The Nucleus and Nuclear Transport

The “brain” of the cell

Form relating to function:

Spherical nucleus is approximately 10% of the whole cell’s volume, making it the central prominent feature.

Functions (Same in all cells)

Ensures that cell reproduction is carried out correctly

Controls all of the functions of the cell

Houses genetic material, DNA

Organizes the uncoiling of DNA for replication

Organelle malfunction:

The nucleus is the brain of the cell, it is used to carry out all cell activities. If it is not working properly the cell will die.

If the nucleus does not function properly the cell will not survive.

Human Diseases:

Any complication with DNA synthesis in the nucleus will result

in a genetic defect.

Can you give any example ?

Example of a Genetic Disease: Trisomy 21

When every somatic cell of the person has three copies of chromosome 21.

Also known as ―Down Syndrome‖

If the chromosomes do not split properly, genetic defects (such as trisomy 21) occur.

Components of the organelle:

Nuclear Envelope (porous double membrane surrounding the

nucleus)

Pores: provide a direct passage for the exchange of material between the cytoplasm and the nucleus

Nucleoplasm (fluid substance in which solutes of nucleus are

dissolved)

Nuclear matrix (protein containing fibrillar network)

Nucleolus (irregular shaped electron-dense structure, no

membrane, involved in the production of ribosomes, cells can have one or more nucleoli)

Chromatin (highly extended nucleoprotein fibers)

Structure of Nucleus

Structure

The Nuclear Envelop

Composed of two membranes

Each is a lipid bilayer with

proteins attached

The two layers are separated

by a space of 10-50 nm

Pores cover the membrane,

100 nm in diameter

At the pores the two membranes

are fused together and contain

complex assemblies of proteins

Average mammalian cell possess several thousands of nuclear pores

Outer membrane is studded with ribosomes and is continuous with

rough endoplasmic reticulum (ER)

The space between the membranes is continuous with ER lumen

The inner surface of the nuclear envelop of animal cells is bound by integral membrane proteins to a thin filamentous meshwork – the NUCLEAR LAMINA

Provides mechanical support to the nuclear envelop

Serves as site of attachment for chromatin fibers at the nuclear periphery

Role in DNA replication and transcription (poorly understood)

The filaments of the nuclear lamina are approximately 10 nm in diameter and composed of polypeptide, called LAMINS

Members of superfamily polypeptides that assemble into intermediate filaments of cytoplasm

Integrity is regulated by phosphorylation (disassembly of lamina) and dephosphorylation.

Nuclear Envelop

Nuclear lamina A-type lamins are inside, next to nucleoplasm; B-type lamins are near the nuclear membrane (inner). They may bind to integral proteins inside that membrane.

The lamins may be involved in the functional organization of the nucleus. They may play a role in assembly and disassembly before and after mitosis. After they are phosphorylated, this triggers the disassembly of the lamina and causes the nuclear envelope to break up into vesicles. Dephosphorylation reverses this and allows the nucleus to reform.

Mutations in one of the Lamin genes (LMNA) can cause several human

diseases

E.g. EDMD2 (Emery-Dreifuss muscular dystrophy) patients have

exceptionally fragile nuclei in their muscle cells

Nuclear Pore Complexes (NPCs) The nuclear envelope is perforated with thousands of pores. Each NPC is huge, supramolecular complex – 50 to 30 times the mass of ribosomes. Exhibits octagonal symmetry due to eightfold repetition of a number of structures NPCs contain different proteins called nucleoporins (30 in yeast; probably around 50 in vertebrates). The entire assembly forms an aqueous channel connecting the cytosol with the interior of the nucleus ("nucleoplasm"). Transport through the nuclear pore complexes is active

NPC: Structure Formed at sites where the inner and outer membranes of the nuclear envelope are joined. Contains 8 subunits that "clamp" over region of the inner and outer membrane where they join. Each subunit projects a spoke-like unit into the center so that the pore looks like a wheel with 8 spokes from the top. The projected spoke is directed towards the central "plug' or granule.

Nuclear Pore Complex

Import into the nucleus

All proteins are synthesized in the cytosol and those needed by the

nucleus must be imported into it through the NPCs (Having

characteristic sequence of amino acids — called a nuclear localization

sequence (NLS) — that targets it for entry).

They include:

All the histones needed to make the nucleosomes

All the ribosomal proteins needed for assembly of ribosomes

All the transcription factors (e.g., the steroid receptors)

needed to turn genes on (and off)

All the splicing factors needed to process pre-mRNA into

mature mRNA molecules; that is, to cut out intron regions and

splice the exon regions.

The classical NLS consists of one or two short stretches of positively charged amino acids

e.g. – Pro – Lys – Lys – Lys – Arg – Lys – Val –

If one of the AA from NLS is replaced by a non-polar AA ???

If NLS is fused to nonnuclear protein e.g. serum albumin ???

Export from the nucleus

Molecules and macromolecular assemblies exported from the nucleus include:

Ribosomal subunits containing both rRNA and proteins

Messenger RNA (mRNA) molecules (accompanied by proteins)

Transfer RNA (tRNA) molecules (also accompanied by proteins)

Transcription factors that are returned to the cytosol to await reuse

Both the RNA and protein molecules contain a characteristic nuclear export sequence (NES) needed to ensure their association with the right carrier molecules to take them out to the cytosol.

How proteins with NLS are transported to nucleus and proteins with NES are exported out ?

A family of proteins, karyopherins, is responsible for transport of molecules across the nuclear envelop and function as mobile transport receptors

Importins (Nuclear Import Receptors)

Exportins (Nuclear Export Receptors)

Transport of Molecules Between Nucleus and Cytosol

Ran GTPase= molecular switch Drives directional transport in appropriate direction Conversion Between GTP and GDP bound states mediated by Ran specific

regulatory proteins GAP converts RNA-GTP to Ran-GDP via GTP hydrolysis GEF promotes exchange of GDP for GTP converting Ran-GDP to Ran-GTP Ran GAP in cytosol thus more Ran-GDP in cytosol Ran GEF (RCC1) in nucleus thus more Ran-GTP in nucleus

Nuclear Import of a Protein (Nucleoplasmin) Four proteins are mainly required:

• Ran (monomeric G protein that exists in two conformations, one when complexed with GTP and an alternative one when the GTP is hydrolyzed to GDP)

• Nuclear transport factor 2 (NTF2), • importin alpha & Beta.

The two importins form a heterodimeric nuclear-import receptor:

• The subunit α binds to a basic NLS in a ―cargo‖ protein to be transported into the nucleus, and

• Subunit β interacts with a class of nucleoporins called FG-nucleoporins (line the channel of the nuclear pore complex)

Receptor guides the protein cargo to the outer surface of the nucleus and dock with cytoplasmic filaments (extended from the outer ring of the NPC)

Movement of complex through nuclear pore

Interaction with Ran-GTP and dissociation from complex

Importin ß subunit in association with Ran-GTP is transported back to cytoplasm where it is hydrolyzed to Ran-GDP.

Importing proteins from the cytoplasm into the nucleus

Mechanism for nuclear Import- export of cargo proteins NLS and NES.

Controlling Transport of Molecules

Controlling rates of import and export determines steady state location, especially for shuttle proteins

Phosphorylation/dephosphorylation of adjacent aa may be required for receptor binding

Cytosolic anchor (bound to inhibitory cytosolic proteins) or mask nuclear localization signals, blocking interaction w/ receptors

Protein made and stored in inactive form as ER transmembrane protein

Examples: 1. The control of nuclear import during T-cell activation

The nuclear factor of activated T cells (NF-AT) is a gene regulatory protein that, in the resting T cell, is found in the cytosol in a phosphorylated state.

T-Cell Activation When T cells are activated, the intracellular

Ca2+ concentration increases.

In high Ca2+, the protein phosphatase, calcineurin, binds to NF-AT.

Binding of calcineurin dephosphorylates NF-AT, exposing one or more nuclear import signals, and it may also block a nuclear export signal.

The complex of NF-AT bound to calcineurin is then imported into the nucleus, where NF-AT activates the transcription of numerous cytokine and cell-surface protein genes that are required for a proper immune response.

During the shut-off of the response, decreased Ca2+ levels lead to the release of calcineurin.

Rephosphorylation of NF-AT inactivates the nuclear import signal, and it re-exposes the nuclear export

signal of NF-AT causing NF-AT to relocate to the cytosol.

Immunosuppressive drugs, such as cyclosporin A and FK506, inhibit the

ability of calcineurin to dephosphorylate NF-AT; these drugs thereby block

the nuclear accumulation of NF-AT.

Other gene regulatory proteins are bound to inhibitory cytosolic proteins that

either anchor them in the cytosol (through interactions with the cytoskeleton or with specific organelles), or

mask their nuclear localization signals so that they are unable to interact with nuclear import receptors.

When the cell receives an appropriate stimulus, the gene regulatory protein is released from its cytosolic anchor or mask and is transported into the nucleus.

Example

The latent gene regulatory protein that controls the expression of proteins involved in cholesterol metabolism.

The protein is made and stored in an inactive form as a transmembrane protein in the ER.

When deprived of cholesterol, the cell activates specific proteases that cleave the protein, releasing its cytosolic domain. This domain is then imported into the nucleus, where it activates the transcription of genes required for cholesterol import and synthesis.

Export of mRNA from the Nucleus

Pre-mRNA (primary transcript) contains exons and introns.

Splicing to form mature mRNA.

Mature mRNA contains exon junction complex (EJC) deposited on mRNA 20-24 nucleotide upstream of each exon-exon junction.

One of the subunit of EJC is protein Aly which plays role in mRNA export by binding to transport receptors such as TAP (Nuclear RNA export factor 1) and forms complex.

The complex moves through the NPC into the cytoplasm.

After passage through NPC, Aly and TAP are shed from mRNP and EJC is released during first round of translation.

Unspliced mRNA is retained in the nucleus.

Export of mRNA from the Nucleus

Chromatin The nucleus contains the chromosomes of the cell. Each chromosome

consists of a single molecule of DNA complexed with an equal mass of

proteins. Collectively, the DNA of the nucleus with its associated

proteins is called chromatin.

Most of the protein consists of multiple copies of 5 kinds of histones. These

are basic proteins, bristling with positively charged arginine and lysine

residues.

The positively charged amino acids bind tightly to the negatively-charged

phosphate groups of DNA.

Chromatin also contains small amounts of a wide variety of nonhistone proteins.

Most of these are transcription factors (e.g., the steroid receptors) and their

association with the DNA is more transient.

Coiled chromatin fibers

Thick enough to see as separate structures

Occur in preparation of cell reproduction

Material made of proteins and DNA

Looks like fiber

Loose in the nucleus

DNA’s ability to function is reliant upon being loose in the nucleus not ―crammed‖ like a ball of string

Chromatin vs. Chromosomes

Inside the nucleus of every human

cell is 6 feet of DNA!!!

Nucleosomes The orderly packaging of eukaryotic DNA depends on HISTONES Two copies of each of four kinds of histones

• H2A • H2B • H3 and • H4

form a core of protein, the nucleosome core. Around this is wrapped about 147 base pairs of DNA. 20–60 bp of DNA link one nucleosome to the next. Each linker region is occupied by a single molecule of histone 1 (H1). The binding of histones to DNA does not depend on particular nucleotide sequences in the DNA but does depend critically on the amino acid sequence of the histone. Histones are some of the most conserved molecules during the course of evolution. Histone H4 in the calf differs from H4 in the pea plant at only 2 amino acids residues in the chain of 102.

Why form NUCLEOSOME The formation of nucleosomes helps somewhat To make the DNA sufficiently compact to fit in the nucleus In order to fit 46 DNA molecules into a nucleus, only 10 µm across, requires more extensive folding and compaction. Interactions between the exposed "tails" of the core histones causes nucleosomes to associate into a compact fiber 30 nm in diameter. These fibers are then folded into more complex structures whose precise configuration is uncertain and which probably changes with the level of activity of the genes in the region.

Formation of Nucleosomes

Histone Modifications Although the amino acid sequence (primary structure) is unvarying, individual

histone molecules do vary in structure as a result of chemical modifications

that occur later to individual amino acids.

These include adding:

acetyl groups (CH3CO-) to lysines

phosphate groups to serines and threonines

methyl groups to lysines and arginines

Although 75–80% of the histone molecule is incorporated in the core, the

remainder — at the N-terminal — hangs out from the core as a "tail".

The chemical modifications occur on these tails, especially of H3 and H4.

Most of theses changes are reversible.

1. Addition of acetyl groups (CH3CO-) to lysines

added by enzymes called histone acetyltransferases (HATs) and

removed by histone deacetylases (HDACs)

Oftenly, acetylation of histone tails occurs in regions of chromatin that become active in gene transcription.

This makes a kind of intuitive sense as adding acetyl groups neutralizes the positive charges on Lys thus reducing the strength of the association between the highly-negative DNA and the highly-positive histones.

Acetylation of Lys-16 on H4 prevents the interaction of their "tails" needed to form the compact 30-nm structure of inactive chromatin.

But this case involves interrupting protein-protein not protein-DNA interactions.

2. Addition of phosphate groups Causes the chromosomes to become more compact as they get ready for mitosis and meiosis.

3. Methylation (neutralizes the charge on lysines and arginines) Can either stimulate or inhibit gene transcription e.g

Methylation of lysine-4 in H3 is associated with active genes while Methylation of lysine-9 in H3 is associated with inactive genes. (These include those imprinted genes that have been permanently inactivated in somatic cells)

Histones are a dynamic component of chromatin and not

simply inert DNA-packing material.

Chromosome Territories During interphase, little can be seen of chromatin structure Although each chromosome is greatly elongated, it tends to occupy a discrete region within the nucleus called its territory. Euchromatin versus Heterochromatin The density of the chromatin that makes up each chromosome (that is, how tightly it is packed) varies along the length of the chromosome. dense regions are called heterochromatin less dense regions are called euchromatin.

Heterochromatin

is found in parts of the chromosome where there are few or no genes, such as

centromeres and telomeres

is densely-packed;

is greatly enriched with transposons and other "junk" DNA;

is replicated late in S phase of the cell cycle;

has reduced crossing over in meiosis.

Those genes present in heterochromatin are generally inactive; that is, not transcribed and show

increased methylation of the cytosines in CpG islands of the DNA

decreased acetylation of histones and

increased methylation of lysine-9 in histone H3, which now provides a binding site for heterochromatin protein 1 (HP1), which blocks access by the transcription factors needed for gene transcription.

Euchromatin

Is found in parts of the chromosome that contain many genes;

Is loosely-packed in loops of 30-nm fibers.

These are separated from adjacent heterochromatin by insulators.

The loops are often found near the nuclear pore complexes. (This would seem to make sense making it easier for the gene transcripts to get to the cytosol, but there is evidence that as gene transcription proceeds, the active DNA actually moves into the interior of the nucleus.)

The genes in euchromatin are active and thus show

decreased methylation of the cytosines in CpG islands of the DNA

increased acetylation of histones and

decreased methylation of lysine-9 in histone H3.

CpG Islands?

CpG islands are genomic regions that contain a high frequency of CG dinucleotides.

In mammalian genomes, CpG islands are typically 300-3,000 base pairs in length. They are in about 70% of human promoters.

The CpG sites in the CpG islands of promoters are unmethylated if genes are expressed.

CpG islands typically occur at or near the transcription start site of genes, particularly housekeeping genes, in vertebrates.

Normally a C (cytosine) base followed immediately by a G (guanine) base (a CpG) is rare in vertebrate DNA because the cytosines in such an arrangement tend to be methylated.

The Nucleolus During the period between cell divisions, when the chromosomes are in their extended state, 1 or more of them (10 in human cells) have loops extending into a spherical mass called the nucleolus. Nucleolus is the site of synthesis of three (of the four) kinds of RNA molecules (28S, 18S, 5.8S) used in the assembly of the large and small subunits of ribosomes. 28S, 18S, and 5.8S ribosomal RNA is transcribed (by RNA polymerase I) from hundreds to thousands of rDNA genes distributed (in humans) on 10 different chromosomes. The rDNA-containing regions of these 10 chromosomes cluster together in the nucleolus. Once formed, rRNA molecules associate with the dozens of different ribosomal proteins used in the assembly of the large and small subunits of the ribosome. The nucleolus is organized from the "nucleolar organizing regions" on different chromosomes

"Nucleoplasm"

The term "nucleoplasm" is still used to describe the contents of

the nucleus. However, the term disguises the structural

complexity and order that seems to exist within the nucleus. For

example, there is evidence that DNA replication and

transcription occur at discrete sites within the nucleus.

Anucleated and polynucleated cells

Although most cells have a single nucleus, some cell types have no nucleus, and others have many nuclei. This can be a normal process, as in the maturation of mammalian red blood cells, or an anomalous result of faulty cell division. Anucleated cells contain no nucleus and are therefore incapable of dividing to produce daughter cells. The best-known anucleated cell is mammalian red blood cell, or erythrocyte, which also lacks other organelles such as mitochondria and serves primarily as a transport vessel to ferry oxygen from the lungs to the body's tissues. Erythrocytes lose their nuclei, organelles, and ribosomes during erythropoiesis. The nucleus is expelled during the process of differentiation from an erythroblast to a reticulocyte, the immediate precursor of the mature RBCs. The mutagens may induce the release of some "micronucleated" erythrocytes Anucleated cells can also arise from flawed cell division in which one daughter lacks a nucleus and the other is binucleate.

Polynucleated cells contain multiple nuclei. Most Acantharean species of protozoa and some fungi in mycorrhizae have naturally polynucleated cells. In humans, skeletal muscle cells, called myocytes, become polynucleated during development; the resulting arrangement of nuclei near the periphery of the cells allows maximal intracellular space for myofibrils. Multinucleated cells can also be abnormal in humans; for example, cells arising from the fusion of monocytes and macrophages, known as giant multinucleated cells, sometimes accompany inflammation and are also implicated in tumor formation.

HEMATOPOIESIS: Differentiation from

STEM CELLS to mature cells

Erythropoiesis