PT 3: FRET

Question 1

What are the current methods of measuring molecular tension based upon?

Answer 1

FRET
Photo-quenching
Loss of fluorescence
Change in fluorophore emission properties

Question 2

Describe the main mechanism/principle of FRET?

Answer 2

FRET is a collision-free distance-dependent photophysical process used for molecular force/tension measurements.

Energy is transferred from an excited donor fluorophore to a suitable acceptor protein or fluorophore via long-range dipole-dipole coupling mechanisms.

Question 3

What is the variable Kfret? What variables does it dependent on?

Answer 3

Kfret is the rate coefficient for energy transfer from D* to A.

It depends on the donor fluorescence lifetime (td) and the Forster distance (Ro) [which is the distance where there is a 50% chance of energy transfer].

Question 4

Which spectra must overlap for FRET to occur?

Answer 4

The emission spectrum of the donor should overlap with an area of the excitation spectrum of the acceptor.

Question 5

What is the equation for Kfret?

Answer 5

Kfret = 1/td * (Ro/r)^6

Question 6

What is the equation for the efficiency of energy transfer (E)?

Answer 6

E = Kfret / (Kfret + Krad + KNrad) = Ro^6/ (Ro^6 + R^6)

R = distance between donor and acceptor
Krad = coefficient for energy transfer via radiative decay
KNrad = coefficient for energy transfer via nonradiative decay

Question 7

How can Ro (Forster distance) be calculated/what is it proportional to?

Answer 7

Ro can be calculated from:
- refractive index of the medium (n)
- relative orientation of the donor and acceptor fluorophores (kappa)^2
- overlap of the donor emission spectrum and acceptor spectrum.(lambda)
- Quantum yield of the donor (Qd)

From paper: Ro^6 ~ (kappa)^2 * n^(-4) * Qd * J

Question 8

When is the energy from FRET maximized?

Answer 8

If contributions from Krad and Knrad are low, the energy is maximized.

Question 9

What is (kappa)^2?

Answer 9

The orientation factor which describes the relative orientation between the dipoles of the acceptor absorption and donor emission

Question 10

What is vinculin?

Answer 10

It is a mechanosensitive intracellular protein that binds to focal adhesions and connects cellular actin networks to the extracellular matrix

vinculin-based tension sensor

Question 11

What is the general design of a vinculin-based tension sensor?

Answer 11

Vinculin head <-> fluorescent protein 1 <-> elastic domain <-> fluorescent protein 2 <-> vinculin tail

Question 12

What affects the force sensitivity of the sensor?

Answer 12

n = the number of repeats of polypeptide chain

Increasing n leads to higher resolution in lower force ranges (0-6 pN for n = 10), but it will lower the maximum magnitude of force it can sense (<6pN for n = 10).

Efficiency is inversely related to the linker size (n)

Question 13

What are the main overall steps of the FRET procedure listed?

Answer 13

1. Generating vinculin tension sensor genes
2. Cell seeding and substrate preparation
3. Imaging with FRET

Question 14

What does the Acceptor Intensity (Figure 3A) plot tell us?

Answer 14

It shows the 2D intensity plot from the acceptor channel, which correlates with localization of vinculin tension sensors in the vinculin -1 MEF cell imaged.

It is assumed that vinculin tension sensors are bound to focal adhesions, therefore this tells us where the focal adhesions are.

Question 15

What is the total force?

Answer 15

Total force: IFI = sqrt (Fz^2 + Fcrit^2) - Fcrit
Fz = amount of force attributed to extension
(Fcriticial is minimum, Fz is actual)

Question 16

Why did the authors decide on a numerically complex model for the interpolation of P(re) [probability density function of the end-to-end distance]?

Answer 16

• Other existing equations do not work for a wide range of persistence and contour lengths
• Other equations are non stochastic and depend on the averaging of end-to-end distance (ex. worm-like chain model)

Question 17

What is the Fcritical?

Answer 17

Fcritial = threshold of force required to rotate the molecule before effective extension
Fcritical = kbT / rc,o

rc,o = unloaded distance between two chromaphores

Question 18

What is the probability density function of the end-to-end distance with force applied?

Answer 18

P(re, Fz) = P(re) * exp(Fz * re / kb * T)

Question 19

How does FRET intensity relate to the distance between the centers of the two fluorescent proteins?

Answer 19

FRET intensity is dependent on the distance between the two centers due to their finite size and steric effects

Question 20

What are the limitations of FRET?

Answer 20

• Suffers in efficiency due to factors like changes in pH, ionic concentrations, oxidation, temperature, and refractive index affecting the fluorescence of many labels
• Low SNR

Question 21

What is the equation for the FRET efficiency of each pixel? (with the corrected intensity)

Answer 21

E = [ Id + Fc/G] / Ia

Fc = corrected intensity
G = constant that accounts for the increase in Ia due to sensitized emission and decrease in Id due to quenching
Id = donor-channel intensity
Ia = acceptor-channel intensity

Question 22

What is the formula used for image adjustment?

Answer 22

Fc = If - dbt * Id - abt * Ia

If = FRET channel intensity
dbt = mean ratio of If/Id
abt = mean ratio of If/Ia

Question 23

What are the main steps for FRET imaging?

Answer 23

1. Select microscope Olympus IX83
2. Use FITC and TRITC filters (differ for the types of fluorophore excitation and emission)
3. Use three-image sensitized emission acquisition sequence (three-image = acceptor, FRET, donor)

Acceptor channel: mRuby2 excitation & emission
FRET channel: Clover excitation, mRuby2 emission (overlap)
Donor channel: Clover excitation & emission

Question 24

What are the fluorescent proteins used as the donor and acceptor in the PT?

Answer 24

acceptor: mRuby2
donor: Clover

Question 25

What is the equation for the approximate distance between the centers of the two fluorescent proteins?

Answer 25

rc = sqrt( (Rfp1 + Rfp2)^2 + re^2 )
rc = Ro sqrt6( (1-E)^2 /E )

re = end-to-end distance
Rfp1 & Rfp2 = radii of fluorescence proteins

Question 26

Walk through how FRET efficiency leads to the calculation of force

Answer 26

Efficiency (last one learned) –> used to calculated rc –> used to calculate Fcritical –> used to calculate IFI

Use this to create a map between IFI and E
(going backwards in order of shown in presentation)

Question 27

In the paper, what are the steps in the guide to evaluating fret-based tension sensors?

Answer 27

Tension Sensor:
- Which forces are to be measured?
- What is the sensor’s force sensitivity?

Biosensor:
- Are the fluorophores functional after integration into the protein of interest (POI)?
- Is the POI functional after tension sensor module integration?

Effects & Impact:
- Are effects of intermolecular FRET or conformation changes significant?
- What do the FRET efficiency differences mean?

Question 28

In the paper, what are the different existing tension sensing techniques listed?

Answer 28

a) Distance-dependent FRET (explained in PT)
b) Orientation-dependent FRET
c) Fluorescence Loss
d) Distance Dependent Emission Spectrum
e) DIstance-Dependent Fluorescence Quenching
f) DNA Shearing

Question 29

In the paper, what is the equation for FRET efficiency from fluorescence lifetime imaging microscopy (FLIM)?

Answer 29

E = 1 - (tau DA / tau D)

tau DA = donor lifetime with acceptor
tau D = donor lifetime without acceptor

Question 30

What are the 4 main graphs produced in the results section?

Answer 30

1. Acceptor intensity
2. FRET Efficiency
3. Force IFI
4. Extension (rz)