Two proposed methods to produce a mechanosensitive fluorescent probe.
Our goal was to create and better understand tools based on fluorescence changes in response to mechanical cues that may be of use in experimental biological systems where understanding how mechanical force can influence and regulate a system may provide insight into their behavior.
Development, wound healing, and immunology are relevant contexts for these tools[1–15]. In this thesis work we explored two methods that might be used to produce such probes.
One proposed method to modulate a fluorescence signal in response to mechanical tension or strain is to apply force or strain to a fluorescent molecule like the green fluorescent protein (GFP) and look for changes in fluorescence (Figure 1).
Figure 1. A mechanosensitive fluorescent probe. It may be possible to change the fluorescence of a fluorescent molecule directly by mechanically manipulating the fluorescent molecule with different forces. Here we indicate different forces applied to the fluorescent molecule with arrows of different length and the change in fluorescence with different colors. Longer arrows mean increased force and the darker color might mean less fluorescence intensity.
We studied the effect of applying tension to stretch GFP (Figure 2) into a variety of mechanical
“intermediate states”[16–20] by modeling GFP on a computer[19,21–23]. To infer the effect of mechanical tension on the fluorescence of the molecule, we made genetically engineered mutations to the molecule to mimic the structures seen in the computer[24].
Figure 2. The green fluorescent protein (GFP). This is one of the active ingredients in the glowing jellyfish aequorea victoria.
Another method to produce a mechanosensitive fluorescent probe is to use the mechanism of
fluorescence resonance energy transfer (FRET). The energy transfer efficiency between the donor and the acceptor depends on the distance between them[25–29]:
E= 1 1+
(
rr0)
6where r is the distance between the donor and the acceptor and r0 is the Förster radius. When the distance between the fluorophores is less than the Förster radius, which is typically on the order of five nanometers, the probability of the energy being transferred from the donor to the acceptor is greater than the probability that the donor fluorophore loses the energy of the excited state by emitting light.
Figure 3. A mechanosensitive FRET probe – two fluorescent molecules connected with a spring. With low tension, the donor (green) and acceptor (red) are closer together and more energy is transferred to the acceptor. This causes the donor to become dimmer and the acceptor to become brighter. With more tension, the donor and acceptor are farther apart and less energy is transferred resulting in a brighter donor and a dimmer acceptor. By measuring the intensities of the donor and acceptor, one may obtain information about the distance between them. Distances that can be practically measured are on the order of 1 to 10 nm. If the spring connecting the fluorescent molecules is well characterized, one may obtain information about the force being applied to the probe.
In principle, one can measure the energy transfer efficiency between a donor and an acceptor molecule by measuring the emission intensity of the donor molecule and the emission intensity of the acceptor
molecule (Figure 4). From these intensities one may obtain information about how the distances between the donor and acceptor are changing. In practice, distances that can be measured with this technique are on the order of 1 to 10 nm By connecting the donor and acceptor molecules with a spring made from protein, one can make the response of the sensor depend on the applied force[30,31].
By using a sharp pulse of light to excite the donor molecule and carefully measuring when fluorescence photons are emitted, one can measure on the average how long the donor stays in the excited state[32–
35]. The average arrival time of the donor photons for a single FRET pair is known as the lifetime of the excited state of the donor. Having a FRET acceptor nearby provides another way for the energy to disappear from the donor's excited state which causes the excited state to last for a shorter period of time than it would without a FRET partner. The relationship between energy transfer efficiency and the lifetime of the excited state of the donor is given by:
E=1− ττ0
where τ is the lifetime of the excited state of the donor in the presence of a FRET acceptor and τ0is the lifetime of the excited state of the donor in the absence of a FRET acceptor. Thus by measuring changes in the lifetime of the excited state of the donor, one can measure FRET efficiency and how distances between the donor and acceptor molecules are changing.
In principle, this approach to FRET measurements has significant advantages over conventional FRET measurements based on the intensities of FRET donors and acceptors. The most obvious advantage is that one measures time. Time can be measured with great precision and reliability. Measurements of time are measurements of a fundamental quantity that can easily be compared with external standards and which can be repeated between labs.
A less obvious advantage which is often overlooked is that with fluorescence lifetime measurements one measures light of a single color. This eliminates chromatic aberrations that are the result of the fact that light of different wavelengths can interact with the measurement system or the sample differently.
Even if the measurement system is perfectly corrected for chromatic aberrations, samples typically aren't. Most samples scatter light to at least some degree. The amount of light that is scattered depends
on its wavelength. This is why on a clear sunny day, the sky appears to be blue. So there will be sample dependent variations in the intensities of donor and acceptor fluorescence that are independent of changes in FRET efficiency. In principle one might correct for these effects if a sample is
homogeneous, but most samples of interest are not homogeneous. Estimating the magnitude of these intensity variations between donor and acceptor fluorescence and determining if they are significant compared to intensity variations caused by changes in FRET efficiency can be a problem.
Another often overlooked advantage of detecting light of a single color is that there is a narrower window of wavelengths where autofluorescence in the sample can affect the measurements. This can make it easier to determine when autofluorescence in the sample is significant compared to
fluorescence from one's FRET probe.
For a FRET based mechanosensor, we studied FRET labeled fibronectin[36] (Figure 4), a molecule that has been shown to be mechanosensitive[37,38]. In this case, the “spring” regulating changes in FRET between the donors and the acceptors is the fibronectin molecule itself. Conformational changes that result in changes in FRET may be triggered by changes in mechanical tension applied to the molecule or changes in strain.
Figure 4. Schematic picture of fibronectin, a large multimodular protein from the extracellular matrix[36]. If fibronectin is affinity-purified on gelatin[39,40] and eluted with 4M Urea[41], it has a contour length of approximately 140 nm[42]. Fibronectin has four free cysteine residues that may be specifically labeled with FRET acceptors (red)[43]. There are numerous (~160) lysine residues that may be randomly labeled with FRET donors. The modules colored in yellow are within 2 Förster radii of the nearest acceptor. Donors on these modules may participate significantly in FRET with the nearby acceptors. Modules colored in green may participate in FRET with acceptors if the molecule
~140 nm
bends significantly enough that distant parts of the molecule can participate in FRET with the
acceptors. Fibronectin has two nearly identical monomers joined with disulfide bonds represented by two yellow lines connecting the monomers[44]. These bonds may be eliminated (reduced) with dithiothreitol (DTT) which converts the molecule into its component monomers[41,45–48].
Fibronectin can exist in many different conformations depending on conditions. Extended
conformations of fibronectin, conformations where there is little interaction between the arms of the fibronectin dimer, may be seen when fibronectin in a solution containing 40% glycerol and 0.2 M ammonium carbonate is sprayed onto a hydrophilic mica surface (Figure 5). For fibronectin in solution under physiological conditions, these conformation are rare. There is a significant interaction between the fibronectin monomers which causes a FRET signal between donors on non-local parts of the
protein that are not immediately adjacent to the FRET acceptors[49]. By converting fibronectin into its monomer components, this non-local FRET signal is significantly suppressed so that the FRET signal is dominated by the local interaction between donors on portions of the protein that are immediately adjacent to the FRET acceptors. By modeling our FRET results geometrically as a length, we obtained good agreement with contour length measurements made by direct measurements of fibronectin
sprayed onto a surface and imaged with electron microscopy[41].
Figure 5. Electron micrograph of rotary shadowed fibronectin sprayed onto mica from glycerol
solutions containing 0.2 M ammonium carbonate (Erickson and Carrell 1983)[41]. A conformation of fibronectin such as those shown in this figure where the molecule is significantly stretched out is known as an “extended” conformation. The arms of the fibronectin dimer do not significantly interact with each other and the FRET signal is dominated by FRET donors attached to portions of the molecule that are local to the FRET acceptors. One may approximate the FRET signal of an extended
conformation by splitting the fibronectin dimer into monomers. This significantly suppresses the FRET signal from donors attached to non-local parts of the molecule.
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