural Architecture of the Cell Nucleus
A fundamental question is whether there is an overall framework structure that can serve to position and order components
and macromolecular complexes in the cell nucleus ???
Electron microscopy using EDTA regressive staining have revealed a non chromatin structure in the cell nucleus that resembles
a network of fibrogranular structure. This fibrogranular structure nuclear network is called the in situ nuclear matrix.
(standard) Whole cell
(EDTA)
Isolated nuclear matrix
Whole cell EDTA regressive staining
Procedure of Nuclear Matrix Isolation
Components of Isolated Nuclear Matrix
Isolated nuclear
matrix is composed
of: nuclear lamina,
residual nucleoli and
a fibrogranular
internal nuclear
matrix
High magnification electron microscopy of nuclear matrix
Whole Mount Electron Microscopy Demonstrating
Fibrogranular Structure of the Internal Nuclear Matrix
2-D PAGE of Nuclear Matrix Proteins
Functional Properties Associated with Nuclear Matrix
Functional Properties Associated with Nuclear
Matrix contd..