Site directed mutagenesis of Drosophila flightin disrupts

J Muscle Res Cell Motil (2007) 28:219–230
DOI 10.1007/s10974-007-9120-y
ORIGINAL PAPER
Site directed mutagenesis of Drosophila flightin disrupts
phosphorylation and impairs flight muscle structure
and mechanics
Byron Barton Æ Gretchen Ayer Æ David W. Maughan Æ
Jim O. Vigoreaux
Received: 18 July 2007 / Accepted: 5 September 2007 / Published online: 3 October 2007
Springer Science+Business Media B.V. 2007
Abstract Flightin is a myosin rod binding protein that in
Drosophila melanogaster is expressed exclusively in the
asynchronous indirect flight muscles (IFM). Hyperphosphorylation of flightin coincides with the completion of
myofibril assembly and precedes the emergence of flight
competency in young adults. To investigate the role of
flightin phosphorylation in vivo we generated three flightin
null (fln0) Drosophila strains that express a mutant flightin
transgene with two (Thr158, Ser 162), three (Ser139,
Ser141, Ser145) or all five potential phosphorylation sites
mutated to alanines. These amino acid substitutions result in
lower than normal levels of flightin accumulation and
transgenic strains that are unable to beat their wings. On two
dimensional gels of IFM proteins, the transgenic strain with
five mutant sites (fln5STA) is devoid of all phosphovariants,
the transgenic strain with two mutant sites (fln2TSA)
expresses only the two least acidic of the nine phosphovariants, and the transgenic strain with three mutant sites
(fln3SA) expresses all nine phosphovariants, as the wild-type
strain. These results suggest that phosphorylation of Thr158
and/or Ser162 is necessary for subsequent phosphorylation of other sites. All three transgenic strains show
normal, albeit long, IFM sarcomeres in newly eclosed
adults. In contrast, sarcomeres in fully mature fln5STA and
fln2TSA adults show extensive breakdown while those in
fln3SA are not as disordered. The fiber hypercontraction
phenotype that characterizes fln0 is fully evident in fln5STA
and fln2TSA but partially rescued in fln3SA. Mechanics on
skinned fibers from newly eclosed flies show alterations
in viscous modulus for fln5STA and fln2TSA that result in a
significant reduction in oscillatory power output. Expression of fln5STA and fln2TSA, but not fln3SA, in a wild-type
(fln+/fln+) background resulted in a dominant negative
effect manifested as flight impairments and hypercontracted IFM fibers. Our studies indicate that Thr158
and/or Ser162 are (is) indispensable for flightin function
and suggest that phosphorylation of one or both residues
fulfills an essential role in IFM structural stability and
mechanics.
Keywords Indirect flight muscle Phosphorylation Flightin Thick filaments
B. Barton G. Ayer J. O. Vigoreaux (&)
Department of Biology, University of Vermont, 109 Carrigan
Drive, 120 Marsh Life Science Building, Burlington, VT 05405,
USA
e-mail: [email protected]
G. Ayer D. W. Maughan J. O. Vigoreaux
Cell & Molecular Biology Program, University of Vermont,
Burlington, VT 05405, USA
D. W. Maughan J. O. Vigoreaux
Department of Molecular Physiology and Biophysics, University
of Vermont, Burlington, VT 05405, USA
Abbreviations
IFM
Indirect flight muscle
LMM
Light meromyosin
MyBP-C
Myosin binding protein C
2DE
two-dimensional gel electrophoresis
RLC
Regulatory light chain
MALDIMatrix assisted laser desorption ionization
TOF
time-of-flight
1DE
One-dimensional gel electrophoresis
Ee
Elastic modulus
Ev
Viscous modulus
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Introduction
Flightin is a 20-kDa thick filament protein that was first
identified in Drosophila melanogaster (Vigoreaux et al.
1993). In this organism, as well as in the waterbug Lethocerus indicus (Qiu et al. 2005), flightin expression is
limited to the indirect flight muscle (IFM), a muscle that
relies on a pronounced stretch activation response to produce oscillatory work. Recombinant flightin binds to
myosin in vitro and to a recombinant fragment of the
myosin rod that include the COOH-terminal 600 amino
acids (28 amino acid-repeat zone 19 to tailpiece) (Ayer and
Vigoreaux 2003). The interaction of flightin and myosin is
abolished by the glutamate to arginine change in the
myosin heavy chain (MHC) mutant Mhc13 at position 1e of
zone 27 of the rod (residue 1554) (Ayer and Vigoreaux
2003). These studies, together with the observation that
flightin fails to accumulate in the IFM of Mhc13 mutants
(Kronert et al. 1995), indicate that the myosin rod represents the primary binding site for flightin in the sarcomere.
A recent study (Flashman et al. 2007) has shown that the
CX myosin binding domain of vertebrate myosin binding
protein C (MyBP-C) binds to the light meromyosin (LMM)
region between amino acids 1554 and 1581, a sequence
that is contained within the LMM fragment (residues
1506–1674) that has been shown to bind the M band proteins myomesin and M-Protein (Obermann et al. 1997,
1998). Thus, a small segment of the myosin coiled coil
appears to have been evolutionarily conserved as a site of
myosin binding protein interaction.
Flightin fulfills an essential role in IFM development
and function. Flies with a null mutation in the flightin gene
(fln0) develop IFM with sarcomeres that are *30% longer
than normal that cannot withstand actomyosin generated
forces (Nongthomba et al. 2003). As a result, sarcomeres
degenerate and fibers hypercontract within hours after
eclosion (the pupal to adult transition) resulting in complete loss of flight ability (Reedy et al. 2000). Further
evidence that thick filaments lacking flightin are structurally compromised comes from the observation that IFM
myosin from fln0 and Mhc13 is proteolyzed at a site near the
hinge (Kronert et al. 1995).
The role of flightin in muscle contraction has been
studied using small amplitude sinusoidal length perturbation analysis of fibers isolated from newly eclosed adults
(i.e., within 30 min of emergence from the pupal case),
before the onset of hypercontraction. Fibers from the three
flightless mutants that affect flightin expression (fln0,
Mhc13, and Mhc6) showed deficits in their passive and
active viscoelastic properties that were commensurate with
their effect on flightin expression and resulted in significant
losses of oscillatory power. In particular, fln0 muscle fibers
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J Muscle Res Cell Motil (2007) 28:219–230
are more compliant than wild-type fibers, suggesting that
flightin is a major contributor to myofilament stiffness and
a key determinant of the stretch activation response in
Drosophila flight muscles (Henkin et al. 2004).
Hyperphosphorylation of flightin, beginning during the
late stages of pupal development and proceeding through
the initial hours of adult life, coincides with the culmination of IFM development. Up to nine phosphovariants have
been identified by two-dimensional gel electrophoresis
(2DE) that in adult flies constitute *50% of the total
flightin (Vigoreaux and Perry 1994; Vigoreaux et al. 1993).
In addition to flightin, several contractile proteins in Drosophila IFM have been shown to be multiply
phosphorylated, including the myosin regulatory light
chain (RLC) (Takano-Ohmuro et al. 1990), paramyosin
(Vinos et al. 1991), and troponin T (Domingo et al. 1998).
Phosphorylation of RLC (Dickinson et al. 1997) and paramyosin (Liu et al. 2005) has been shown to be important
for IFM function but appears to play no role in the
assembly or structural stability of myofibrils. As in Drosophila, phosphorylation of contractile proteins plays an
important role in the development and function of vertebrate cardiac and skeletal muscle (Davis et al. 2001;
Layland et al. 2005; Mayans et al. 1998; Sweeney et al.
1993) and in the regulation of myofibrillar protein interactions (Obermann et al. 1997, 1998).
In this study, we investigate the possibility that the
multiple phosphorylations of flightin dictate its dual role in
thick filament assembly and muscle contraction. We generated transgenic Drosophila strains that express mutant
flightin with two (fln2TSA), three (fln3SA), and five (fln5STA)
putative phosphorylation sites changed to alanine. Unlike a
fln+ transgene (Barton et al. 2005), none of the mutant
transgenes was able to rescue the sarcomere defects and
flightless phenotype of fln0 flies.
Materials and methods
Fly stocks
w1118, an otherwise wild-type strain except for a white eye
(w) mutation, was obtained from the Bloomington Stock
Center and used as host for generation of transgenic lines.
Generation of the flightin null strain fln0 has been described
previously (Reedy et al. 2000). w*; T(2;3)apXa, apXa/CyO;
TM3, Sb1 was used for linkage group analysis.
Identification of potential phosphorylation sites
Lyophilized thoraces from 2 to 5 day old wild-type (OR)
flies were homogenized in isoelectric focusing (IEF)
J Muscle Res Cell Motil (2007) 28:219–230
sample buffer and the proteins separated by 2DE as
described in Vigoreaux et al. (1993). Gels were stained
with coomassie blue and spots N1 and P8 were excised and
in-gel trypsin digestion performed as described (Rosenfeld
et al. 1992). The resulting peptides were separated by
HPLC (Michrom BioResource, Auburn CA) equipped
with a Reliasil C18 column (1.0 mm i.d. · 150 mm) at
40 ml/min. The mobile phase for gradient elution were:
A, 0.1% TFA/2%MeCN/water, and B, 0.07% TFA/
98%MeCN/water. Individual fractions were collected,
processed, and analyzed by MALDI-TOF mass spectrometry as described (Kinumi et al. 2000) using a PerSeptive
Biosystems Voyager Elite Biospectrometry workstation.
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0.39 mg/ml of pCasPeR and 0.171 mg/ml of D2–3.
Transformants were identified by yellow or orange eye
color in the G1 generation. Homozygous lines were produced by crossing individuals with a dark eye color based
on the assumption that one copy of the transgene resulted
in yellow or light orange eye color, while two copies
resulted in dark orange or red eye color. Each transgene
was mapped to its resident chromosome using w*;
T(2;3)apXa, apXa/CyO; TM3, Sb1 by standard crossing
techniques. The transgene was crossed into the fln0 strain
using standard crossing techniques.
Gel electrophoresis and western blot analysis
Construction of transformation vector
We used a pCaSpeR transformation vector that contains
part of the wild-type flightin gene (from the start codon to
0.55 kB past the translation stop codon) fused to the Actin
88F promoter (Barton et al. 2005). For mutagenesis of
amino acids S139, S141, and S145 to alanines (i.e., generation of fln3SA), the following primers were used
(changed nucleotides are bold underlined):
50 -ACCTCGCTGGCGCTGACATTGATGCGTATGCACCGGC-30
50 -TGCATACGCATCAATGTCAGCGCCGCGAGGTGGATA-30
For mutagenesis of amino acids T158 and S162 to alanines
(i.e., generation of fln2TSA), the following primers were used:
50 -ATTCAAGCACTGGCGGCCGCGATAAGAACTTA-30
50 -TAAGTTCTTATCGCGGCCGCCAGTGCTTGAA30
For mutagenesis of all five amino acids (i.e., generation
of fln5STA), we used the mutagenized template (S139A,
S141A, and S145A) with the same primers described above
for mutagenesis of T158 and S162.
Nucleotide changes in the transformation vectors were
verified by DNA sequence analysis prior to microinjections.
Generation of transgenic lines
Transformation was performed as described previously
(Spradling and Rubin 1982). The helper plasmid used was
pUChsD2–3 (flybase #0000938), obtained from Margarita
Cervera (Univ. Autonoma, Madrid). The pCasPeR plasmid
vector and helper plasmid were grown in E. coli XL1 Blue
cells in LB broth and purified using Qiagen maxiprep kit
(Valencia, CA). The concentration of DNA injected was
One-dimensional gel electrophoresis (1DE) was performed
using the discontinuous buffer system of Laemmli (1970)
as described previously (Vigoreaux et al. 1991). 2DE was
performed using the Protean IEF cell (BioRad Inc., Hercules, CA). IEF strips (pH 4–7 gradient) and precast 12.5%
gels were purchased from BioRad. Separation in the first
dimension was carried out using a three-step protocol. The
IEF strips were rehydrated in IEF sample buffer (8 M urea,
10 mM DTT, 8% CHAPS, 0.5% BIORAD 3/10 ampholyte) for 12 h at 20C. Step two involved a 2 h rapid volt
ramp to 3,500 volts per hour and step three focused the
strips for 14 h or 50,000 volt hours. To prepare samples for
electrophoresis, flies were placed in acetone for 1 h at
room temperature followed by lyophilization in a speed
vac. The thorax was dissected away from other body parts,
homogenized in IEF sample buffer and spun down to
remove the cuticle debris.
Western blots were performed as described in Vigoreaux et al. (1993) with a rabbit anti-flightin polyclonal
antibody described in Reedy et al. (2000).
Protein expression assays
To determine flightin expression levels in wild type and
transgenic flies, whole thoraces were homogenized as
described above. Protein concentration was determined
using the BioRad DC protein quantification kit using BSA
as a standard and equal amounts of protein (typically
*20 lg) were loaded in individual lanes of a 12% SDS
gel. After electrophoresis, proteins were transferred to
BioRad PVDF membrane and the membranes were
blocked with Aqua Block (East Coast Biologics, Inc.,
Berwick, ME) for 1 h. The primary antibody was an antiflightin polyclonal (Reedy et al. 2000) and Alexaflor 698
fluorescent antibodies (Molecular Probes, Eugene, OR)
were used as secondary antibodies. After incubation and
washing, the membranes were scanned on an Odyssey
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fluorescent scanner (LI-COR Biosciences, Lincoln, NE)
and analyzed with Phoretix 1D software (Nonlinear
Dynamics, Newcastle upon Tyne, UK) as follows. The
image was inverted to a negative in Photoshop and opened
as a new experiment. After automatic selection of lanes, the
bands were manually selected and their borders adjusted
based on the peak profile in the analysis window. Protein
quantity was obtained from band volume after background
subtraction.
Transmission electron microscopy
Fly thoraces were bisected and the separated halves were
fixed for 2 h in 2.5% glutaraldehyde and 0.1% paraformaldehyde. After fixation, samples were stored in 0.1 M
Millonigs phosphate buffer, pH 7.2. Samples were dehydrated through an ethanol series from 35% through
absolute for 10 min in each concentration. The final
dehydration step was in propylene oxide 3· for 5 min each.
Infiltration was performed with propylene oxide and
Spurr’s resin 3:1 for 30 min, 1:1 for 30 min, 1:3 for
45 min, and 100% Spurr’s resin for 45 min. Embedding
was done in 100% Spurr’s resin and polymerized for 24 h
at 70C. Semi-thin sections (1 lm) were cut with glass
knives on a Reichert ultracut microtome, stained with
methylene blue–azure II, and evaluated for areas of interest. Ultrathin sections (60–80 nm) were cut with a diamond
knife, retrieved onto 150 mesh copper grids, contrasted
with uranyl acetate (2% in 50% ethanol) and lead citrate,
and examined with a JEOL 1210 Transmission Electron
Microscope (TEM) (JEOL USA, Inc, Peabody, MA)
operating at 60 kV.
Polarized light microscopy
To examine IFM fiber morphology by polarized light
microscopy (PLM), flies were prepared as described previously (Nongthomba and Ramachandra 1999) except
thoraces were kept intact. Briefly, whole flies were dehydrated through a series of 50, 70, 80, 90, and 100% ethanol
for 1 h in each solution at room temperature. The flies were
then placed in methyl salicylate for 1 h at room temperature, fixed on slides using permount and viewed under
polarized light. Pictures were taken with a digital camera
and Magnafire imaging software.
Flight test and wing beat frequency analysis
Flight test analysis was performed as described in Vigoreaux et al. (1998) using a scoring range from 0 (flightless)
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J Muscle Res Cell Motil (2007) 28:219–230
to 6 (upward flight). Wing beat frequency was measured
using an optical tachometer as described previously (Hyatt
and Maughan 1994).
Sinusoidal analysis of skinned flight muscle fibers
Sinusoidal analysis was performed as described previously
(Dickinson et al. 1997) with the exception that after being
stretched to just taught, each fiber was stretched by 2%
increments until the oscillatory work was maximized (for
details, see Liu et al. (2005)). The solutions used are
described in Henkin et al. (2004).
Results
Transgenic Drosophila expressing mutant flightin are
flightless
MALDI-TOF mass spectrometry was used to identify
potential phosphorylation sites in flightin. We compared
the spectra obtained from the tryptic digest of non-phosphorylated spot N1 to that generated from phosphorylated
spot P8 (Vigoreaux and Perry 1994; also, Fig. 2). A peak at
m/z 1437.65, corresponding to peptide N137-K150 (theoretical MH+ mass 1437.69), was detected in N1 and P8. A
peak at 1677.08 (corresponding to N137-K150 plus three
phosphates) was detected only in P8. Also present in P8
only was a peak at 1616.26 that closely matches the theoretical mass of the partial tryptic peptide D152-R164 with
the addition of two phosphates. Based on these preliminary
results, we selected S139, S141, S145, T158, and S162 for
mutagenesis.
Three P element transformation vectors were constructed that consist of the Actin88F (Act88F) promoter
and part of the flightin gene with five (fln5STA), three
(fln3SA), and two (fln2TSA) potential phosphorylation site
residues changed to alanine (Fig. 1). The Act88F promoter
fragment extends from –1420 to +628 base pairs relative to
the translation initiation site and has been previously
shown to drive transgenic expression of flightin (Barton
et al. 2005) and other genes (e.g., a MHC construct (Cripps
et al. 1999)) in the IFM. The flightin fragment, extending
from +1 to +1136 and including 455 base pairs of 30 noncoding DNA, is identical to that used previously to rescue
the flightin null strain fln0 (Barton et al. 2005) except for
the following changes: fln5STA has five potential phosphorylation sites changed to alanine (S139A, S141A,
S145A, T158A, S162A); fln3SA has three of the five sites
(S139A, S141A, and S145A) changed to alanine, and
fln2TSA has the remaining two of the five sites (T158A and
S162A) changed to alanine (Fig. 1).
J Muscle Res Cell Motil (2007) 28:219–230
223
Fig. 1 Partial amino acid sequence of flightin and schematic
representation of Act88F-fln transgene used in this study. (A) The
portion of flightin sequence from residue 137 to residue 164 that
include the amino acids that were altered by site directed mutagenesis
(bold underlined). (B) The Act88F promoter region is represented by
the gray rectangle, flightin exons (E) with filled boxes, and introns (I)
and the 30 untranslated region (UTR) are represented as empty boxes.
Bar = 100 base pairs
crossed into fln0 to generate w1118; P[w+, Act88F-flnS139A,
S141A, S145A
]; fln0 e (hereafter referred to as P[fln3SA]b1).
Six independent lines were generated using the fln2TSA
transgene. Three were on the third chromosome, one was
on the second, one was on the X chromosome and 1 was
not mapped. A line on the second chromosome with the
darkest eye color, w1118; P[w+, Act88F-flnT158A, S162A]; fln+
(hereafter referred to as P[fln2TSA]a1), was chosen for
characterization and crossed into fln0 to generate w1118;
P[w+, Act88F-fln T158A, S162A]; fln0 e (hereafter referred to
as P[fln2TSA]b1).
None of the transgenes was able to rescue the flightless
phenotype of fln0 and all three transgenic strains were
unable to beat their wings (Table 1).
P[fln5STA]b1 and P[fln2TSA]b1 affect accumulation of
flightin phosphovariants
Thirteen independent fln5STA transgenic lines were generated. Nine were on the 3rd chromosome, one was on the
second, and three were on the X chromosome. One of the
lines with an X chromosome insertion was chosen for full
characterization based on the apparent levels of protein
expression using eye color as a reference. This line, w1118,
P[w+, Act88F-flnS139A, S141A, S145A, T158A, S162A]; fln+, (hereafter referred to as P[fln5STA]a1), was crossed into fln0
to generate w1118, P[w+, Act88F-flnS139A, S141A, S145A, T158A,
S162A
]; fln0 e (hereafter referred to as P[fln5STA]b1).
Seven independent lines were generated using the fln3SA
transgene. Three were on the third chromosome, three were
on the second chromosome, and one was on the X chromosome. Based on the eye color criteria mentioned above,
one line with a 2nd chromosome insertion, w1118; P[w+,
Act88F-flnS139A, S141A, S145A]; fln+ (hereafter referred to as
P[fln3SA]a1), was chosen for characterization and was
We carried out 2DE to determine the flightin phosphorylation profile that results from expression of the mutant
transgenes in flies with no endogenous expression of
flightin (i.e., ‘‘b1’’ transgenic lines). Adult (2–5 days old)
wild-type flies express nine phosphorylated variants, P1–
P9, a major unphosphorylated variant (N1) and a minor
unphosphorylated variant (N2) that is not always detected
(Fig. 2A; see also Vigoreaux and Perry (1994)). All three
transgenic lines show expression of N1 but differ in their
level of phosphovariant expression. P[fln5STA]b1 showed
only weak accumulation of spots P1 and P2 in newly
eclosed flies (not shown) but these phosphovariants fail to
accumulate in the mature adult (Fig. 2B). In contrast, spots
P1, P2, and P3 are present in adult P[fln2TSA]b1 (Fig. 2D).
The phosphovariant distribution in P[fln3SA]b1 flies is
similar to that of wild type (Fig. 2C).
Table 1 Flight and muscle properties of normal, mutant, and transgenic flies
Genotype
Flight index (0–6)
Wing beat
frequency (Hz)
Fiber
morphology
Sarcomere length
(lm) newly eclosed
Sarcomere length
(lm) adult
+/+
5.4 ± 0.1
192 ± 1
N
3.3 ± 0.02
3.3 ± 0.02
P[fln5STA]b1
0*
0*
C
4.0 ± 0.05*
1.9 ± 0.04*
P[fln3SA]b1
0*
0*
P
4.5 ± 0.04
3.6 ± 0.02*
0*
0*
C
4.1 ± 0.06*
2.8 ± 0.07*
fln0
0*
0*
C
3.81
2.3 ± 0.09*
P[fln+]fln0
3.2 ± 0.2*
198 ± 15
N
n/a
3.4 ± 0.03
P[fln
2TSA
]b1
* Denotes a significant difference (P \ 0.05) from wild type (+/+). Denotes a significant difference (P \ 0.05) from wild type, P[fln5STA]b1,
and P[fln2TSA]b1. From Reedy et al. (2000); from Barton et al. (2005). Fiber morphology scores obtained by polarized light microscopy,
N = normal, C = complete hypercontraction and P = partial hypercontraction. Flight index n = 30; wing beat frequency n = 10; fiber morphology score n = 5; sarcomere length newly eclosed 40 \n \ 90; sarcomere length adult 60 \ n \ 90. n/a = not available. Refers to distance
between Z-band remnants for mutants in which sarcomere structure was disrupted. Newly eclosed refers to flies \30 min post-eclosion. Adult
refers to flies 2–5 days old
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J Muscle Res Cell Motil (2007) 28:219–230
filaments is not maintained. Similar to fln0, sarcomeres
from both mutants (i.e., distance from Z-band remnant to
Z-band remnant) have a shorter length than wild type, with
P[fln5STA]b1 approximately thirty percent shorter than
P[fln2TSA]b1. Sarcomeres in adult P[fln3SA]b1 have shortened by almost 1 lm from their length in newly eclosed
and are now closer in length to those in wild-type adult
IFM (Table 1). Adult P[fln3SA]b1 sarcomeres also show
degeneration, but the M-line is still visible and the Z-bands
are more intact than those in P[fln5STA]b1 and P[fln2TSA]b1
sarcomeres. The defects in P[fln3SA]b1 are most intriguing
given that this mutant exhibits almost the same flightin
isovariant profile as wild-type flies (Fig. 2).
Flightin accumulation is reduced in transgenic lines
Fig. 2 Western blot 2DE analysis of adult (2–5 day old) wild type
and transgenic flies. (A) Wild type; (B) P[fln5STA]b1; (C) P[fln3SA]b1;
(D) P[fln2TSA]b1. N1 and N2 are the non-phosphorylated isoelectric
variants and P1–P9 are phosphorylated variants (Vigoreaux and Perry
1994). Wild-type flies express one predominant non-phosphorylated
variant (N1) and up to nine phosphorylated variants (P1–P9). Note
that P[fln5STA]b1 and P[fln2TSA]b1 have abnormal phosphovariant
accumulation. For all blots, the basic end is to the left
P[fln5STA]b1, P[fln3SA]b1, and P[fln2TSA]b1 show fiber
and myofibrillar defects
A characteristic feature of fln0 flies is severe hypercontraction of IFM, where dorsolongitudinal muscle (DLM)
fibers are pulled away from the cuticle (Reedy et al. 2000).
When examined under polarized light, adult flies (2–5 day
old) from all three lines demonstrate hypercontraction
(Table 1). P[fln5STA]b1 and P[fln2TSA]b1 show complete
hypercontraction of all DLM fibers, a phenotype nearly
identical to that of fln0. P[fln3SA]b1 shows partial hypercontraction and some DLM fibers do not appear affected
(data not shown).
Electron micrographs show structurally normal IFM
sarcomeres in newly eclosed transgenic flies (Fig. 3 C, E,
G). The M-lines and Z-bands appear intact. One striking
difference, however, is that the sarcomeres of the three
transgenic lines are significantly longer than normal
(Table 1), a feature also observed in fln0 late stage pupa
(Reedy et al. 2000). Sarcomeres in newly eclosed
P[fln3SA]b1 are significantly longer than sarcomeres in wild
type, P[fln5STA]b1 and P[fln2TSA]b1. Sarcomere structure is
severely disrupted in adult (2–5 day old) P[fln5STA]b1 and
to a lesser but significant degree in P[fln2TSA]b1 and
P[fln3SA]b1 (Fig. 3 D, F, H). P[fln5STA]b1 and P[fln2TSA]b1
myofibrils have similar defects in that the M-line is no
longer visible and the structured lattice of thick and thin
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We measured accumulation of flightin in the IFM by
quantitative western blots. The three transgenic lines
showed reduced levels of flightin expression when compared to wild type in both newly eclosed and mature adult
stages (Fig. 4). Flightin levels increased with adult maturation in both wild-type and P[fln3SA]b1 flies but decreased
in P[fln5STA]b1 and P[fln2TSA]b1. Flightin expression levels
also were found to be reduced in independently generated
transgenic lines for the three constructs suggesting that the
reduction in flightin expression is not due to position
effects (data not shown).
P[fln5STA]b1 and P[fln2TSA]b1 IFM fibers are
mechanically impaired
We used sinusoidal analysis to study the mechanical
properties of IFM skinned fibers. These studies were limited to fibers from newly eclosed adults as fibers from older
adults show structural defects (Fig. 3). The oscillatory
power output profile of P[fln3SA]b1 fibers was similar to
that of wild type over the frequency range tested (0.5–
1,000 Hz), and there was no difference in the amount of
oscillatory power produced at fmax (the frequency at which
maximum power production occurs) (Table 2, Fig. 5A). In
contrast, nearly one half and one third, respectively of the
fibers from P[fln5STA]b1 and P[fln2TSA]b1 were unable to
produce oscillatory power while the remaining fibers produced only minimal power.
The inability of P[fln5STA]b1 and P[fln2TSA]b1 fibers to
produce oscillatory power is reflected in the reduced amplitude
of the viscous modulus (Ev), the out-of-phase component of
dynamic stiffness that is a measure of the oscillatory work
produced (negative values) and the work absorbed (positive
values) by a fiber. Unlike wild-type and P[fln3SA]b1 fibers
whose modulus is negative over the frequency range *25 to
J Muscle Res Cell Motil (2007) 28:219–230
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Fig. 3 Electron microscopy of
newly eclosed and adult IFM
from wild-type and transgenic
flies. A, C, E, G are from newly
eclosed IFM. B, D, F, H are
from 2 to 5 day old adult IFM.
All pictures are longitudinal
sections. (A, B) Wild-type;
(C, D) P[fln5STA]b1; (E, F)
P[fln3SA]b1; (G, H)
P[fln2TSA]b1. The myofibrillar
structure in newly eclosed
adults of the three transgenic
lines is indistinguishable from
wild type, except for longer
sarcomeres (see Table 1). Note
that there is myofibrillar
degeneration in mature adults of
the three transgenics lines,
P[fln5STA]b1 shows the most
severe sarcomere breakdown
and P[fln3SA]b1 the least severe.
Bar = 1 lm
110 Hz, fibers from P[fln5STA]b1 and P[fln2TSA]b1 produce a
plot with a less pronounced dip into the negative quadrant
(Fig. 5B). Note also that P[fln3SA]b1 fibers show a frequency
shift that mimics the one produced by wild-type fibers over the
frequency range tested, while P[fln5STA]b1 and P[fln2TSA]b1
do not show a substantial shift. The frequency of maximum
work is lower in P[fln5STA]b1 and P[fln2TSA]b1 than in wildtype and P[fln3SA]b1 but the differences are not statistically
significant (Table 2).
The elastic modulus (Ee, the in-phase component of the
dynamic stiffness that is a measure of fiber compliance) is
higher in P[fln5STA]b1 but unchanged in P[fln2TSA]b1 and
P[fln3SA]b1 (Fig. 5C, Table 2). While not statistically significant, a gradual increase in the elastic modulus values is
evident in the mutants with two, three, and five phosphorylation site alterations.
fln5STA and fln2TSA transgenes show a dominant negative
effect
Fig. 4 Quantification of flightin accumulation in normal and transgenic IFM in newly eclosed and 2–5 day old adult Drosophila. The
graph represents the relative intensity of flightin bands from 1DPAGE. The y-axis is arbitrary units representing intensity. Each lane
was loaded with an equal amount of protein (n = 4)
Wild-type flies (fln+) expressing the fln5STA transgene
(P[fln5STA]a1) and the fln2TSA transgene (P[fln2TSA]a1)
displayed a dominant negative phenotype. Both
123
226
J Muscle Res Cell Motil (2007) 28:219–230
Table 2 Mechanical parameters of IFM skinned fibers from newly eclosed flies
Genotype
fmax (Hz)
N
Power Output (W/m3)
+/+
9
96 ± 4
15 ± 2
P[fln5STA]b1
9€
49 ± 4
1.4 ± 1
P[fln3SA]b1
9
91 ± 4
17 ± 4
P[fln
2TSA
]b1
fln0
€
Ev (kN/m2)
–40 ± 7
–6 ± 6
–46 ± 8
Ee (kN/m2)
Dynamic stiffness (kN/m2)
173 ± 20
177 ± 21
325 ± 63**
325 ± 63**
253 ± 34
258 ± 35
11
57 ± 4
0±1
3±2
194 ± 40*
194 ± 40*
14
77 ± 38
–4 ± 3
10 ± 3
127 ± 23
128 ± 23
Values are at maximum Ca2+ activation (pCa 5). fmax is the frequency of maximum power output. * Denotes a significant difference (P \ 0.05)
from P[fln5STA]b1; ** Denotes a significant difference (P \ 0.05) from wild type and P[fln2TSA]b1; Denotes a significant difference (P \ 0.05)
from wild type and P[fln3SA]b1; € four fibers did not produce positive work. From Henkin et al. (2004)
P[fln5STA]a1 and P[fln2TSA]a1 show flight and fiber defects
(Table 3). While the fibers are not hypercontracted to the
extent seen in P[fln5STA]b1 and P[fln2TSA]b1, there was on
average three DLM fibers whose length were shortened and
were detached from the cuticle (Table 3 and data not
shown). This is in direct contrast to P[fln3SA]a1, which was
indistinguishable from wild type.
Electron micrographs of adult IFM showed normal
myofibrils in P[fln3SA]a1 (Figs. 6E, F and 7C) and myofibrillar defects in P[fln5STA]a1 (Figs. 6C, D and 7B) and
P[fln2TSA]a1 (Figs. 6G, H and 7D). Newly eclosed myofibrils were normal in both P[fln3SA]a1 and P[fln2TSA]a1
(Fig. 6E, G), and nearly normal in P[fln5STA]a1, although
slight abnormalities such as atypical M-lines were
observed (Fig. 6C). In adult IFM, sarcomere length was
shorter in all three transgenic lines than in newly eclosed
flies (Table 3).
Discussion
In this study we have shown that transgenic flies expressing
flightin with specific amino acid substitutions are flight
impaired due to mechanical and structural defects of their
flight muscles. Unlike the Act88F-fln+ construct used previously (Barton et al. 2005), none of the three Act88F-fln
mutant transgenes tested in this study were able to fully
rescue the flight and muscle defects engendered by fln0.
The results suggest that phosphorylation plays an essential
role in dictating flightin’s functional properties in myofibril
assembly and contractile mechanics. An alternative
explanation is that the amino acid replacements affect
protein folding or stability and the resulting phenotypes are
due to reduced levels of flightin accumulation. The dominant negative effects seen in wild-type flies that express the
5STA and 2TSA mutant transgenes argues against underexpression as the sole mechanism responsible for the
contractile and structural defects. Regardless of the
mechanisms underlying the defects, the results validate
previous studies that showed flightin is essential for flight
123
muscle development and function in Drosophila (Henkin
et al. 2004; Reedy et al. 2000).
We had shown in a previous study that an Act88F-fln+
transgene construct, similar to the ones used here, drives
expression of nearly wild-type levels of flightin in the IFM
and rescues the mutant phenotype of fln0 flies (Barton et al.
2005). P-element mediated transgenes insert randomly into
the Drosophila genome occasionally resulting in an insertion that is not favorable for transcription. The reduced
flightin expression levels in the transgenic lines reported
here are not due to position effects since independently
generated transgenic lines for each of the three constructs
showed similar levels of expression (data not shown).
Another plausible explanation is that the amino acid
replacements affect flightin binding to myosin, and
unbound flightin is degraded. Mhc13, a single amino acid
mutation in the myosin rod that prevents flightin binding
in vitro, is known to prevent flightin accumulation in the
IFM in vivo (Ayer and Vigoreaux 2003; Kronert et al.
1995). The amino acids mutated in this study could (i) be
part of the myosin binding domain, (ii) affect binding
indirectly (and non-specifically) by altering flightin conformation, or (iii) be phosphorylation sites that regulate the
binding of an adjacent domain to myosin, as has been
shown for M-protein (Obermann et al. 1998). The C-terminal region of flightin that includes S139 through S162 is
poorly conserved across the Insecta, making this region an
unlikely candidate for a myosin-binding domain. That
T158 and S162 are important for flightin function is supported by the observation that these two amino acids,
unlike S139, S141, and S145, are strictly conserved among
14 species of Drosophila spanning *65 million years of
evolution (Soto and Vigoreaux, in preparation).
The dominant negative phenotype of P[fln5STA]a1 and
P[fln2TSA]a1 is unlikely to result from over-expression of
flightin. Excess wild-type flightin gene copy number does
not negatively affect IFM structure or function (Barton
et al. 2005), unlike what has been reported for other contractile protein genes (e.g., Arredondo et al. 2001; Cripps
et al. 1994). A more likely explanation for the dominant
J Muscle Res Cell Motil (2007) 28:219–230
Fig. 5 Mechanical parameters of maximally Ca2+ (pCa 5.0) activated
IFM fibers from newly eclosed adults. All measurements are plotted
as a function of oscillation frequency. (A) Power output; (B) viscous
modulus (Ev); (C) elastic modulus (Ee). Values are means ± SE for
wild-type, P[fln5STA]b1, P[fln3SA]b1, and P[fln2TSA]b1
negative phenotype is that expression of mutant protein
lacking potential phosphorylation sites disrupts the normal
stoichiometry between phosphorylated and dephosphorylated flightin required for function. Adult wild-type
flies express up to nine phosphorylated and two
227
non-phosphorylated flightin variants whose relative abundance change during IFM development and maturation
(Vigoreaux and Perry 1994). Since the mutant transgenes
are regulated by the Act88F promoter whose expression is
activated earlier in development than the expression of the
endogenous flightin promoter (Barton et al. 2005), it is
possible that the mutant protein out-competes the wild-type
protein during the initial stages of myofibril assembly. This
would result in a greater proportion, relative to expression
levels, of the mutant protein being incorporated in the
myofibril. That a dominant negative effect is not seen in
P[fln3SA]a1 demonstrate that premature expression of a
flightin transgene does not impair flight muscle development, a fact borne out from our previous study (Barton
et al. 2005). Altogether, these results suggest that phosphorylation of T158 and/or S162 is necessary for flight
muscle function.
In addition to the dominant negative phenotype of
P[fln5STA]a1 and P[fln2TSA]a1, results from the EM analyses and the fiber mechanic experiments support our
conclusion that the phenotypes of the transgenic strains are
not due solely to reduced levels of flightin expression. One
of the characteristic features of fln0 is that pupal IFM sarcomeres are, on average, longer than normal (Reedy et al.
2000). Likewise, sarcomeres of newly eclosed
P[fln5STA]b1, P[fln3SA]b1, and P[fln2TSA]b1 adults are also
longer than those of wild-type IFM. Sarcomeres of
P[fln3SA]b1 were significantly longer than either of the two
other transgenic strains despite the fact that P[fln3SA]b1
flies had higher levels of mutant protein expression than
P[fln5STA]b1 and P[fln2TSA]b1. Thus the absence of flightin,
as well as the presence of a mutant form, affects length
regulation of thick filaments and sarcomeres.
Skinned fibers from fln0 and Mhc13, which are devoid of
flightin, showed an *59% and 82% reduction, respectively, in Ee when compared to fibers from wild-type flies
(Henkin et al. 2004). In contrast, the elastic moduli of
skinned fibers from P[fln3SA]b1 and P[fln2TSA]b1 was not
different from wild type, while that of P[fln5STA]b1 was
higher than wild type (Table 2). Similar trends are
observed in dynamic stiffness, with fln0 and Mhc13 showing
reductions of *61% and 83%, respectively, P[fln5STA]b1
showing an increase, and P[fln3SA]b1 and P[fln2TSA]b1
showing no change. P[fln5STA]b1 is the first described flight
muscle mutant to show an increased elastic modulus.
Mutants in paramyosin (Liu et al. 2005), projectin (Moore
et al. 1999), myosin regulatory light chain (Dickinson et al.
1997), as well as the aforementioned flightin and MHC
mutants, all demonstrate a decrease in Ee. The reason for
this is not presently known but the results suggest that the
mutant flightin may be interacting differently with the
myosin rod, or that the presence of a disproportionately higher amount of unphosphorylated or partially
123
228
J Muscle Res Cell Motil (2007) 28:219–230
Table 3 Flight and muscle properties of normal and transgenic flies
Genotype
Flight index (0–6)
Fiber morphology
Sarcomere length (lm) newly eclosed
Sarcomere length (lm) adult
3.3 ± 0.01
+/+
5.4 ± 0.1
N
3.3 ± 0.02
P[fln5STA]a1
0.7 ± 0.01*
P
3.4 ± 0.01#
3.3 ± 0.1
P[fln3SA]a1
4.9 ± 0.1
N
3.2 ± 0.01
3.0 ± 0.1§
P[fln2TSA]a1
0.6 ± 0.2*
P
3.3 ± 0.02
3.0 ± 0.1§
* Denotes a significant difference (P \ 0.05) from wild type and P[fln3SA]a1; Denotes a significant difference (P \ 0.05) from wild type,
P[fln5STA]a1, P[fln2TSA]a1. # Denotes a significant difference (P \ 0.05) from wild type and P[fln3SA]a1 and P[fln2TSA]a1. § Denotes a significant
difference (P \ 0.05) from wild type and P[fln5STA]a1. Fiber morphology score N = normal and P = partial hypercontraction. Flight index
n = 30, fiber morphology score n = 15, sarcomere length newly eclosed 24 \ n \ 65, sarcomere length adult 35 \ n \ 90
Fig. 6 Electron microscopy of
newly eclosed and adult IFM
from wild-type and transgenic
flies. (A, B) Wild-type; (C, D)
P[fln5STA]a1; (E, F)
P[fln3SA]a1; (G, H)
P[fln2TSA]a1. A, C, E, and G are
from newly eclosed adults. B,
D, F, and H are from 2 to 5 day
old adults. Newly eclosed adults
of all strains have normal
myofibrils except for minor
defects in P[fln5STA]a1, such as
the incomplete M line (arrow in
E). In older adults, myofibrillar
structure remains intact in
P[fln3SA]a1 but not in
P[fln5STA]a1 and P[fln2TSA]a1.
All pictures are longitudinal
sections. Bar = 1 lm
phosphorylated flightin alters the biomechanical properties
of thick filaments.
The expression of the 5STA and 2TSA transgenes had a
more disruptive effect on the fiber mechanical properties
than did the expression of the 3SA transgene. Likewise,
P[fln5STA]b1 and P[fln2TSA]b1 fibers are more hypercontracted and myofibrils show more structural damage than
the corresponding fibers and myofibrils in P[fln3SA]b1. On
2DE of IFM fibers, P[fln5STA]b1 and P[fln2TSA]b1 show a
complete absence of phosphovariants while the
123
phosphovariant distribution of P[fln3SA]b1 is more similar
to that of wild type. We conclude from these results that
T158 and/or S162 define a function that is distinct from the
one that involves S139, S141, and S145. One scenario that
is consistent with the results presented here is that phosphorylation of T158 and/or S162 precedes phosphorylation
of the other three sites. T158 and/or S162 are important for
the structural integrity and the mechanical performance of
IFM fibers, while the role of S139, S141, and S145 in
mechanical performance does not appear to be as critical.
J Muscle Res Cell Motil (2007) 28:219–230
229
Fig. 7 Electron microscopy of
adult (2–5 day old) IFM from
wild-type and transgenic flies.
(A) Wild-type; (B) P[fln5STA]a1;
(C) P[fln3SA]a1; (D) P[fln2TSA]a1.
Note the peripheral disruption and
loose myofilaments in
P[fln5STA]a1 and P[fln2TSA]a1.
P[fln3SA]a1 appears normal
(compare to A). All pictures are
cross sections. Bar = 1 lm
Recent studies have examined the role of phosphorylation of various thick filament proteins. Disruption of up
to four phosphorylation sites of Drosophila paramyosin
(Liu et al. 2005) and two phosphorylation sites of myosin
regulatory light chain (Dickinson et al. 1997; Tohtong
et al. 1995) had no discernible effects on muscle ultrastructure. In contrast, transgenic mice expressing a
mutant cardiac MyBP-C with five phosphorylation sites
changed to alanine showed slightly misaligned sarcomeres and other minor structural defects (Sadayappan
et al. 2005). These results contrast sharply with the
pronounced alterations in muscle ultrastructure that result
from the expression of a mutant flightin. Thus, while
phosphorylation of cardiac MyBP-C and IFM flightin
appear to be essential for maintaining sarcomere integrity, our results point to a more indispensable role for
flightin. Despite their lack of sequence identity, flightin
and MyBP-C bind to the same site in the LMM and are
necessary for normal thick filament assembly and contractile mechanics. The absence of flightin in vertebrate
genomes and MyBP-C in Drosophila raises the possibility that these two distinct muscle proteins may perform
similar structural and functional roles in the thick
filament.
In summary, the results presented here demonstrate that
phosphorylation of flightin is essential for its structural and
functional properties. The results are consistent with
models that have proposed a role for flightin in dictating
thick filament and sarcomere length in developing muscle
and maintaining the structural integrity of myofibrils in
adult IFM (Reedy et al. 2000).
Acknowledgements We are indebted to William Barnes and Mark
Miller for help in mechanics, Nicole DeLance for assistance with
electron microscopy imaging, and members of the Maughan and
Vigoreaux labs for advice and helpful discussions. We thank Hiroyuki
Matsumoto for facilitating the mass spectrometry analysis. Supported
by NSF grants 0090768 and 0315865 to JOV, and a predoctoral fellowship of the Vermont Genetics Network (BB) through NIH Grant
Number 1 P20 RR16462 from the BRIN program of the National
Center for Research Resources.
References
Arredondo JJ, Mardahl-Dumesnil M, Cripps RM, Cervera M,
Bernstein SI (2001) Overexpression of miniparamyosin causes
muscle dysfunction and age-dependant myofibril degeneration in
the indirect flight muscles of Drosophila melanogaster. J Muscle
Res Cell Motil 22:287–299
Ayer G, Vigoreaux JO (2003) Flightin is a myosin rod binding
protein. Cell Biochem Biophys 38:41–54
Barton B, Ayer G, Heymann N, Maughan DW, Lehmann FO,
Vigoreaux JO (2005) Flight muscle properties and aerodynamic
performance of Drosophila expressing a flightin transgene. J Exp
Biol 208:549–560
123
230
Cripps RM, Becker KD, Mardahl M, Kronert WA, Hodges D,
Bernstein SI (1994) Transformation of Drosophila melanogaster
with the wild-type myosin heavy-chain gene: rescue of mutant
phenotypes and analysis of defects caused by overexpression. J
Cell Biol 126:689–699
Cripps RM, Suggs JA, Bernstein SI (1999) Assembly of thick
filaments and myofibrils occurs in the absence of the myosin
head. EMBO J 18:1793–1804
Davis JS, Hassanzadeh S, Winitsky S, Lin H, Satorius C, Vemuri R,
Aletras AH, Wen H, Epstein ND (2001) The overall pattern of
cardiac contraction depends on a spatial gradient of myosin
regulatory light chain phosphorylation. Cell 107:631–641
Dickinson MH, Hyatt CJ, Lehmann F-O, Moore JR, Reedy MC,
Simcox A, Tohtong R, Vigoreaux JO, Yamashita H, Maughan
DW (1997) Phosphorylation-dependent power output of transgenic flies: an integrated study. Biophys J 73:3122–3134
Domingo A, Gonzalez-Jurado J, Maroto M, Diaz C, Vinos J, Carrasco
C, Cervera M, Marco R (1998) Troponin-T is a calcium-binding
protein in insect muscle: in vivo phosphorylation, musclespecific isoforms and developmental profile in Drosophila
melanogaster. J Muscle Res Cell Motil 19:393–403
Flashman E, Watkins H, Redwood C (2007) Localization of the
binding site of the C-terminal domain of cardiac myosin-binding
protein-C on the myosin rod. Biochem J 401:97–102
Henkin JA, Maughan DW, Vigoreaux JO (2004) Mutations that affect
flightin expression in Drosophila alter the viscoelastic properties
of flight muscle fibers. Am J Physiol Cell Physiol 286:C65–C72
Hyatt CJ, Maughan DW (1994) Fourier analysis of wing beat signals:
assessing the effects of genetic alterations of flight muscle
structure in Diptera. Biophys J 67:1149–1154
Kinumi T, Niwa H, Matsumoto H (2000) Phosphopeptide sequencing
by in-source decay spectrum in delayed extraction matrixassisted laser desorption ionization time-of-flight mass spectrometry. Anal Biochem 277:177–186
Kronert WA, O’Donnell PT, Fieck A, Lawn A, Vigoreaux JO,
Sparrow JC, Bernstein SI (1995) Defects in the Drosophila
myosin rod permit sarcomere assembly but cause flight muscle
degeneration. J Mol Biol 249:111–125
Laemmli UK (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 270:680–685
Layland J., Solaro RJ, Shah AM (2005) Regulation of cardiac
contractile function by troponin I phosphorylation. Cardiovasc
Res 66:12–21
Liu H., Miller MS, Swank DM, Kronert WA, Maughan DW,
Bernstein SI (2005) Paramyosin phosphorylation site disruption
affects indirect flight muscle stiffness and power generation in
Drosophila melanogaster. Proc Natl Acad Sci U S A 102:10522–
10527
Mayans O, van der Ven PF, Wilm M, Mues A, Young P, Furst DO,
Wilmanns M, Gautel M (1998) Structural basis for activation of
the titin kinase domain during myofibrillogenesis. Nature
395:863–869
Moore JR, Vigoreaux JO, Maughan DW (1999) The Drosophila
projectin mutant, bentD, has reduced stretch activation and
altered indirect flight muscle kinetics. J Muscle Res Cell Motil
20:797–806
Nongthomba U, Cummins M, Clark S, Vigoreaux JO, Sparrow JC
(2003) Suppression of muscle hypercontraction by mutations in
123
J Muscle Res Cell Motil (2007) 28:219–230
the myosin heavy chain gene of Drosophila melanogaster.
Genetics 164:209–222
Nongthomba U, Ramachandra NB (1999) A direct screen identifies
new flight muscle mutants on the Drosophila second chromosome. Genetics 153:261–274
Obermann WM, Gautel M, Weber K, Furst DO (1997) Molecular
structure of the sarcomeric M band: mapping of titin and myosin
binding domains in myomesin and the identification of a
potential regulatory phosphorylation site in myomesin. EMBO
J 16:211–220
Obermann WM, van der Ven FP, Steiner F, Weber K, Furst DO
(1998) Mapping of a myosin-binding domain and a regulatory
phosphorylation site in M-protein, a structural protein of the
sarcomeric M band. Mol Biol Cell 9:829–840
Qiu F, Brendel S, Cunha PM, Astola N, Song B, Furlong EE, Leonard
KR, Bullard B (2005) Myofilin, a protein in the thick filaments
of insect muscle. J Cell Sci 118:1527–1536
Reedy MC, Bullard B, Vigoreaux JO (2000) Flightin is essential for
thick filament assembly and sarcomere stability in Drosophila
flight muscles. J Cell Biol 151:1483–1499
Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P (1992) In-gel
digestion of proteins for internal sequence analysis after one- or
two-dimensional gel electrophoresis. Anal Biochem 203:173–
179
Sadayappan S, Gulick J, Osinska H, Martin LA, Hahn HS, Dorn WG
2nd, Klevitsky R, Seidman EC, Seidman GJ, Robbins J (2005)
Cardiac myosin-binding protein-C phosphorylation and cardiac
function. Circ Res 97:1156–1163
Spradling AC, Rubin MG (1982) Transposition of cloned P elements
into Drosophila germ line chromosomes. Science 218:341–347
Sweeney HL, Bowman FB, Stull TJ (1993) Myosin light chain
phosphorylation in vertebrate striated muscle: regulation and
function. Am J Physiol 264:C1085–C1095
Takano-Ohmuro H, Takahashi S, Hirose G, Maruyama K (1990)
Phosphorylated and dephosphorylated myosin light chains of
Drosophila fly and larva. Comp Biochem Physiol 95B:171–177
Tohtong R, Yamashita H, Graham M, Haeberle J, Simcox A,
Maughan D (1995) Impairment of muscle function caused by
mutations of phosphorylation sites in myosin regulatory light
chain. Nature 374:650–655
Vigoreaux JO, Hernandez C, Moore J, Ayer G, Maughan D (1998) A
genetic deficiency that spans the flightin gene of Drosophila
melanogaster affects the ultrastructure and function of the flight
muscles. J Exp Biol 201:2033–2044
Vigoreaux JO, Perry ML (1994) Multiple isoelectric variants of
flightin in Drosophila stretch-activated muscles are generated by
temporally regulated phosphorylations. J Muscle Res Cell Motil
15:607–616
Vigoreaux JO, Saide JD, Pardue ML (1991) Structurally different
Drosophila striated muscles utilize distinct variants of Z bandassociated proteins. J Muscle Res Cell Motil 12:340–354
Vigoreaux JO, Saide JD, Valgeirsdottir K, Pardue ML (1993)
Flightin, a novel myofibrillar protein of Drosophila stretchactivated muscles. J Cell Biol 121:587–598
Vinos J, Domingo A, Marco R, Cervera M (1991) Identification and
characterization of Drosophila melanogaster paramyosin. J Mol
Biol 220:687–700