exploring proteins, localisation and targeting

Question 1

Why is protein localisation important?

Answer 1

Incorrect localisation may result in disease

Examples include a-syn aggregation in Parkinson’s disease and beta-amyloid plaques in Alzheimer’s disease.

Question 2

What are the consequences of mutations in signal sequences?

Answer 2

Mutations can lead to diseases such as Swyer syndrome and retinitis pigmentosa

These mutations can affect nuclear localisation signals (NLS) and cause protein retention in the endoplasmic reticulum (ER).

Question 3

What role do microtubules play in the cell?

Answer 3

Microtubules organise the cell and allow correct localisation of organelles, proteins, and mRNA

They are long filaments made of globular tubulin subunits.

Question 4

What are the two types of motor proteins that move along microtubules?

Answer 4

• Kinesins - move towards the + end
• Dyneins - move towards the - end

Question 5

How does a protein get to the correct cellular location?

Answer 5

Proteins contain targeting signals in their amino acid sequence

These signals direct proteins to their appropriate organelles.

Question 6

What are the three groups of proteins based on their location?

Answer 6

• Secretory proteins
• Organelle proteins (chloroplasts, mitochondria, nuclei, peroxisomes)
• Cytosolic proteins

Question 7

What is the role of the Signal Recognition Particle (SRP) in protein transport to the ER?

Answer 7

SRP binds to the ER signal sequence and pauses translation

This process is crucial for trans-membrane transport into the ER.

Question 8

What is the classic example of localised mRNAs?

Answer 8

• oskar
• bicoid
• nanos

Question 9

Why is localising mRNA important?

Answer 9

• Local production of protein
• Rapid and local response to stimuli
• Facilitate assembly of protein complexes

Question 10

What techniques can be used to determine protein localisation?

Answer 10

• Immunofluorescence (fixed cells)
• Generate a fusion protein with a fluorescent tag (live cells)

Question 11

What are the limitations of immunofluorescence?

Answer 11

• Cells are dead
• Fixing and permeabilisation may affect epitope availability
• Cannot determine protein mobility or kinetic analysis

Question 12

What are the advantages of live cell imaging?

Answer 12

• Follow sequential events in real time
• No fixation artefacts

Question 13

What is the significance of Green Fluorescent Protein (GFP)?

Answer 13

GFP can be used as a luminous genetic tag for various biological phenomena

First isolated from Aequorea victoria by Osamu Shimomura.

Question 14

What are the three ways to make GFP fusion proteins?

Answer 14

• Protein X with GFP
• Protein Y with GFP
• GFP alone

Question 15

What does FRAP stand for?

Answer 15

Fluorescent Recovery After Photobleaching

Question 16

What can FRAP show?

Answer 16

• Protein/molecule movement and diffusion
• Compartmentalization and connections between intracellular compartments
• Binding characteristics between proteins

Question 17

What is FRET?

Answer 17

Fluorescence Resonance Energy Transfer, a process of energy transfer between two fluorescent molecules

Question 18

What is required for FRET to occur?

Answer 18

The distance between donor and acceptor must be less than approximately 10 nm

Question 19

What are applications of FRET?

Answer 19

• Studying protein-protein interactions
• Ca2+ signalling in specific cellular compartments
• Detection of protein dimers

Question 20

What does FRET stand for?

Answer 20

Fluorescence Resonance Energy Transfer

FRET is a technique used to study interactions between molecules at the nanoscale.

Question 21

What can FRET be used to show?

Answer 21

Applications of FRET include:
* Interactions between integrins
* Ca2+ signalling in specific cellular compartments
* Detection of protein dimerisation (e.g., EGF receptor)
* Real-time PCR monitoring protein synthesis in live cells

Integrins are transmembrane receptor proteins involved in cell signalling and adhesion.

Question 22

What are integrins?

Answer 22

Transmembrane receptor proteins involved in cell signalling and cell adhesion

Integrins play a key role in connecting the extracellular matrix to the cytoskeleton.

Question 23

How does FRET relate to protein dimerisation?

Answer 23

FRET can detect protein dimerisation, such as the EGF receptor

This is important for understanding receptor interactions and cell signaling pathways.

Question 24

What is the role of Ca2+ in cellular compartments?

Answer 24

Ca2+ signalling is crucial in specific cellular compartments

This signalling is involved in various cellular processes, including muscle contraction and neurotransmitter release.

Question 25

What does real-time PCR monitor?

Answer 25

Real-time PCR monitors protein synthesis in live cells ## Footnote This technique allows researchers to observe dynamic cellular processes as they happen.

Question 26

Who authored the reference material titled 'Fluorescent protein FRET: the good, the bad and the ugly'?

Answer 26

Piston and Kremers ## Footnote This publication discusses the challenges and advantages of using FRET in biochemistry.

Question 27

What is the significance of stable expression of FRET biosensors in cancer research?

Answer 27

It provides new insights in cancer research ## Footnote This allows for real-time monitoring of cellular processes in cancer cells.