Week 3 - Nucleus and gene expression

ANHB3323

How do Cells respond to their environment? (4 things)

1. Signalling

2. Receptors

3. Intermediaries (cascade of events triggered by signalling)

4. Nuclear translocation and import

[all 4 lead to gene regulation]

Chemical Messangers (4 categories/examples)

Have effect on local or far away tissue

Examples:

• growth factors

• hormones

• neurotransmitters

• extracellular matrix components

Must interact with plasma membrane

Receptors are either...

Membrane bound proteins

or

Intracellular receptors, lipid soluble molecules bind to them

Estrogen signalling (5 basic steps)

1. estrogen enters cell

2. binds to receptor in cytoplasm

3. receptor dimerises

4. receptor translocates to nucleus

5. activates gene expression

Dimerised receptor

two molecules come together

• changes shape

• once shape is changed, can pass through nuclear pore and affect gene expression

Examples of Surface Receptors (membrane bound receptors)

1. GPCR

• e.g. Frizzled

2. Ion Channels

3. Receptor Tyrosine kinases

[all for non lipid soluble molecules]

G protein coupled receptor (GPCR)

1. Ligand binds extracellularly

2. activates g protein

   • g protein = heterotrimeric protein complex in the plasma membrane. 

3. The G protein undergoes GDP-GTP exchange

4. Alpha subunit dissociates from beta-gamma dimer

5. Both subunits react with downstream effectors

e.g. Frizzled receptor in Wnt/β-catenin pathway

ion channel

1. Messenger binds to receptor

2. Receptor changes shape

3. Ions pass through via conc. gradient

Tyrasine Kinase

Kinase is an enzyme that adds phosphate groups onto molecules

1. Ligand binds to receptor

2. Receptor dimerises

3. Tyrasine kinase tails are phosphorylated

4. triggers secretion of secondary messengers

example: epidermal growth factor/EGFR)

Non-lipid soluble signalling

cell surface receptors are required

• ligand binds to receptor

• initiates secondary receptors

Signal Transduction cascades

Transmission of signal to the nucleus:

• via Secondary messengers

• there can be multiple secondary messengers

What does a signal transduction result in?

Post-translational modifications (usually phosphorylation)

Protein-Protein interactions

• e.g. dimerisation

Secondary messenger example

PIP3

(generated by PI3K)

PI3K

generates secondary messenger PIP3

resulting in:

• activation of AKT = cell growth/proliferation

• mechanism used by cells of the immune system

JAK-STAT signalling (7 steps)

• example of secondary messengers

1. binding of ligand = dimerisation

2. receptor phosphorylation

3. STAT binds

4. JAK phosphorylates STAT

5. STAT dimerises

6. translocation to nucleus (STAT)

7. transcriptioin activated

How does Nuclear Translocation occur?

Phosphorylation and dimerisation

• e.g. JAK-STAT

or

Increased levels of messenger molecules

• Via concentration gradient

Apoptosis vs Necrosis

Apoptosis = programmed cell death

Necrosis = not programmed cell death (in response to injury)

what does Apoptosis activate?

proteases + nucleases

Necrosis (what happens)

Progressive injury to normal cell resulting in:

• inflammation

• breakdown of membrane, organelles and nucleus

• leakage of cellular contents

• and cell death

Apoptosis (what generally happens)

condensation of chromatin + membrane begins to bleed

=

cellular fragmentation + apoptotic bodies release

(then phagocytes remove apoptotic bodies and fragments)

Apoptosis signalling

Mitochondrial release of Cytochrome C = capsase activation

Capsase activation = break down cell

Cause of Apoptosis signalling (3 things)

Either:

1. FAS ligand

2. ER stress

3. DNA damage

P53 location (in cell)

Nucleus

at low levels (because it is degraded by MDM2 )

P53 functions

Tumour suppressant

• prevents cell division

• prevents cell growth

What happens inside the Nucleus (4 things)

1. transcription (DNA to mRNA)

2. processing/export of mRNA

3. DNA replication

4. DNA integrity/repair

Birth of Daughter Nuclei

Each cell division:

• nuclear envelope breaks down and is reformed

Nuclear factors have to be re-imported via nuclear pores

Nuclear organisation has to be re-created

• Each cell is established by the formation of a new nucleus

Nuclear envelope

Double Membrane:

1. Outer = continuous with Rough ER

2. Inner = associates with nuclear lamina

has pores for import/export, molecules sit in the space between the two sheets of membrane

Nuclear import/export

All molecules pass through nuclear pores

Passive diffusion:

- for molecules under 20-30kDa

Large proteins:

- have export/import signals

- need cargo molecules which act as carriers

Nuclear lamina

Composed of lamina proteins:

• gives structural stability to nucleus

Mutations in Lamin Genes

Cause/lead to:

• progeria (premature ageing)

• other muscle diseases

Lamin Mutations

1. Nucleus cannot withstand mechanical stress

2. Alters nuclear organisation = gene expression changes

What is found inside the Nucleus?

Chromatin = DNA + packaging proteins

What is Chromatin connected to?

the Nuclear Lamina

Chromosome Territories

defined location of each chromosome:

- some at the periphery

- some in the centre

What influences Chromosome Territories?

Cell type + shape

Size of chromosome (larger chromosome are usually at the periphery)

Fluorescent in Situ Hybridisation (FISH)

Used to localise DNA sequences

How:

• short fragments ('probe') of DNA complement sequence of interest

• probe is labelled with fluorescent dye

• target DNA is deanatured, allowing probe to anneal

• It is possible to FISH on multiple chromosomes are the same time

Nucleolus (general info)

Forms around ribosomal DNA repeat

• Densest part of cell

Nucleolus function

Site of:

• ribosome production

• subnuclear sequestration of regulatory molecules

Paraspeckles (general info)

Stress-induced subnuclear bodies

Built around long noncoding RNA (NEAT1)

Paraspeckles function

Regulate gene expression by:

- Sequestration of paraspeckle proteins

Paraspeckle substructure

Has distinct zones:

- Core

- Shell

- Patch

Molecular movement

Despite crowding, molecules move rapidly throughout the nucleus

• inactive genes in the middle

• Actively transcribed genes are found at the edges or outside the territory

• genes cluster together to be transcribed

How to determine how fast molecules move in living cells

Photodynamics, via fluorescent protein fusion:

- bleach protein

- image recovery of fluorescence (how long it takes to recover)