Mechanical interactions at the levels of cells, organs and organisms are responsible for such familiar physiological functions as motor function, hearing [18], touch [19], and the regula
Trang 1sensor for specific proteins in situ
Fanjie Meng, Thomas M Suchyna and Frederick Sachs
Center for Single Molecule Biophysics, Department of Physiology and Biophysics, State University of New York at Buffalo, NY, USA
Mechanical stress is one of the most influential
physical factors in biology and one of the least
charac-terized Whereas it is obvious from molecular
dyna-mics [1–4] and force spectroscopy [5–12] that forces
deform molecules, the mechanics of cells are much
more complicated, involving the interaction of
hetero-geneous polymers and membranes and their interaction
with both two-dimensional heterogeneous liquid
mem-branes [13,14] and three-dimensional cytoplasmic
solu-tions, where signaling factors can vary in time and
space [15–17] Mechanical interactions at the levels of
cells, organs and organisms are responsible for such familiar physiological functions as motor function, hearing [18], touch [19], and the regulation of blood pressure [20], but the interactions are also deeply embedded in the biochemistry of the cell, affecting such varied processes as the phenotype of stem cells [21], DNA transcription [22,23], translation of cellular components by motor proteins such as kinesin [5], stress-induced changes of structure, such as occur in shear stress modulation of the cytoskeleton of the endothelia [24,25], and more general interactions due
Keywords
Cerulean; fluorescence resonance energy
transfer; relative orientation factor; Venus;
a-helix linker
Correspondence
F Sachs, Center for Single Molecule
Biophysics, Department of Physiology and
Biophysics, State University of New York at
Buffalo, 3435 Main Street, Buffalo, NY,
14214 USA
Fax: +1 716 829 2569
Tel: +1 716 829 3289 ext 105
E-mail: sachs@buffalo.edu
(Received 15 December 2007, revised 9
April 2008, accepted 11 April 2008)
doi:10.1111/j.1742-4658.2008.06461.x
To measure mechanical stress in real time, we designed a fluorescence reso-nance energy transfer (FRET) cassette, denoted stFRET, which could be inserted into structural protein hosts The probe was composed of a green fluorescence protein pair, Cerulean and Venus, linked with a stable a-helix
We measured the FRET efficiency of the free cassette protein as a function
of the length of the linker, the angles of the fluorophores, temperature and urea denaturation, and protease treatment The linking helix was stable to
80C, unfolded in 8 m urea, and rapidly digested by proteases, but in all cases the fluorophores were unaffected We modified the a-helix linker by adding and subtracting residues to vary the angles and distance between the donor and acceptor, and assuming that the cassette was a rigid body,
we calculated its geometry We tested the strain sensitivity of stFRET by linking both ends to a rubber sheet subjected to equibiaxial stretch FRET decreased proportionally to the substrate strain The naked cassette expressed well in human embryonic kidney-293 cells and, surprisingly, was concentrated in the nucleus However, when the cassette was located into host proteins such a-actinin, nonerythrocyte spectrin and filamin A, the labeled hosts expressed well and distributed normally in cell lines such as 3T3, where they were stressed at the leading edge of migrating cells and relaxed at the trailing edge When collagen-19 was labeled near its middle with stFRET, it expressed well in Caenorhabditis elegans, distributing simi-larly to hosts labeled with a terminal green fluorescent protein, and the worms behaved normally
Abbreviations
CFP, cyan fluorescent protein; COL-19, collagen-19; D ⁄ A ratio, donor emission to acceptor emission ratio; DIC, differential interference contrast; E, fluorescence resonance energy transfer energy transfer efficiency; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; HEK, human embryonic kidney; YPF, yellow fluorescent protein.
Trang 2to the physical chemistry of concentrated protein
solu-tions [26] To dissect which stresses affect which
func-tions, we need labels that are sensitive to mechanical
stress and that can be attached to specific proteins
To meet that need, we designed a cassette
(denoted stFRET) that can be inserted into
struc-tural proteins and reports molecular strain via
changes in fluorescence resonance energy transfer
(FRET), and, with appropriate calibration, molecular
stress The cassette consists of the green fluorescent
protein (GFP) monomers Cerulean and Venus
[27–31], linked by a stable a-helix [32] This article
characterizes the properties of the probes, and shows
that they can be efficiently incorporated into
struc-tural proteins such as collagen-19 (COL-19),
nonery-throcyte spectrin, a-actinin and filamin A within
living cells, and that the FRET from this cassette
changes with stress in situ
The efficiency of energy transfer for a FRET pair
is E 1 ⁄ [1 + (R ⁄ RO)6], where R is the distance
between the dipoles and RO is the characteristic
distance for 50% energy transfer [33] The maximal
sensitivity for changes in R occurs at R = RO For
Venus and Cerulean, RO is 5 nm [34], so we linked
them with a 5 nm a-helix The efficiency is affected
by the angle between the transition dipoles as well as
the distance between them, and we estimated the
probe geometry by varying the number of residues in
the linker Removing one residue caused a large
change in angle with a small change in distance, and
adding or removing a full turn produced a change in
distance with no change in angle We used six
mutants to solve for the three relevant angles of the
dipoles, assuming that the cassette was rigid stFRET
was stable over temperature and mild urea denaturing
conditions, but with 8 m urea, the linker unfolded
and the fluorophores remained stable Thus, stFRET
is robust
stFRET expressed well in various biological systems,
including 3T3 and human embryonic kidney
(HEK)-293 cells and in Caenorhabditis elegans After insertion
into a variety of structural host proteins such as
colla-gen, filamin, actinin and spectrin, it distributed in the
same manner as the same hosts with terminal GFP
tags stFRET changed FRET with the spontaneous
movement of motile cells, decreasing efficiency in
regions under tension and increasing it in regions
expected to be free of significant stress By axially
stretching C elegans, we could demonstrate acute
reversible changes in FRET associated with tension
and relaxation stFRET opens the door to studying in
real time many physiological processes that are
modu-lated or driven by mechanical stress
Results
General configuration and FRET spectra of stFRET and its variants
Figure 1 is a diagram of stFRET geometry as deduced from the procedure described in Modeling and calibra-tion in the Experimental procedures Figure 2A shows the general configuration of six stFRET variants The inward arrows show the excitation wavelength, and the outward arrows show the emission wavelength Width
of arrows denotes light intensity Figure 2B shows the alignment of the DNA sequence of the linker with five modified versions (the predicted geometrical changes are shown in Table 1) As shown in Table 1, according
to the general property of a-helices, one amino acid deletion produces a change ()100) in angle with negli-gible change ()1.5 A˚) in length A five residue deletion
of the helix rotates the structure by 360 but shrinks the helix by 2.7 nm Deletion or addition of two and a half turns of the helix twists the structure by)180 or +180 and decreases or increases the length by 1.35 nm Figure 2C gives the amino acid sequence and the segments of the helix linker that we modified Deletion of 18 amino acids eliminates five turns of the helix, and a nine amino acid deletion eliminates two and half turns
Figure 3A shows the emission spectrum of stFRET with excitation at 433 nm There are peaks at 475 nm
Table 1 Changes in stFRET geometry caused by adding and delet-ing amino acids Positive symbols indicate an increasdelet-ing amount, and negative symbol indicate a decreasing amount.
No amino acids added or subtracted
Change in length
of linker (nm)
Change in angle
of linker (radians)
Fig 1 Geometry of stFRET D and A are donor and acceptor dipole vectors, and r is the length of the linker The three angles (h A , h D , U) are the unknown parameters R A–D is the distance between acceptor and donor chromophores.
Trang 3and 527 nm, with the 475 nm emission from the donor
Cerulean and 527 nm from the acceptor Venus having
robust energy transfer A 100 lm solution of unlinked
donor and acceptor (1 : 1 mixture, green filled squares
and line with 433 nm excitation) had a small emission
at 527 nm due to the bleed-through from Cerulean,
the donor (blue filled inverted triangle and line) and
some direct excitation of the acceptor Venus by
433 nm (black triangle and line) The donor and
accep-tor mixture had E = 0 and donor emission to accepaccep-tor
emission ratio (D⁄ A ratio) = 2.47 ± 0.05 (Fig 3B)
However, for stFRET, E = 44 ± 2.5% and D⁄ A
ratio = 0.47 ± 0.02, showing efficient energy transfer (for E and D⁄ A ratio calculation, see Experimental procedures)
Calibration of three angles and j2
Confident in the origin of stFRET energy transfer, we purified the other five variants and measured their flu-orescence (Fig 4A) All mutants exhibited robust FRET (Table 2) stFRET itself had 44 ± 2.5% energy transfer, and the 5T construct had the highest effi-ciency, E = 56 ± 4.5%, the 2.5T construct increased
A
B
C
Fig 2 Construction of stFRET protein and five variants (A) Schematic structure of stFRET Cyan is the donor, Cerulean; yellow
is the acceptor, Venus The height of the b-can structure is 4.2 nm The black helix is the linker, and it nominal length is 5.0 nm Incoming arrows indicate excitation, and outgoing arrows indicate emission, with the wavelength marked next to them; the width
of the arrows is proportional to the light intensity (B) Alignment of the primary and modified linker DNA sequences (C) Modifi-cations to the linker with DNA and amino acid sequences.
Trang 4E to 47 ± 2.1%, whereas the 2.5I construct decreased
E to 37 ± 0.9% FT1AA and FT2AA, presumably
only having their angles changed, decreased E to
29 ± 7.1% and 38 ± 4.3%, respectively (Fig 4B)
Table 2 summarizes the apparent change of angles and
distances obtained by modifying the linker and the
corresponding energy transfer efficiency
If we assume that a single residue alters the linker
length by a translation of 0.15 nm and 100, and that
the structure is rigid, we can use the data in Table 2 to
solve for the probe geometry (see Experimental
proce-dures) The numerical solutions gave hA= 3.83,
hD=)0.78 and U = 1.97 radians, and j2= 0.86,
which is 30% higher than 2⁄ 3, the j2value that one
would obtain assuming random rotation of the donor
and acceptor (Fig 1) However, it should be pointed
out that a value of j2 2 ⁄ 3 does not necessarily imply
that the probes are moving randomly
Stability of the linker as perturbed by urea,
temperature, and proteinase K
We did a number of tests to assess linker integrity If
the linker was an a-helix, then melting would increase
the end–end spacing and the efficiency would decrease
With urea as a denaturant [35,36], Fig 5A shows that
the efficiency of stFRET declined with concentration
up to 8 m, and the previously quenched donor
emis-sion recovered Remarkably, the fluorophore spectra
were almost unaffected by urea, with < 10–15%
change in amplitude (Fig 5C,D) Figure 5B shows
that 1–8 m urea caused the D⁄ A ratio to increase
from 0.46 to 1.21, as expected if the helix unfolded
into a random coil allowing the donor and acceptor to
move further apart and reducing energy transfer
(Fig 5E)
As a second test of the helix stability, we tried to
melt stFRET at elevated temperatures, but the protein
proved stable up to 80C Figure 6A shows the
tem-perature dependence of fluorescence of 100 lm
stFRET protein excited at 433 nm from room
temper-ature to 80C Donor and acceptor emission both
declined somewhat as the temperature increased,
prob-ably due to a direct change in quantum efficiency, but
there was no significant change in transfer efficiency
from 60C to 80 C, the upper limit of our
measure-ments, so that the linker structure can be considered to
be quite robust
As a final test of linker integrity, we digested
stFRET with proteases that cut the linker but left the
fluorophores intact Figure 7 shows that proteinase K
led to a rapid fall in efficiency that was complete
within 1 min The D⁄ A ratio changed from 0.42 to
1.95 over 30 min (Fig 7B), as compared to a change from 0.46 to only 1.21 when the protein was treated with 8 m urea (Fig 5B) Similar behavior was found for all six constructs (data not shown) The donor and acceptor fluorophore spectra were unaffected by pro-teinase K after 30 min of digestion (Fig 7C,D) Figures 5E and 7E are diagrammatic models summa-rizing the energy transfer between donor and acceptor under different treatments (the width of the arrows represents signal intensity)
In vitro measurement of strain sensitivity
To verify the strain responsiveness, we bonded the ends of derivatized stFRET to a silicone rubber sheet using StreptagII–Streptactin and stretched the sheet equibiaxially on the fluorescence microscope When the C-terminal and N-terminal ends of stFRET were derivatized so that it would be stretched with the sheet, there was a reversible 11% decrease in the D ⁄ A ratio (Fig 8) As a control, we measured FRET from stFRET that was derivatized at one end only so that it was simply immobilized but not stretched and there was no significant change in FRET with strain (Fig 8) Nonspecific binding of double-tagged stFRET
to an untreated silicone surface also produced no sig-nificant change in FRET with strain Thus, stFRET is sensitive to strain, as expected from the solution assays and the design of the probe
Eukaryotic expression and targeting property
of stFRET Before inserting stFRET into host proteins, we placed the gene under a eukaryotic promoter (human cyto-megalovirus) and transiently transfected HEK cells with stFRET alone Control transfections with Venus
or Cerulean monomers showed no preferential locali-zation and no obvious energy transfer (Fig 9A–F) Cells transfected with stFRET displayed significant energy transfer (Fig 9I) stFRET localized to the nucleus with an extremely high density in the nucleoli (Fig 9K)
Nuclear targeting proteins have a consensus amino acid sequence of lysine⁄ arginine [K ⁄ R(4–6)] or smaller clusters separated by 10–12 amino acids: [K⁄ R(2)X(10–12)K ⁄ R(3)] [37] The linker has multiple arginine clusters similar to the nuclear targeting sequence, but simply removing one or the other fluoro-phores from stFRET produced a uniform cytoplasmic distribution showing that the linker’s sequence alone was not sufficient for targeting These unexpected nuclear targeting properties of stFRET may provide
Trang 5a useful tool for understanding nuclear protein
transport
Host proteins of stFRET with normal expression
showing stress sensitivity
We inserted stFRET into various host proteins,
inclu-ding COL-19 (Fig 10G), nonerythrocyte spectrin
(Fig 10E), filamin A (Fig 10C), and a-actinin
(Fig 10A), and expression systems including HEK-293,
3T3 and C elegans, and the insertion locations were
optimized to obtain protein distributions similar to
those observed for the host protein C-terminus tagged
with GFP or Cerulean (Fig 10B,D,F,H) Inserting
stFRET into host proteins eliminated nuclear targeting
The fluorescence of stFRET in cultured cells was
located in the cytoplasm and⁄ or the cell membrane, depending on the host (Fig 10A,C,E) We expressed the construct of the most abundant collagen in C elegans, COL-19, and the protein was properly assem-bled, showing the typical striated pattern, and the worms behaved normally When we stretched the worm with micromanipulators, the labeled COL-19 showed a decrease in FRET efficiency with stretch, and in convex regions as it actively wiggled (Fig 10G,H)
Figure 11 indicates that stFRET integrated into actinin and filamin can sense tension in situ Migrating
A
B
C
Fig 4 Modification of the linker change FRET efficiency of six con-structs (A) Fluorescence spectra of stFRET and its five variants (scan parameters as in Fig 3) (B) FRET efficiency of the six con-structs (C) SDS ⁄ PAGE gel of the purified proteins, and Cerulean and Venus monomers FT stands for stFRET; 5T and 2.5T are con-structs with five-turn or 2.5-turn deletions from the linker; 2.5I is the construct with a 2.5-turn insert; FT1AA and FT2AA are the con-structs with one amino acid or two amino acid deletions All values are means ± SD, and the data were obtained with proteins from three separate purifications.
A
B
Fig 3 FRET efficiency and D ⁄ A ratio (mean ± SD) (A) Spectra of
stFRET, Cerulean and Venus monomers and Cerulean and Venus in
a 1 : 1 mixture Venus + Cerulean mixture, green filled squares.
Donor Cerulean, blue inverted triangles and line Acceptor Venus,
black triangles and line Pure stFRET protein, red filled circles and
line (B) FRET efficiency and D ⁄ A ratio of stFRET with Cerulean
and Venus in a 1 : 1 mixture Data were obtained with protein from
three separate purifications CV is Cerulean and Venus in a 1 : 1
mixture FT, stFRET Excitation 433 nm; emission 460–550 nm.
Trang 63T3 cells have a characteristic leading and lagging
edge, and Fig 11A–C shows the donor, acceptor and
FRET images from three confocal microscopy
chan-nels stFRET was distributed evenly across the cyto-plasm as visualized with a 16-color pseudocolor map (Fig 11C,D) Transfection with actinin–stFRET
Table 2 Values of parameters in Eqn (8) of six stFRET variants as described in Fig 4.
Protein
constructs
Energy transfer
efficiency (E) (%)
r (linker length) (nm)
Z (H ⁄ 2 of b-Can) (nm)
h A (unknown parameter 1)
h D (unknown parameter 2)
U (unknown parameter 3)
Fig 5 Melting the linker (A) Spectra from
stFRET treated with 1–8 M urea (scan
parameters as in Fig 3B) (B) D⁄ A ratio of
stFRET after treatment with different
con-centrations of urea (means ± SD, n = 3 in
each treatment); increasing D ⁄ A ratio
indi-cates the recovery of donor emission and
decrease of energy transfer (C) Cerulean
monomer fluorescence with urea
treat-ments (scan parameters as in Fig 3) (D)
Venus monomer fluorescence with urea
(excitation at 515 nm and scan 520–
600 nm) (E) Urea melts the linker and
leaves the donor and acceptor intact,
decreasing FRET energy transfer as donor
emission recoverers and D ⁄ A ratio increases
(definitions as in Fig 2A).
Trang 7revealed that during migration, the lagging edge
showed higher energy transfer than the leading edge
(Fig 11E,F), i.e it was relaxed We measured the
effi-ciency of various domains in the lagging and leading
edges from 14 confocal image stacks The lagging
edges (the red-outlined domain) nearly doubled the
FRET efficiency as compared to the leading edge
(blue- and green-outlined domains) Multiple cells had
the same behavior, but because of the complexity of
the various shapes it was difficult to arrive at any
use-ful statistic for frequency We have shown a typical
cell with different domains as an internal control The
same phenomenon was observed in
filamin–stFRET-transfected 3T3 cells (Fig 11G–L) Figure 11G–I
shows three confocal image channels, and Fig 11J is
the pseudocolor image of stFRET protein distribution
Figure 11K is the FRET efficiency image in which three domains were selected The efficiency in the red-outlined domain is twice as high as that in the blue and green domains (Fig 11L) These data suggest that tension in both actinin and filamin is lower in domains close to the lagging edge (where adhesion to the sub-strate is released), and higher at the leading edge where adhesions pull the cell forward
Discussion
Designed to be an in situ stress sensor, stFRET has robust and predictable energy transfer both in vitro and in vivo We were able to explore the geometry of stFRET by perturbing the linker length and terminal angles using the known properties of a-helices FRET efficiency changed in a predictable manner with the postulated geometry, suggesting that the fluorophores are not free to rotate A recent molecular dynamics simulation study of FRET in lysozyme found that j and ROcould be correlated by as much as 0.8, so that FRET measurements that assume random rotational freedom are likely to be in error [38] The ability to change angle and distance by varying the linker can be used in vivo to examine the effect of host proteins on probe geometry Regardless of the coupling of the flu-orophores to the linker, all of the host proteins that
we studied were coiled-coiled dimers or trimers, so that the fluorophores of stFRET would not be able to rotate freely
Figure 2A shows the predicted mean structure of free stFRET The three unknown angles of Eqn (8) (see Experimental procedures) were solved using data for the six mutants using the least squares equation solver in maple The solutions were stable to perturba-tions of the starting values, suggesting that we were measuring a constrained system Our final solution was
hA= 3.83, hD=)0.78, and U = 1.97, yielding
j2= 0.86 There will be bending and flexing motions
of the structure in solution, but we obtained consistent answers from the overdetermined set of equations, sug-gesting that the calculated mean values are at least self-consistent The geometric values that we have cal-culated would represent mean values weighted by the efficiency Fluctuations that bring the dipoles closer are more heavily weighted than those that move them further away, although the probability of occupancy of these conformations is another weighting factor A detailed molecular dynamics simulation would be use-ful, but is not essential for the use of stFRET as a probe of molecular stress, as the most important vari-ables are the differences in efficiency, i.e the gradients
of stress
A
B
Fig 6 a-Helix linker in stFRET is resistant to temperature melting.
(A) stFRET spectra obtained at 60 C for 2 min, 60 C for 5 min,
70 C for 5 min and 80 C for 5 min (scan parameters as in Fig 3).
(B) stFRET D ⁄ A ratio after different temperature treatments
(means ± SD, n = 3 in each treatment) Temperatures are given in
degrees Celsius; roomtem, room temperature.
Trang 8The robust nature of stFRET was clear from the
melting experiments stFRET was thermally stable up
to at least 80C, with the FRET efficiency being
virtu-ally unchanged Melting the linker with urea (Fig 5)
[39] left the fluorophores untouched (Fig 5C,D), but
decreased the energy transfer, consistent with
unfold-ing of the linker (Fig 5B,E) Two models have been
proposed for urea-induced protein denaturation: the
binding model, in which the denaturant binds weakly
but specifically to sites exposed by the unfolded
pro-teins [40], and a solvent exchange model, in which the
interaction of the solvent and the denaturant is a
one-for-one substitution reaction at particular sites [41]
stFRET might serve as a useful probe to examine these
alternatives
The sensitivity of stFRET to protease cleavage has
both positive and negative implications If proteases
are accidentally present in situ, they could cleave
stFRET and provide misleading results We saw no evidence of protease activity in HEK or 3T3 cells
or C elegans However, the presence of intracellular proteases has been associated with acute pancreatitis, proposed to arise from trypsin overactivation in large endocytotic vacuoles of acinar cells [42] Thus, to study pancreatitis, stFRET may be a useful probe (Fig 7)
Having established the basic physical properties of stFRET, we expressed it in HEK cells (Fig 9) and evaluated the energy transfer by Xia’s method [43], using confocal microscopy The surprising localization
of stFRET to the nucleus was proved not to be a result of the linker possessing a consensus nuclear tar-geting sequence, as deletion of either fluorophore from the construct destroyed localization This adaptability suggests that stFRET can serve as a useful probe of nuclear targeting
Proteinase K
C
E
D
Fig 7 Two units of proteinase K
(1 unitÆlL)1) digests the linker but not
Cerulean or Venus (A, C, D) Spectra of
stFRET protein (A), Cerulean (C) and Venus
(D) digested for 20 s, 1 min, 2 min, 3 min,
5 min, 10 min, 15 min and 30 min at room
temperature with 200 lL of 100 l M protein.
(B) Time course of D ⁄ A ratio for
protein-ase K digestion of stFRET (E) Proteinprotein-ase K
cleaved the linker and eliminated FRET
in stFRET protein PK, proteinase K; S,
seconds; M, minutes; n = 3.
Trang 9Knowing how native stFRET itself distributes,
we incorporated it into host proteins, including nonerythrocyte spectrin, filamin A and a-actinin (Fig 10A,C,E) in 3T3 cells, and COL-19 in C elegans (Fig 10G) The distribution of the probe depended upon where the cassette was placed within the host When it was inserted towards the middle, the fluores-cence distribution appeared similar to that of the host protein tagged with GFP or cyan fluorescent protein (CFP) at the C-terminus (Fig 10B,D,F,H) Insertion
of the cassette towards the termini of the host led to different spatial distributions There is no gold stan-dard for the proper localization of proteins in cells, as fixation and exposure to various tracer ligands can produce changes in structure, but, to first order, the stFRET probes placed in the middle of the hosts appeared to cause minimal perturbation
Under physiological conditions, FRET efficiency varied in different regions of the cells (Fig 11), and these seemed to be correlated with the anticipated distribution of stress Efficiency should be reduced when the host is under tension Actinin–stFRET and filamin–stFRET generally showed lower efficiency than free stFRET (Fig 3B), suggesting that those proteins were normally under tension (Fig 11E,F,K,L,
green-1.5
2.0
2.5
3.0
Untreated - double Strep-Tag2 Strep-tactin treated - double Strep-Tag2 Strep-tactin treated - single Strep-Tag2
11%
1 s
100 mmHg
Fig 8 Double Streptag II-tagged stFRET shows a decrease in
FRET ratio when stretched on silicone rubber disks Single and
dou-ble Streptag II-tagged stFRETs were allowed to bind to either
untreated or Strep-tactin-modified silicone disks The FRET ratio
was monitored in 10 spots on each disk during application of the
suction stimulus shown Only the disks with Strep-tactin-treated
surfaces and stFRET proteins with Streptag tags at both the
C-ter-minus and N-terC-ter-minus showed a significant change in FRET ratio
when stretched.
Fig 9 stFRET expressed in HEK-293 cells exhibits efficient FRET (A–C) Confocal refer-ence image of Cerulean taken from the CFP channel (A) and the DIC channel (B), with the overlap in (C) (D–F) Reference image of Venus from the YFP (D) and DIC channels (E), with the overlap in (F) (G–K) Images of stFRET using the CFP channel (G), YFP channel (H), FRET channel (I) and DIC nel (J), with the overlap of these four chan-nels in (K) (L) The vFRET index was calibrated pixel by pixel using Xia’s method [43] Hollow black regions were excluded from the calculation because of intensity saturation stFRET is localized in the nucleus and especially concentrated in the nucleoli (arrowheads).
Trang 10line and blue-line domains) However, as cells migrate,
the stress in the leading and trailing edges changes
Connections to the extracellular matrix in the lagging
edge must be disengaged and the connections at the
leading edge put under tension Figure 11E,F,K,L
shows increased FRET efficiency in the lagging edge
as the filopodia were released from the substrate and
tension decreased (red-line domains), and decreased
efficiency associated with increased tension in the lead-ing edge as the cell was pulled forward
To turn stFRET from a strain sensor into a stress sensor, we need to measure its force–distance properties
At the current time, we only have estimates from pub-lished atomic force microscopy data on the stretching
of the coiled-coil myosin II [44] Schweiger et al [45] obtained a three-phase force–distance relationship: a linear phase of 1 mNÆm)1, a plateau of 25 pN, and
a wormlike chain phase as the helices were stretched closer to the contour length The presence of a force plateau implies that if monomeric stFRET was sub-jected to a force of > 10–25 pN, it would unfold in an all-or-none manner for about 3 nm, producing a large drop in FRET We do not see this, probably in part because the in situ probes are not homomers, but are coiled coils where the stress is shared with labeled and unlabeled neighbors It may be possible to knock down the background hosts to at least create homogeneously labeled hosts In addition, stress is shared between different proteins within the cell, and at the current time, we are only probing one of those components stFRET can be applied to any biological system with large covalently bonded proteins It is possible to examine the role of stress in selected proteins within cells or even within free-ranging organisms With organ targeting in small organisms such as C elegans and zebrafish, it should be possible to develop high-contrast video images of specific parts of the organism during controlled or natural behavior We look for-ward to finding out how mechanical stress is coupled
to biochemistry and to cell biology
Experimental procedures
Gene construction and protein purification pEYFP-C1 Venus and pECFP-C1 Cerulean plasmids were generous gifts from D W Piston (Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, USA) [45] The Cerulean gene was subcloned from pECFP-C1 with the primers 5¢-GCAGGTGTGAATTCCATGGTGAGCAAGGGCGAG GAGC-3¢ and 5¢-CCAGATCGCGGCCGCCTTGTACAG CTCGTCATGCCGAGAG-3¢; EcoRI and ApaI restriction enzyme sites were introduced into the 5¢-end and 3¢-end of the Cerulean DNA fragment This DNA fragment was inserted into multiple cloning sites of pEYFP-C1 Venus by EcoRI and ApaI digestion and ligation The resulting vector has Venus followed closely by Cerulean, and between them there are two restriction enzyme sites, BglII and EcoRI, which then were employed to insert the a-helix linker The a-helix linker DNA, 5¢-GGCCTGCGCAAGCGCTTACG
A
C
B
D
Fig 10 Normal expression of stFRET in various host proteins.
a-Actinin–stFRET (A), a-actinin–GFP (B), filamin A–stFRET (C),
filam-in A–CFP (D), spectrfilam-in–stFRET (E) and spectrfilam-in–CFP (F) filam-in 3T3
fibro-blast cells; Collagen-19–stFRET (G) and COL-19–GFP (H) in
C elegans (with assistance of R Gronostajski; Biochemistry
Department, State University of New York at Buffalo, NY, USA).
Arrowheads indicate the striated expression pattern and central line
in the worm cuticle.