Cell Motility and the Cytoskeleton 65: 353–367 (2008)
Tracking Changes in Z-Band Organization
During Myofibrillogenesis With FRET Imaging
Andrea L. Stout,1 Jushuo Wang,2 Jean M. Sanger,2 and Joseph W. Sanger2*
1
Department of Cell and Developmental Biology, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania
2
Department of Cell and Developmental Biology, SUNY Upstate Medical
University, Syracuse, New York
There are a large number of proteins associated with Z-bands in myofibrils, but the
precise arrangements of most of these proteins in Z-bands are largely unknown.
Even less is known about how these arrangements change during myofibrillogenesis. We have begun to address this issue using Sensitized Emission Fluorescence
Resonance Energy Transfer (SE-FRET) microscopy. Cultured skeletal muscle cells
from quail embryos were transfected to express fusions of alpha-actinin, FATZ,
myotilin, or telethonin with cyan and yellow fluorescent proteins in various pair
wise combinations. FATZ and myotilin were selected because previous biochemical studies have suggested that they bind to alpha-actinin, the major protein of the
Z-band. Telethonin was selected for its reported ability to bind FATZ. Statistical
analysis of data from FRET imaging studies yield results that are in agreement with
published biochemical data suggesting that FATZ and myotilin bind to alpha-actinin near its C-terminus as well as to each other and that a region near the amino-terminus of FATZ is responsible for its interaction with telethonin. In addition, our
analysis has revealed changes in the arrangement of alpha-actinin and FATZ that
take place during the transition as the z-bodies of premyofibrils fuse to form the Zbands of mature myofibrils. There was no evidence for a change in the arrangement
of myotilin as z-bodies transformed into Z-bands. Myotilin is one Z-band protein
that does not exhibit decreased dynamics as z-bodies fuse to form Z-bands. These
FRET results from living cells support a stepwise model for the assembly of myofibrils. Cell Motil. Cytoskeleton 65: 353–367, 2008. ' 2008 Wiley-Liss, Inc.
Key words: fluorescence; microscopy; alpha-actinin; myotilin; FATZ; telethonin; z-bodies; Z-bands
INTRODUCTION
Striated muscle is notable for its ability to generate
and sustain coordinated mechanical forces. Behind this
lies a remarkable degree of organization that arises during
development of the embryo and that is maintained through
an organism’s lifetime by the orderly exchange of bound
sarcomeric proteins with those in the cytoplasmic pool
[Wang et al., 2005a, 2007, 2008]. Inside a muscle fiber,
the myofibril is the basic structural unit comprising the
force generation machinery [Clark et al., 2002]. Myofibrils are comprised of several types of filaments that are
organized into distinct bands. We have proposed a threestage mechanism, depicted in Fig. 1, for the assembly of a
mature myofibril [Rhee et al., 1994; Sanger et al., 2002,
' 2008 Wiley-Liss, Inc.
2005]. First, alpha-actinin and a few other proteins organize into premyofibrils. These premyofibrils are composed
of minisarcomeres formed by alpha-actinin-enriched
Contract grant sponsors: National Institutes of Health, Muscular Dystrophy Association.
*Correspondence to: Joseph W. Sanger, Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse,
NY 13210, USA. E-mail: [email protected]
Received 28 August 2007; Accepted 14 January 2008
Published online 10 March 2008 in Wiley InterScience (www.
interscience.wiley.com).
DOI: 10.1002/cm.20265
354
Stout et al.
punctate structures called z-bodies and interdigitating filaments comprised of actin and non-muscle myosin IIB
[Dabiri et al., 1997; Wang et al., 2005b]. Then, these zbodies align to form nascent myofibrils with the addition
of titin and overlapping muscle myosin II filaments
[Dabiri et al., 1997]. Finally, the aligned z-bodies fuse to
form the characteristic Z-bands and the non-muscle myosin II filaments are lost, while the overlapping arrays of
muscle myosin II filaments align with each other to form
A-bands in the mature myofibrils (Fig. 1). A number of
questions about the process of myofibrillogenesis remain
to be answered, such as how z-bodies are nucleated, in
what order do z-body and Z-band proteins appear, and
how is the arrangement of proteins different in z-bodies
and Z-bands. These questions are of import since other
models of myofibrillogenesis suggest no changes in the
formation of Z-bands [reviewed in Sanger et al., 2005].
To address some of these questions, we have
turned to live cell imaging of cultured quail myotubes
expressing fluorescent protein fusions with a number of
proteins known to localize to the Z-band. Multicolor fluorescence microscopy is often used as a tool to examine
distributions of molecules in living cells, and in our laboratory we have used this technique extensively to gain
insight into the development of skeletal and cardiac muscle [Dabiri et al., 1997; Du et al., 2003; Wang et al.,
2005a,b]. When investigating complicated protein networks and protein-protein interactions within such networks, however, conventional fluorescence imaging cannot always provide the desired information due to the
lower limit (!200 nm) on spatial resolution. In recent
years the phenomenon of FRET (Fluorescence Resonance Energy Transfer), in conjunction with wide-field
and confocal microscopy, has been harnessed to provide
a means of surmounting this resolution limit [LippincottSchwartz et al., 2001; Jares-Erijman et al., 2003]. FRET
is a non-radiative exchange of energy between two fluorophores, an excited state ‘‘donor’’ and a ground-state
‘‘acceptor’’. The probability that this energy transfer
will take place is highly dependent on the degree to
which the donor emission spectrum overlaps with the
acceptor excitation spectrum as well as on the separation
between and relative orientation of the two molecules.
For most fluorophores commonly used in biological
applications, this probability drops to nearly zero for separations greater than about 10 nm [Clegg, 1996; Gordon
et al., 1998; Berney and Danuser, 2003]. The strong distance-dependence of FRET has made it a valuable tool
for revealing subtle changes in both intra- and intermolecular structures inside living cells [Day et al., 2001;
Zal and Gascoigne, 2004]. There are several approaches
to measuring FRET, but for mapping energy transfer in
living cells one of the most popular is sensitized emission FRET (SE-FRET) microscopy [Berney and Dan-
user, 2003]. In SE-FRET imaging, the illumination is
tuned to the donor’s absorption maximum while fluorescence is collected near the acceptor’s emission maximum. Signal from non-FRET sources is subtracted away
to leave an image where intensity is correlated with the
strength of the FRET response.
We have used this method to look for changes in the
arrangement of Z-band proteins during myofibrillogenesis, to see if the large-scale morphological changes (zbodies to Z-bands) visible in fluorescence images are
accompanied by smaller-scale changes in the arrangement
of proteins, especially with alpha-actinin, the major protein of z-bodies and Z-bands. We also set out to investigate reports of interactions among several recently discovered Z-band proteins, namely two alpha-actinin binding
proteins FATZ (also known as myozenin and calsarcin-2),
telethonin (or T-cap), and myotilin [Faulkner et al., 2001].
Recent FRAP (Fluorescence Recovery After Photobleaching) experiments comparing mobilities of proteins in zbodies and Z-bands have found that not only do different
proteins have different replacement rates, but also that
these rates can change as development proceeds from the
z-body to the Z-band stage [Wang et al., 2005a]. For
example, in FRAP experiments examining YFP-fused
alpha-actinin, an actin-filament cross-linker that is one of
the major components of the Z-band, Wang et al., [2005a]
found that there is a larger exchangeable fraction of the
population in premyofibrils than in mature myofibrils,
with slightly faster exchange rates in z-bodies than in Zbands. In contrast to the decreased dynamics of alpha-actinin and FATZ, there was no change in the dynamics of
myotilin in the transition from z-bodies to Z-bands [Wang
et al., 2005a]. We chose to examine various combinations
of four Z-band proteins: alpha-actinin, FATZ, myotilin,
and telethonin, and we found statistically significant differences in the FRET signal between early-stage z-bodies
and later-stage Z-bands for some, but not all, of the protein pairs. While there was evidence for differences in
FRET signals for alpha-actinin/alpha-actinin pairs and
alpha-actinin/FATZ pairs, there was no evidence for a
change in the arrangement of myotilin molecules with either alpha-actinin or FATZ as z-bodies transformed into
Z-bands. In addition, by examining the effect of fluorophore location on the FRET response, we have increased
our understanding of how these four proteins are arranged
in the complicated network comprising the Z-band.
MATERIALS AND METHODS
Skeletal Muscle Cell Culture and Transfection
Mononuclear myoblasts were isolated from the
breast muscle of 9-day quail embryos and plated on collagen-coated 35 mm glass-bottom culture dishes using
the method of Dabiri et al. [1999]. Myoblasts were
FRET Imaging of Z-Band Proteins
grown in minimal essential medium (MEM) containing
horse serum and chicken embryo extract in a tissue culture incubator (378C, 5% CO2) for 36–48 h before transfection. During this time, the cells begin to fuse and
form multinucleate elongated myotubes. Immediately
prior to transfection, the medium was replaced with antibiotic-free, low-serum medium. Muscle cells were transfected with 1–3 lg plasmid DNA per dish using FuGene
6 (Roche Diagnostics Corporation, Indianapolis, IN)
transfection reagent according to the manufacturer’s
instructions. Cells were allowed to grow for at least 24 h
more before changing back to the higher-serum medium
in preparation for imaging. Premyofibrils and nascent
myofibrils containing fluorescent proteins are usually
visible 24 h after transfection; mature myofibrils appear
36–48 h after transfection.
Constructs
The plasmids used in this study were assembled
using previously reported methods [Ayoob et al., 2000;
Wang et al., 2005a]. For alpha-actinin fusions, cDNA for
human skeletal muscle alpha-actinin was cloned into vectors encoding mutants of ECFP and EYFP that were kind
gifts of Dr. Roger Tsien (University of California, San
Diego, CA). Referred to here as mCFP and mYFP (mYFP
is sometimes referred to in the literature as mCitrine),
these variants differ from ECFP and EYFP by a single
point mutation (A206K). They are particularly useful for
FRET studies because they have spectral properties and
quantum yields very similar to EYFP and ECFP, but they
are less prone to dimerization, which could interfere with
native intermolecular interactions [Zacharias et al., 2002].
In experiments with CFP-FATZ and CFP-myotilin, the
cDNAs encoding human skeletal muscle mRNAs were
cloned into a vector containing another CFP mutant, ‘‘cerulean’’, which was a kind gift of Dr. David Piston (Vanderbilt University, Nashville, TN). Like mCFP, cerulean
also contains the A206K mutation. Cerulean is superior to
mCFP, however, in that it is significantly brighter and less
prone to photobleaching, and it exhibits a single fluorescence lifetime [Rizzo and Piston, 2005]. Experiments
involving YFP-telethonin and YFP-FATZ used a commercial plasmid, pEYFP (BD Biosciences Clontech,
Mountain View, CA).
Microscopy
All images were acquired on a DeltaVision Spectris imaging workstation (Applied Precision LLC, Isaquah, WA) consisting of an Olympus IX-70 microscope
(Olympus America, Melville, NY), a CoolSNAP HQ
camera (Photometrics, Tucson, AZ), with Deltavision
softWoRx software controlling filter wheel positions,
camera, shutters, and stage position. Photodetector readings were used to normalize images in order to correct
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for fluctuations in illumination that may occur during
imaging.
SE-FRET imaging was done using the standard
‘‘3-cube method’’ [Gordon et al., 1998; Zal and Gascoigne, 2004], which is described briefly here. Images
were acquired in three-channels: the donor (D) channel
(excitation filter 5 436 nm/10 nm, emission filter 5 470
nm/30 nm), the FRET (F) channel (excitation filter 5
436 nm/10, emission filter 5 535/30 nm), and the
acceptor (A) channel (excitation filter 5 500 nm/20,
emission filter 5 535/30 nm). All filters were from
Chroma Technology Corporation (Rockingham, VT).
Even if no energy transfer takes place, a sample containing both CFP and YFP will produce a large signal in the
FRET channel due to direct acceptor excitation by the
435 nm light and to spectral bleedthrough of unquenched
donor fluorescence passed by the acceptor’s emission filter. Mathematically, this can be written as
FTotal ðkDA Þ ¼ FTF þ FA ðkDA Þ þ FD ðkDA Þ þ Fauto ðkDA Þ
þ Fback ðkDA Þ
ð1Þ
FTF refers to ‘‘true FRET’’ fluorescence captured in the
FRET channel, the second and third terms refer to the
bleedthrough terms described above, the fourth to cellular autofluorescence excited and detected in the FRET
channel, and the last to all other sources of background
signal (camera noise and fluorescence from the coverslip
and medium, for example). kDA refers to the FRET channel filter conditions, with illumination and detection
wavelengths suitable for donor and acceptor, respectively. Fback was measured by averaging over a cell-free
region in each image after subtracting the contribution
from camera noise. FA(kDA) and FD(kDA) were calculated from acceptor and donor channel images using
FA ðkDA Þ ¼ aA ðkDA Þ & FA ðkAA Þ
FD ðkDA Þ ¼ aD ðkDA Þ & FD ðkDD Þ
ð2Þ
aA and aD are empirically determined parameters relating the detected ‘‘bleedthrough’’ fluorescence to the
amount of fluorescence detected in the appropriate channel for each fluorophore. These expressions are correct
as long as the camera response is linear and as long as
FA(kDD) and FD(kAA) are both negligible (which they
were for our filter sets). The desired quantity, FTF, is
therefore given by
FTF ¼ FTotal ðkDA Þ ' aA FA ðkAA Þ ' aD FD ðkDD Þ
' Fauto ðkDA Þ ' Fback ðkDA Þ
ð3Þ
To measure aA and aD, images of myotubes expressing
cyan or yellow fusion proteins only are acquired in the
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Stout et al.
same three A, F, and D channels described above, and intensity profiles along myotube axes were extracted from
these images using custom software written in the IDL
programming environment (Research Systems Inc, Boulder, CO). After smoothing each profile, intensities at local
maxima (corresponding to centers of Z-bands or Z-bodies)
were measured in each channel, then subjected to a linear
least-squares fit to extract the slopes, aA and aD:
FA ðkDA Þ ¼ aA ðkDA ÞFA ðkAA Þ þ F1 ðkDA Þ
FD ðkDA Þ ¼ aD ðkDA ÞFD ðkDD Þ þ F2 ðkDA Þ
ð4Þ
The average of the two intercepts, F1 and F2, was used
to estimate the autofluorescence component of the FRET
channel image of Z-bands, Fauto(kDA).
To obtain FRET images, the correction described
in Eq. 3 was applied on a pixel-by-pixel basis to F-channel images of doubly transfected cells. For quantitative
analysis of Z-band fluorescence, however, this correction
was applied to intensity profiles extracted from these
images. Prior to subtraction, the three profiles were manually aligned if necessary, as chromatic aberration and/
or cell movement could sometimes result in slight shifting of images. If the profiles could not be aligned (due to
distortion), they were rejected. To compare the FRET
response between samples, we converted the FRET intensity, FTF, to a dimensionless quantity, the apparent
FRET efficiency, defined as
Eapp ( 1 '
FDA
FD alone
ð5Þ
where FDA refers to fluorescence emitted by donors in
the presence of acceptors while FD alone is the fluorescence that would be emitted by the same donors in the
absence of acceptors [Clegg, 1996]. It is not possible to
measure FD alone in a sample containing both donors and
acceptors; however, as shown by Gordon et al., [1998],
Zal and Gascoigne [2004], we can define a constant of
proportionality g relating the FRET-induced acceptor
emission to the FRET-induced loss of donor fluorescence:
FTF ¼ g & ðFD alone ' FDA Þ:
ð6Þ
This g factor is a function of many things: the quantum
yields of donor and acceptor, the spectral response of the
CCD camera, and the throughput of the microscope
optics at donor and acceptor emission wavelengths
[Gordon et al., 1998; Elangovan et al., 2003; Zal and
Gascoigne, 2004]. Substituting this expression into the
previous one yields a method for calculating an apparent
FRET efficiency from measurable quantities:
Eapp ¼
FTF
FTF þ gFDA
ð7Þ
g may be estimated from measured quantum efficiencies
and from known specifications of the imaging system. It
may also be measured for a particular apparatus - see Zal
and Gascoigne [2004] for one method. While accurate
values of g are required for comparisons of efficiency
data obtained on different instruments, precise determination of this factor is less crucial in situations where a
single instrument and the same fluorophores are used for
all experiments to be compared, as was the case for our
studies. We focused primarily on treatment-dependent
differences in observed Eapp values between samples,
not absolute efficiencies. Therefore, we used an estimated value of g, based on manufacturer-supplied specifications for the microscope filters and the camera, and
on values for the quantum yields of cerulean and mYFP
reported in the literature (13, 14). Finally, this apparent
efficiency was converted to a percent, E%, using E% 5
Eapp 3 100.
It is important to note that the apparent efficiency
measured from sensitized acceptor emission, Eapp, is not
necessarily identical to the ‘‘true’’ FRET efficiency that
is often used to infer donor-acceptor separation. The latter, defined as
R6
ð8Þ
E¼ 6 0 6
R0 þ R
is equal to the former only if each donor is paired with
exactly one acceptor with the same donor-acceptor separation, R [Lakowicz, 1999]. In Eq. 8, R0 is the characteristic Förster distance for the donor and acceptor fluorophores in question. In the case of intermolecular FRET
between fusion proteins in living cells, there is no way to
ensure that this is indeed the case. For example, if a Zband contains a mixture of labeled and unlabeled proteins, so that only a fraction f of the donors are a distance
R away from an acceptor, the measured Eapp will be
lower than E by the same factor f [Lakowicz, 1999]. The
situation could be further complicated if there is a distribution of donor-acceptor distances that may or may not
have a single mean. In this case, even if all donors are
near acceptors the measured Eapp will represent a
weighted average of multiple ‘‘true’’ efficiencies. These
potential complications underscore the need for care
when using SE-FRET as a tool for investigating protein
separations, particularly when using transfected cells
expressing fluorescent fusion proteins.
Analysis
While it is inconvenient that the value of E% as
calculated above is potentially confounded by uncontrol-
FRET Imaging of Z-Band Proteins
lable factors such as donor and acceptor concentrations,
there are statistical methods available for teasing apart
the effects of multiple variables on an observed parameter. One such method is multiple linear regression
(MLR). MLR provides us with a systematic way to identify factors affecting the apparent FRET efficiency, E%,
and it allows us make predictions about the effect of a
particular treatment such as CFP location while controlling for any variability in the response due to all other
factors. For each peak in an intensity profile, the following information was recorded: E%, acceptor and donor
intensities, unquenched donor intensity (FD alone in Eq. 5
calculated from quenched donor and corrected FRET
values), structure type (z-body or Z-band), CFP location
(C- or N-terminus), image name, and experiment date.
The statistical software package JMP (SAS Institute Inc.,
Cary, NC) was used for all statistical analyses. We found
that within a single image, E% and intensity data were
usually distributed normally around a single mean, but
that there was often image-to-image variation in these
means. Therefore, each image was represented by mean
values for each of the numerical parameters listed above.
In MLR, it is assumed that the response (here, the image
mean E%) can be described by a linear combination of
the predictors (date, acceptor and unquenched donor
intensities, donor location, and structure type). Interaction terms (represented by products of predictors) were
also included in the model to allow the effects of predictors to be different for the various treatments; this
improves the predictive power of the model. For consistency, the same model was used for each pair of proteins.
Once the regression coefficients were determined for a
particular experiment, the resulting linear model was
used to calculate the least-squares mean E% for a given
combination of predictors. Visual examination of normal
quantile plots of residuals was used to assess the validity
of the linearity assumption. A Student’s T-test on the
least squares mean E% values inferred from the model
yielded a mean difference attributable to each
‘‘treatment’’ (N- or C- terminal CFP; z-body or Z-band)
and was used to assess whether or not a particular difference was significant.
RESULTS
Empty Vectors
If the constants aD and aA are determined correctly, application of Eq. 3 should yield images that
accurately reflect the contribution of FRET-generated
acceptor fluorescence to the FRET channel image. To
test this, myoblasts were transfected with empty CFP
(cerulean) and YFP vectors, yielding myotubes containing the two fluorescent proteins distributed uniformly
357
throughout their cytoplasm. The predicted true FRET efficiency for a three-dimensional population of immobile,
randomly-distributed donors and acceptors can actually
be quite substantial if the concentration of acceptors is
above a characteristic concentration specific to that particular donor-acceptor pair, and diffusion will further
enhance the FRET efficiency [Lakowicz, 1999; Corry
et al., 2005]. For CFP and YFP, whose R0 is ! 4.9 nm
[Patterson et al., 2000], this characteristic concentration,
at which E% 5 76%, is 3.8 mM, which is quite high.
The predicted efficiency drops rapidly, to less than 0.02,
when the acceptor concentration decreases 10-fold. In
repeated experiments with cells co-expressing the two
fluorescent proteins, we found that both acceptor and donor concentrations influenced the apparent FRET efficiency, as predicted by the simple random model (data
not shown). While we did observe a FRET signal in cells
expressing CFP and YFP at very high levels, the subset
of cells whose YFP and CFP intensities were comparable
to those observed in cells expressing fluorescent fusion
proteins showed little or no FRET response after correction for bleedthrough. The myotube shown in Fig. 2 is a
typical example of such a cell. Analysis of regions of interest inside the nine cells comprising this subset yielded
an average apparent E% of 0.04 6 0.52%, lower than average values measured in any of the experiments involving fusion proteins.
Fusion Proteins
Our imaging experiments involving fusion proteins
had two primary goals. First, we hoped to find evidence
supporting or refuting purported interactions (proximities) between several recently identified Z-band proteins
in living cells. Earlier work by other investigators has
relied on in vitro assays to reveal potential interactions
between proteins. FATZ and myotilin were selected
because previous studies have suggested that they bind
to alpha-actinin, the major protein of z-bodies and Zbands [Faulkner et al., 2001; Gontier et al., 2005]. Telethonin was selected for its purported ability to bind
FATZ [Faulkner et al., 2001], Moreover, telethonin is
absent in the z-bodies of premyofibrils or nascent myofibrils; rather, it is a late assembling protein in the Z-bands
of mature myofibrils [Wang et al., 2005a]. Little is
known about these various interactions in living muscle
cells, and even less is known about developmental
changes in these interactions. Our second goal was to
determine whether or not SE-FRET imaging could be
used to detect any molecular-scale changes that might
accompany the transition from premyofibrils to mature
myofibrils proposed in Fig. 1. As mentioned in the Introduction, myofibrillogenesis can be monitored through
changes in the morphology of structures containing Zband proteins: in the earliest stages, these proteins are
Fig. 1. Diagram of the transition from premyofibril to mature myofibril. In this model, assembly begins at the edges of myotubes with premyofibrils composed of minisarcomeres comprised of alpha-actininenriched z-bodies (which also contain FATZ and myotilin) as well as
thin filaments of actin and associated proteins (tropomyosin, troponins). The isoform of myosin II in the premyofibril is a non-muscle or
cytoplasmic one. Nascent myofibrils form when premyofibrils align
their z-bodies, and titin and muscle-specific myosin II are incorporated
into the fibrils. However, the muscle myosin II filaments are not
aligned in A-bands and thus produce a continuous staining pattern,
whereas the non-muscle myosin II is in periodic bands. As mature
myofibrils form, muscle myosin II filaments align to form A-bands;
non-muscle myosin II is no longer associated with the fibrils; aligned
z-bodies fuse to form Z-bands, and myosin-binding proteins (C-Protein and M-band Protein) are incorporated into the A-bands [Sanger
et al., 2004].
Fig. 2. Cultured chick myotubes
expressing both CFP (donor) and
YFP (acceptor) fluorescent proteins alone (not fusion proteins).
Merge 5 RGB merge of donor
(green) and acceptor (red) images
showing apparent colocalization in
yellow. E% image is prepared by
correcting the raw FRET channel
image for donor and acceptor
bleedthrough according to the text.
The E% image indicates that neither CFP nor YFP molecules are
close enough to each other, on average, to produce a FRET
response. Note also that neither
CFP nor YFP are incorporated into
any regions of the myofibril. Scale
bar 5 10 lm.
FRET Imaging of Z-Band Proteins
localized in small, punctate objects called z-bodies lying
along actin filaments. As maturation progresses, the zbodies in adjacent myofibrils align and fuse to form Zbands or Z-lines [Dabiri et al., 1997; Du et al., 2003]. A
single myotube may contain both z-bodies and Z-bands
at the same time, with bodies located near the ends and
spreading edges of the cells and bands near the middle of
the myotubes. For analysis purposes we classified each
intensity profile extracted from a FRET image as belonging to either z-bodies or Z-bands.
Alpha-Actinin and Alpha-Actinin
Because alpha-actinin is one of the primary components of z-bodies and Z-bands, we chose to use it as
the centerpiece of several experiments. The z-bodies of
premyofibrils are located at the leading edges of the
myotubes whereas the Z-bands of the mature myofibrils
are best studied in the central shafts of the same myotubes (Fig. 1) [Wang et al. 2005a, 2007, 2008]. To look
for changes in molecular organization associated with Zband maturation, mYFP-alpha-actinin was co-expressed
with mCFP-alpha-actinin in cultured quail skeletal myotubes. In both cases, the fluorescent protein was fused to
the carboxy-terminus of the alpha-actinin monomer.
Dabiri et al., [1997] have previously reported that the
GFP moiety must be on the C-terminus of alpha-actinin
to obtain proper localization of the fusion protein to zbodies and Z-bands. Since this protein readily forms
antiparallel homodimers, this results in a fluorophore on
either end (or both ends) of an alpha-actinin crosslink.
As seen in Fig. 3, there was a FRET signal detected in
both z-bodies and Z-bands. Expression levels for CFPand YFP-alpha-actinins were remarkably consistent
from experiment to experiment, and a simple Student’s
T-test found a significant difference between the two
population mean E% even without taking other factors
into consideration. After applying the linear model
described in Methods, we found there was a significant
increase of 0.55% 6 0.23% in the least-squares mean
E% (two-sided p-value 5 0.016) that could be assigned
to the z-body - Z-band distinction alone (Table II).
FATZ and Alpha-Actinin
With CFP-FATZ and alpha-actinin-mYFP transfections of myotubes (Fig. 4) we were able to examine
the effect of developmental stage and fluorophore fusion
site on FATZ on the FRET signal. MLR analysis of these
data reveal that the apparent FRET efficiency is affected
by both the location of the CFP (e.g. whether it was on
the C- or N-terminus of the FATZ molecule) and the developmental stage of the myofibril (z-body versus Zband) and that the two ‘‘treatments’’ have different
effects depending upon how they are combined. Specifically, our model predicts that in z-bodies, CFP location
359
on FATZ has no significant effect on E%, whereas in Zbands, replacing N-terminal CFP with C-terminal CFP
leads to predicted values of E% that are 23% lower (Table I). Furthermore, the model predicts that E% does not
change during the Z-body to Z-band transition if CFP is
on the N-terminus of FATZ; however, if CFP is on the
C-terminus, E% is actually 26% higher for z-bodies than
for Z-bands (Table II).
To ascertain whether or not the difference in FRET
response was due to a difference in functionality of the
two varieties of CFP-FATZ, we performed FRAP (Fluorescence Recovery After Photobleaching) experiments
on myotubes expressing either FATZ with N-terminal
CFP or FATZ with C-terminal CFP. Previously, we have
used mobile fractions and recovery rates measured in
FRAP experiments to characterize the dynamic properties of Z-bands proteins [Wang et al., 2005a]. As summarized by Fig. 5, we found that the total mobile fraction
of the population of CFP-FATZ was unaffected by the
location of the CFP fluorophore: with CFP on the N-terminus, 93 6 8% of bleached CFP-FATZ was replaced
after 20 min, whereas with CFP on the C-terminus, 95 6
7% was replaced. This result suggests that the location
of the CFP fluorophore has no appreciable effect on the
ability of FATZ to incorporate into and exchange properly in and out of Z-bands.
FATZ and Telethonin
Previous in vitro work has suggested that FATZ
also interacts with telethonin (also known as T-cap)
[Faulkner et al., 2001]. Here and in previous studies
[Wang et al., 2005a], we have found that telethonin only
localizes to the Z-bands characteristic of more mature
myofibrils, not to z-bodies in premyofibrils or nascent
myofibrils. Whereas there was no FRET signal between
FATZ and telethonin in the z-bodies, there was a FRET
signal in the Z-bands of the same myotubes. The present
results support the model presented in Fig. 1. Using
EYFP-telethonin with the acceptor fused to its C-terminus, we found that as with the FATZ-alpha-actinin pair,
there was a significant effect of donor location on the
measured FRET response in the Z-bands of the mature
myofibrils. The model resulting from MLR analysis of
our data predicts that moving CFP from the N-terminus
(Fig. 6) to the C-terminus of FATZ leads to a 33%
decrease in predicted E% (Table I).
Myotilin and Alpha-Actinin
Myotilin is another protein purported to interact
with alpha-actinin, and indeed, we found a significant
FRET signal in the Z-bands of myotubes expressing
YFP-alpha-actinin and CFP-myotilin (Fig. 7). As with
FATZ, our MLR analysis predicts that the magnitude of
the observed E% is affected by both CFP location on
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Fig. 3. Images of skeletal myotubes cotransfected with CFP-alphaactinin (donor) and YFP-a-actinin (acceptor). Merged images are
RGB merges of donor (green) and acceptor (red) showing colocalization in the Z-bands and z-bodies. E% images were prepared as
described in the text and smoothed for presentation purposes. Top
row: Z-bands in four-day-old myotubes. Bottom row: z-bodies in
three-day-old myotubes. Note that in contrast to the uniform distribution displayed by CFP and YFP alone as shown in Fig. 2, the fusion
proteins localize to z-bodies and Z-bands. Moreover, the FRET
response shown in the E% images provide evidence that the donor and
acceptor fluorophores fused to alpha-actinin are maintained in close
proximity in both z-bodies and Z-bands.
Fig. 4. Images of Z-bands in skeletal myotubes cotransfected with
CFP-FATZ (donor) and alpha-actinin-mYFP (acceptor). Merged
images are RGB merges of donor (green) and acceptor (red) showing
colocalization in the Z-bands. The E% image was prepared as
described in the text and smoothed for presentation purposes. The
FRET response shown in the E% image provides evidence that the donor and acceptor fluorophores fused to FATZ and alpha-actinin
respectively are maintained in close proximity in the Z-bands.
myotilin and developmental stage. Moving the donor
from the N-terminus to the C-terminus of myotilin
results in a significant decrease in E% in both z-bodies
and Z-bands, as shown in Table I. Looking at the predictions in a slightly different way we find that regardless of
donor location on myotilin, there is not a significant
change in predicted E% as z-bodies mature into Z-bands
(Table II).
nus of FATZ) and CFP-myotilin, varying the CFP fusion
site on myotilin (Fig. 8). Recently, in vitro experiments
have suggested that these two proteins may interact
[Gontier et al., 2005], and other work has shown that
myotilin and FATZ bind to the same region of the alphaactinin molecule, spectrin-like repeats R3 and R4
[Faulkner et al., 2001; Salmikangas et al., 1999]. Our
FRET results agree with this: we observed a FRET signal with these two proteins comparable in magnitude to
that observed with YFP-alpha-actinin and myotilin.
MLR analysis of the FRET data obtained from images of
mature Z-bands revealed a significant effect of CFP location, with an 35% decrease in E% of predicted when
Myotilin and FATZ
Finally, we carried out SE-FRET measurements on
myotubes expressing YFP-FATZ (EYFP on the C-termi-
FRET Imaging of Z-Band Proteins
361
TABLE 1. Summary of the CFP Location Effect for Four Protein Pairs
LSMean E% 6 SE N-term CFP
LSMean E% 6 SE C-term CFP
%difference (2-sided P-value)
7.34 6 0.38
6.92 6 0.33
3.90 6 0.29
10.36 6 0.60
9.33 6 0.35
12.90 6 0.76
6.78 6 0.39
5.36 6 0.31
2.61 6 0.24
5.38 6 0.67
6.42 6 0.65
8.35 6 1.04
NS (0.162)
223% (<0.0001)
233% (0.0003)
248% (<0.0001)
231% (<0.0001)
235% (0.0002)
Alpha-actinin-YFP 1 FATZ, z-bodies
Alpha-actinin-YFP 1 FATZ, Z-bands
Telethonin-YFP 1 FATZ, Z-bands
Alpha-actinin-YFP 1 Myotilin, z-bodies
Alpha-actinin-YFP 1 Myotilin, Z-bands
FATZ-YFP 1 myotilin, Z-bands
LSMean E% is the least squares mean E% predicted by the MLR model with all factors other than CFP location fixed to the same ‘‘neutral’’
value. NS, no significant difference for a 5 0.05 significance level. In all cases the acceptor (YFP) was located at the C-terminus of its protein.
%difference 5 (C-term – N-term)/N-term.
TABLE 2. Summary of Body-Band Effect
Alpha-actinin-YFP 1 Alpha-actinin-CFP
Alpha-actinin-YFP 1 CFP-FATZ
Alpha-actinin-YFP 1 FATZ-CFP
Alpha-actinin-YFP 1 CFP-Myotilin
Alpha-actinin-YFP 1 Myotilin-CFP
LSMean E% 6 SE z-bodies
LSMean E% 6 SE Z-bands
%difference (2-sided P-value)
3.22 6 0.25
7.34 6 0.38
6.78 6 0.39
10.36 6 0.60
5.38 6 0.67
3.77 6 0.22
6.92 6 0.33
5.36 6 0.31
9.33 6 0.35
6.42 6 0.65
117% (0.016)
NS (0.236)
226% (0.0007)
NS (0.0582)
NS (0.084)
LSMean E% is the least squares mean E% predicted by the MLR model. NS, no significant difference for a 5 0.05. %difference 5 (Z-bands –
z-bodies)/z-bodies.
analysis to generate predictions for the effect of fusion protein on E%. Table III displays the result of this analysis,
the main point of which is that the N-terminus of myotilin
appears to have better access to the C-termini of FATZ
and alpha-actinin than does the C-terminus of myotilin.
DISCUSSION
Fig. 5. Fluorescence recovery after photobleaching (FRAP) experiments were used to compare the mobility of the two variants of CFPFATZ used in SE-FRET experiments: CFP on the C-terminus of
FATZ versus CFP on the N-terminus of FATZ. Results were obtained
using the method previously reported in Wang et al. [2005a]. We
found that the percent of photobleached CFP-FATZ that recovered
after 20 min (‘‘Mobile Fraction’’) was not significantly affected by the
CFP fusion site, i.e., N- versus C-termini.
moving the CFP from the N-terminus to the C-terminus
of myotilin (Table I).
Because the CFP-myotilin experiments (varying
CFP fusion site) with FATZ and with alpha-actinin (YFP
fused only to the C-termini of both proteins) were performed on the same days, it is also possible to use MLR
While intensity-based FRET techniques such as
sensitized emission are easily implemented, care must be
taken to avoid potential artifacts when doing comparative studies. For example, proper correction for all of the
non-FRET contributions to the ‘‘FRET’’ channel hinges
on the linearity of the imaging system and on the accurate measurement of the bleedthrough constants aA and
aD in Eq. 2, as well as on the characterization of autofluorescence. It is interesting to note that while the value
of aD did not vary much from experiment to experiment,
the value of aA did change significantly over time, even
when neutral density filters were left unchanged. This
was likely due to changes in the mercury arc lamp’s
spectrum associated with aging, since the ratio of CFP to
YFP excitation intensity is one of the factors contributing to aA. Measuring the bleedthrough constants every
time an experiment is performed is the best way to minimize errors due to incorrect bleedthrough subtraction.
Another factor that may affect sensitized emission
results is the dependence of the overall intensity of
FRET-induced emission on the composition of the donor/acceptor mixture. This complication does not arise
in situations where donor and acceptor are attached to
the same molecule. However, when cells are expressing
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Stout et al.
Fig. 6. Images of Z-bands in skeletal myotubes cotransfected with
CFP-FATZ (donor) and telethonin-mYFP (acceptor). Merged images
are RGB merges of donor (green) and acceptor (red) showing colocalization in the Z-bands. The E% image was prepared as described in
the text and smoothed for presentation purposes. The FRET response
shown in the E% image provides evidence that the donor and acceptor
fluorophores fused to FATZ and telethonin respectively are maintained in close proximity in the Z-bands. Bar 5 10 microns.
Fig. 7. Images of Z-bands in skeletal myotubes cotransfected with
CFP-myotilin (donor) and alpha-actinin-mYFP (acceptor). Merged
images are RGB merges of donor (green) and acceptor (red) showing
colocalization in the Z-bands. The E% image was prepared as
described in the text and smoothed for presentation purposes. The
FRET response shown in the E% image provides evidence that the donor and acceptor fluorophores fused to myotilin and alpha-actinin
respectively are maintained in close proximity in the Z-bands. Bars 5
10 microns.
Fig. 8. Images of Z-bands in skeletal myotubes cotransfected with
CFP-myotilin (donor) and FATZ-mYFP (acceptor). Merged images
are RGB merges of donor (green) and acceptor (red) showing colocalization in the Z-bands. The FRET response shown in the E% image
provides evidence that the donor and acceptor fluorophores fused to
myotilin and FATZ respectively are maintained in close proximity in
the Z-bands.
fusion proteins with donor and acceptor on different
molecules, there is no way to control the concentration
of either fluorophore. As explained in (Zal and Gascoigne. 2004), the energy transfer in this type of setting
is difficult to interpret, as the donor-acceptor distance is
not necessarily uniform, and there may be more than one
acceptor near each donor. Theoretical models have been
developed for random distributions of unlinked donors
and acceptors, either fixed or diffusing freely in two and
three dimensions. Such models indicates that the apparent efficiency measured using SE-FRET will depend on
acceptor and donor concentrations in a rather complicated manner. Because the three-dimensional arrangement (and stoichiometry) of Z-band proteins is largely
unknown, extracting actual distances from the apparent
efficiency, E%, is difficult if not impossible.
FRET Imaging of Z-Band Proteins
363
TABLE 3. Acceptor Protein Effect for CFP-Myotilin, Z-Bands Only
CFP-Myotilin
Myotilin-CFP
LSMean E% 6 SE Alpha-actinin
LSMean E% 6 SE FATZ
%difference (2-sided P-value)
11.87 6 0.68
5.51 6 0.79
8.44 6 0.97
4.3 6 1.5
229% (0.002)
NS (0.422)
The YFP acceptor was located on the C-termini of FATZ and alpha-actinin. LSMean E% is the least squares mean E% predicted by the MLR
model. NS, no significant difference for a 5 0.05. %difference 5 (FATZ – alpha-actinin)/alpha-actinin.
In spite of these limitations, SE-FRET imaging can
provide answers to interesting questions. By using multiple linear regression (MLR) to account for variability in
the FRET signal due to uncontrollable factors (such as
expression levels), and by directing our attention at
changes in E% correlated with factors we could either
control (fluorophore fusion site) or characterize (z-body
vs Z-band), we were able to gain some insight into how
Z-band proteins are arranged and how those arrangements change during myofibrillogenesis. Changing the
location of the donor fluorophore on the proteins of interest and quantifying any correlated changes in E% can
provide information about how the two ends of the protein differ in their proximity to the fixed acceptor-linked
protein. This, in turn, allows us to construct a simple
model of the spatial arrangement of these two proteins.
Similarly, quantifying differences in E% connected to
changes in the maturity of the myofibril can give us
insight into how arrangements might evolve during development.
Alpha-Actinin
Experiments using mCFP- and mYFP-alpha-actinin were designed to address a developmental question.
The premyofibril stage is characterized by punctate
structures, z-bodies, distributed as minisarcomeres along
the lengths of filaments comprised of actin and non-muscle myosin II (Fig. 1), which lie at the ends and sides of
growing myotubes. As the myofibrils mature, these zbodies, which contain alpha-actinin, FATZ, myotilin,
and actin, align and fuse to form the distinctive Z-bands
of the mature myofibril, incorporating new proteins such
as telethonin as they do so (Fig. 1). Electron microscopic
studies of mature Z-disks in skeletal muscle have
revealed a striking degree of organization, with diametrically opposed pairs of !22-nm-long alpha-actinin crosslinks lying at regular !19 nm intervals along each 6–
8 nm diameter actin filament [Young et al., 1998; Luther,
2000]. According to this model, we should expect energy
transfer only between donors and acceptors that are
directly across an actin filament from each other, since
all other distances are too large (Fig. 9). The organization of alpha-actinin and other proteins in the Z-bodies
of premyofibrils, on the other hand, is still a mystery.
Using Fluorescence Recovery After Photobleaching
(FRAP) to study the dynamics of Z-band proteins, Wang
et al. [2005a] found that in cultured myotubes, YFPalpha-actinin fusion proteins incorporated into z-bodies
and Z-bands underwent exchange with a cytoplasmic
pool. In both cases, the fluorescence recovery curves
could be characterized by two components; however, in
z-bodies, the faster component dominated the recovery
more than in Z-bands. This result prompts speculation
regarding the organization and plasticity of Z-band protein organization. Our SE-FRET results show that energy
transfer is possible in z-bodies, albeit with a slightly
lower overall efficiency than in Z-bands. This suggests
that even in z-bodies, alpha-actinin crosslinks may already be fairly well organized with their characteristic
spacing, although not to the same degree as in Z-bands.
Figure 10 is a simple model illustrating how a smaller
FRET signal might arise from z-bodies than Z-bands.
FATZ, Telethonin
We also hoped to add to what is known about the
arrangement of several recently discovered Z-band proteins, namely FATZ (also known as myozenin and calsarcin-2), myotilin, and telethonin (T-cap) [Salmikangas
et al., 1999; Faulkner et al., 2001; Gontier et al., 2005;
Zou et al., 2006]. These proteins and their purported
interactions were identified through genetic screens of
muscle cDNA libraries along with yeast 2-hybrid screenings, GST pulldown and immunoprecipitation assays
[Faulkner et al., 2000; Takada et al., 2001; Salmikangas
et al., 2003]. Other than their primary structures, little
else is known about how these proteins arrange themselves during the course of muscle development. Using
the C-terminus of alpha-actinin as the acceptor location
and spatial reference point, we explored the effect of donor location and developmental stage on the FRET
response with myotilin and FATZ. Both of these proteins
localize to z-bodies and Z-bands regardless of the fluorescent protein fusion site.
FRET experiments with CFP-FATZ and mYFPalpha-actinin tell a somewhat complicated story. We
found that donor location had a significant effect on the
observed FRET response in Z-bands: when CFP was
fused to the N-terminus of FATZ, the average FRET efficiency was significantly higher than when it was fused to
the C-terminus. However, this difference disappeared
when looking only at z-body data. Because little is
known about the tertiary structure of FATZ, we can only
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Stout et al.
Fig. 9. Diagram depicting the expected locations of CFP and YFP
moieties in myotubes coexpressing CFP-alpha-actinin and YFP-alphaactinin. Because fluorophores on the same dimer crosslink are main-
tained at a separation of about 24 nm, no FRET signals should arise
from this source. Any FRET detected should instead be due to dimers
on opposite sides of the actin filament.
Fig. 10. Model proposing how subtle differences in filament arrangement could result in a slight increase in the FRET response during the
transition from Z-bodies (left of arrow) to Z-bands (right of arrow). The
relatively small change in E% we observed suggests that the spacing
between adjacent alpha-actinin crosslinks is similar in Z-bodies and Zbands. The difference between the two may lie in the extent to which the
actin filaments are aligned and ordered by the alpha-actinin molecules.
EM studies have shown that in mature Z-bands alpha-actinin crosslinks
occur at regular !19 nm intervals, with adjacent crosslinks rotated 908,
and that alpha-actinin dimers line up on opposite sides of an actin filament as shown. Not shown are crosslinks perpendicular to the page. This
drawing is not to scale, and differences between the two proposed configurations have been exaggerated for clarity.
speculate about the source of this difference. FATZ is a
relatively small protein, with a molecular mass of only
!57 kDa. From sequence analysis, it is thought to consist of two alpha-helical domains connected by a flexible
glycine-rich domain [Takada et al., 2001], with its alphaactinin binding activity confined to the C-terminal
region. Taken together with our FRET results, we propose that in z-bodies, both ends of FATZ are rather mobile and close enough to the end of alpha-actinin to allow
energy transfer (modeled in Fig. 11). As z-bodies fuse to
form Z-bands, the N-terminus of FATZ remains mobile
enough and close enough to alpha-actinin to permit the
same degree of energy transfer. The C-terminus, however, may be pulled further away, leading to a smaller
FRET response in Z-bands than in z-bodies (Fig. 11). It
is possible to look at the data in a slightly different way
and come to the same conclusion: if we confine our
attention to cells with N-terminal FATZ, we find no difference in E% between z-bodies and Z-bands. However,
in cells with C-terminal FATZ, the MLR model predicts
a drop in E% of about 26% as z-bodies fuse into Zbands. Again, this unexpected result suggests that after
z-bodies have fused to form Z-bands, FATZ becomes
locked in to a configuration where its C-terminus has
less access to the end of alpha-actinin than it did before.
This could be due to the addition of other proteins, such
as telethonin (Figs. 1 and 11) or filamin, to the network
that change the shape and/or flexibility of the FATZ molecule.
The FATZ-telethonin interaction was the subject of
another series of our SE-FRET experiments. Unlike
alpha-actinin and FATZ, telethonin does not localize to
z-bodies in premyofibrils of myotubes; rather, it is uniformly distributed throughout the cytoplasm at the ends
of the myotubes where the premyofibrils are located
[Wang et al., 2005a]. Only after z-bodies have fused to
form the Z-bands of the mature myofibrils [Wang et al.,
2005a] does localization of telethonin to Z-bands
FRET Imaging of Z-Band Proteins
365
Fig. 11. Model depicting possible changes in Z-band proteins during
z-body (left) to Z-band (right) transition, as suggested by the results of
SE-FRET experiments and the model proposed by Faulker et al.
(2001). Not shown are many other Z-band proteins such as filamin
and cypher. Drawing is not to scale and the fluorescent tagging proteins (CFP, YFP) are not shown. If myotilin dimerizes in the staggered
antiparallel fashion shown, as proposed by Salmikangas et al. (2003),
its amino terminus could be closer to or have more access to the end
of an alpha-actinin dimer than its carboxyl terminus. Telethonin, a
FATZ binding protein, and titin, a telethonin binding protein, are
absent from the z-bodies of premyofibrils. The addition of these two
latter proteins may be involved in the decrease in the proximity of the
C-termini of FATZ molecules with respect to alpha-actinin molecules
in the Z-bands of mature myofibrils.
become apparent (Fig. 1). Thus, there was no FRET signal between telethonin and FATZ in z-bodies. Because
we relied on a relatively crude, binary classification of
Z-band organization in this study, we were unable to
explore subtle, time-dependent changes in FRET
between YFP-telethonin and CFP-FATZ. We did, however, investigate the effect of CFP location on the FRET
response. As in the previous two examples, we found
that fusing CFP to the N-terminus of FATZ resulted in a
significantly higher apparent FRET efficiency than fusing it to the C-terminus, suggesting that the N-terminus
of FATZ is closer to telethonin than the C-terminus is
(Fig. 11). This agrees nicely with the results of immunoprecipitation experiments by [Frey and Olson, 2002],
which suggest that a stretch of 17 amino acids near the
N-terminus of calsarcin-3, a member of the same gene
family as FATZ, was responsible for its telethonin-binding activity.
Unlike FATZ , we found that regardless of CFP location
on myotilin, there were only very small changes in the
FRET response with mYFP-alpha-actinin that could be
correlated with the fusion of z-bodies into Z-bands (Table II). This suggests that the arrangement and conformation of myotilin does not change substantially as premyofibrils mature into myofibrils. However, CFP location did have a profound effect on the observed E% in
both z-bodies and Z-bands, with an decrease in apparent
FRET efficiency attributable to moving the CFP from
the N-terminus to the C-terminus of myotilin in both
cases (Table I). Work by Salmikangas et al. [2003] has
provided biochemical evidence suggesting that myotilin
forms antiparallel dimers, with the carboxy-terminal
region the site of dimerization. Yeast 2-hybrid experiments by the same investigators show that alpha-actinin
binding activity is located in the amino-terminal region
of myotilin. Our FRET results are consistent with this,
for they suggest that the C-terminal portion of myotilin
is held further away from the end of alpha-actinin than
the N-terminal portion in both z-bodies and Z-bands.
This implies that the myotilin homodimer may be maintained in a relatively long, straight conformation, rather
than a more compact conformation.
Myotilin
As in the FATZ experiments, our myotilin experiments add to a growing body of information about this
protein [Wang et al., 2005a; Carlsson et al., 2007].
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Stout et al.
Initially, we were somewhat surprised to observe a
strong FRET signal in the Z-bands of myotubes coexpressing CFP-myotilin and YFP-FATZ. However,
three independent researchers have used yeast twohybrid experiments to show that both FATZ and myotilin interact with alpha-actinin somewhere within spectrin-like repeats 3 and 4 [Faulkner et al., 2000; Takada
et al., 2001; Salmikangas et al., 2003], so energy transfer
to a YFP on the C-terminus of FATZ (near its alpha-actinin binding site) from a CFP on the N-terminus of myotilin (near its alpha-actinin binding site) is not entirely
unexpected. More recent yeast two-hybrid and GST pulldown assays [Gontier et al., 2005] provide even more
evidence supporting the validity of our FRET results, for
they suggest that the C-terminus of FATZ does indeed
interact with myotilin. Our observation that moving CFP
to the C-terminus of myotilin was correlated with a
decrease in apparent FRET efficiency is also consistent
with the published results, as it has been suggested that
the C-terminus is responsible for myotilin dimerization,
which may place it further from alpha-actinin than the
N-terminus [Salmikangas et al., 2003] or at least make it
less accessible for energy transfer (Fig. 11).
In an attempt to combine these FRET results into a
single picture, we have created a single model illustrating
a possible arrangement of alpha-actinin, FATZ, myotilin,
and telethonin in the Z-band (Fig. 11). Our model is
based on one presented by Faulkner et al. [2001], with
additional details about molecular orientations that are
suggested by our FRET results. In particular, our comparison of the myotilin/lalpha-actinin FRET response with
the myotilin/FATZ response suggests that the N-terminus
of myotilin is equidistant from the C-termini of alphaactinin and FATZ. Based on this, we propose that the
binding site for myotilin is closer to the C-terminus of
alpha-actinin than the binding site for FATZ, as shown in
Fig. 11. This arrangement would result in similar FRET
efficiencies for the two pairs of Z-band proteins.
SUMMARY
Our approach to SE-FRET imaging is similar to
that of Muller et al. [2005], who used information from
numerous pair wise FRET experiments to build a model
of the spindle pole body in yeast. Rather than attempt to
measure absolute FRET efficiencies for the purpose of
inferring distances, we have instead chosen to look at
how alterations in the fluorophore locations affect the
FRET response in order to construct a picture of the
arrangements of several Z-band proteins myofibrillogenesis. Because of the large number of protein pairs possible in a complicated network such as the Z-band, an
approach like this requires a large number of experiments: if one varies both acceptor and donor locations,
each pair represents up to four FRET experiments that
must be done. Experiments aimed at discovering stepwise changes associated with myofibril development add
yet another layer of complexity to an already large data
array [Rhee et al., 1997; Sanger et al., 2004, 2005; Golson et al., 2004; Wang et al. 2005a,b; Wang et al., 2007].
We have shown that careful statistical analysis, in our
case, multiple linear regression, can be used to extract
meaningful information from multifaceted data sets.
This approach gives us a powerful tool for exploring the
formation and architecture of the Z-band and of other
multi-protein complexes in muscle and non-muscle cells.
There are a number of muscle diseases involving point
mutations of Z-band proteins that lead to myofibrillar
disarray [Selcen and Engel, 2004]. The use of FRET
(this work) and FRAP [Wang et al., 2005a] techniques
on Z-bands in living cells expressing fluorescent protein
fusions to mutated Z-band proteins should yield novel
insights into the molecular-level changes underlying the
myofibrillar disarray observed in these type of muscle
diseases.
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