Lecture 20

Question 1

What is a nematode? What can fluorescence imagery tell us about them?

Answer 1

Nematode is a small organism that biologists like to study.
- By using fluorescent markers, we can visualize their two different neurons as they have one that is very short and another that is very long.
- We can use this information to learn more about them as a species!

Question 2

What is the visible light spectrum

Answer 2

The visible light spectrum is the segment of the electromagnetic spectrum that the human eye can view.

Question 3

In which direction of the light spectrum does energy increase?

Answer 3

Energy increases as we move from the red to the purple (or from larger to smaller)
- more energy at 400nm than at 700nm.

Question 4

In which direction of the light spectrum does wavelength increase?

Answer 4

Wavelength increases as we move from purple to red (smaller values to larger values)
- Longer wavelength at 700nm than at 400nm

Question 5

What is fluorescence?

Answer 5

Fluorescence is the emission of light by a substance that has absorbed light or other radiation.
- They absorb one colour and emit another colour

Question 6

What is an example of an insect that can be visualized with fluorescence?

Answer 6

We can visualize scorpions with fluorescence, as they will emit a different colour than the one that thy absorb/

Question 7

What do fluorescent molecules do?

Answer 7

Fluorescent molecules absorb light at one wavelength and emit it at another, longer wavelength.

Question 8

Ground state to excited state back to ground state?

Answer 8

Fluorophore goes from ground state where it absorbs a photon, therefore putting it in its exited state.
- Following this, the photon is emitted at a longer wavelegnth, putting the fluorophore back into its ground state.

Question 9

What are the two phases called?

Answer 9

The two phases of a fluorophore are excitation and emission

Question 10

What can occur to an orbital electron of a fluorochrome?

Answer 10

A orbital electron of a fluorochrome molecule can be raised to an excited state after the absorption of a photon

Question 11

When does fluorescence occur?

Answer 11

Fluorescence occurs when the electron return to its ground state and emits a photon of light at a longer wavelength (than what was absorbed)

Question 12

What is photobleaching?

Answer 12

Photobleaching is a process in with too much exposure to light destroys the fluorochrome molecule.

Question 13

What is immunofluorescence?

Answer 13

Immunofluorescence is a commonly used method in molecular and cell biology labs. It is a robust and simple method to reliably localize molecules on fixed (dead) cells or tissues.

Question 14

How does immunofluorescence work (simple)

Answer 14

1. Antigens are added to a cell/tissue
2. A primary antibody directed against our antigen is added
3. A secondary antibody directed against primary antibodies is added
4. The markers on the secondary antibody are activated and emit light

Question 15

What are antibodies?

Answer 15

Antibodies are proteins produced by the vertebrate immune system as a defence against infection

Question 16

What is Osamu Shimomura discover?

Answer 16

Osamu Shimomura worked on jellyfish to isolate GFP proteins (fluorescent!)

Question 17

What did Martin Chalfie discover?

Answer 17

Martin Chalfie discovered that GFP can be applied to specimens that we want to study.

Question 18

What did Rogen Tsien discover?

Answer 18

Roger Tsien discovered that changing select amino acids of GFP would allow us to have differently coloured fluorescent markers.

Question 19

What are the two fluorescent molecules that come from jellyfish?

Answer 19

• Aequorin is a blue-light emitting protein that responds to Calcium
• GFP is a green-light emitting protein
• Together they form a complex where GFP associates with Aequorin

Question 20

How do Aequorin and GFP work together?

Answer 20

Aequorin and GFP work together to convert Ca2+ induced luminescent signals into the green luminescence

Question 21

What does Aequorin need in order to be fully functional?

Answer 21

Aequorin requires the binding of a chromorphore prosthetic group in order to work and actually emit light.

Question 22

What does the light emitted from Aequorin cause?

Answer 22

The light that is emitted from the Aequorin excites the chromophore of GFP

Question 23

Does GFP require a Chromophore prosthetic group?

Answer 23

GFP doesnt have a chromophore prosthetic group, but it instead has a naturally built chropmohore within it (contaiing Ser, Tyr and Gly)
- The excitred state of the GFP chromophore emits green light

Question 24

GFP absorbs and emits?

Answer 24

GFP absorbs blue light and emits green light
- The absorption/excitation curve (peak at 400) is before the emission curve (peak at 500)

Question 25

GFP genetically encoded?

Answer 25

GFP does nit need a prosthetic group or any other cofactor so it can be ENTIRELY genetically encoded. - The chromophore in GFP is made from amino acids

Question 26

How do we directly visualize a protein?

Answer 26

`Fusing GFP to the coding sequence of a gene allows the direct visualization of the protein.

Question 27

Peptide location signal added to GFP does what?

Answer 27

Peptide location signals can be added to the GFP to direct it to a particular cel compartment, such as the endoplasmic reticulum or a mitochondrion.

Question 28

Examples of peptide localization signals?

Answer 28

NLS - GFP goes to nucleus Mitochondria - GFP KDEL - GFP goes to ER and more!

Question 29

What do GFP mutations cause?

Answer 29

Mutations in GFP change the absorption and emission colours. - These result from small changes in the amino acid sequences

Question 30

What else can mutations change about fluorescent molecules?

Answer 30

Mutations can also change other fluorscence properties including brightness stability and maturation time and more.

Question 31

How can we track movement of proteins?

Answer 31

We can track movement of proteins and cells in vivo (test tube) by using fluorescent markers! - Histone (RFP) - Actin (GFP) - Other cells (magenta)

Question 32

How can we visualize intracellular Ca2+ conc?

Answer 32

We can experimentally visualize intracellular Ca2+ concentrations by using a fluorescent indicator - GCaMP iOS a GFP calmodulin protein that measures the Ca concentration

Question 33

Calcium imaging technique?

Answer 33

Calcium imaging is a powerful technique for monitoring the activity of distinct neurons in brain tissues in vivo.

Question 34

Example of calcium imaging technique?

Answer 34

Changes in calcium = neuronal activity - We can observe the cold neuron line rise drastically, meaning it is activated - At the same time, the heat neuron line drops, meaning it is not firimg

Question 35

GFP and pH?

Answer 35

GFP fluorescence responds rapidly and reversibly to pH changes, allowing us to understand at which pH the cells become activated, therefore allowing us to determine where it might be localized - Lysosomal cells might need to have a lower pH for their job, so they would be active at lower pH

Question 36

What is the technique of photobleaching and what is FRAP?

Answer 36

Photobleaching is the photochemical alteration of a fluorophore that makes it unable to fluoresce. - FRAP is fluorescence recovery after photobleaching - This indicates the dynamics of a protein in a living cell (how fast they are able to move in and recover.

Question 37

What are photoactivatable fluorescent proteins?

Answer 37

Photoactivatable fluorescent proteins are fluorescent proteins that display unique changes in their spectral properties upon exposure to a specific wavelength of light - We can measure changes in cell behaviour by observing colour changes in fluorescence

Question 38

Fluorescent colour change?

Answer 38

Some fluorescent proteins slowly change their colour so we can test the "age" of proteins and cells using these proteins.

Question 39

What is FRET?

Answer 39

Fluorescence resonance energy transfer (FRET) is a special technique used to gauge the distance between two chromophores. - we can test how close two proteins are in a sample - we can test a protein to protein binding between 1 and 10 nm

Question 40

How can FRET be used?

Answer 40

FRET can be used to make genetically encoded fluorescent biosensors many molecular sensors. - For example, without cAMP the yellow might be emitted because FRET is present - With cAMP the bond might dissociate and create a bend in the protein, meaning there is nio FRET and blue light is emittef - Therefore, the chages in the yellow fluorescence estimate the amount of certain molecules

Question 41

how does split GFP indicate protein association?

Answer 41

Split GFP proteins into 2 fragments that spontaneouslty assemble into a functional protein when in very close proximity. - These indicate protein-protein interactions or cell-cell contacts - When the two proteins/cells marked with GFP assemble, the GFP will emit light