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Review articles
Genetics of the Drosophila flight
muscle myofibril: a window into the
biology of complex systems
Jim O. Vigoreaux
Summary
This essay reviews the long tradition of experimental
genetics of the Drosophila indirect flight muscles (IFM). It
discusses how genetics can operate in tandem with
multidisciplinary approaches to provide a description, in
molecular terms, of the functional properties of the
muscle myofibril. In particular, studies at the interface
of genetics and proteomics address protein function at
the cellular scale and offer an outstanding platform with
which to elucidate how the myofibril works. Two generalizations can be enunciated from the studies reviewed.
First, the study of mutant IFM proteomes provides insight
into how proteins are functionally organized in the
myofibril. Second, IFM mutants can give rise to structural
and contractile defects that are unrelated, a reflection
of the dual function that myofibrillar proteins play as
fundamental components of the sarcomeric framework and biochemical ``parts'' of the contractile
``engine''. BioEssays 23:1047±1063, 2001.
ß 2001 John Wiley & Sons, Inc.
Introduction
Functional genomics is making possible the description of
biological processes from a broad, systems perspective.
Genome sequence information reveals the identity of the
principal molecular players that define life. As we embark into
the ``post-genome'' era, great efforts are being expended in
trying to elucidate when and where these molecular players
are active and how their interactions determine the fate of the
cell and the organism.
The genome era has unleashed a new data-driven, largescale style of molecular science. Many biologists are facing the
imminent task of consolidating their ``bits'' of information,
derived from painstaking years of reductionist research, with
``terabyte'' datasets being generated daily by new, expansive
technologies. The culmination of these efforts will be the grand
realization of how genotype dictates phenotype. For this goal,
model experimental systems that are amenable to interdisciplinary functional validation are highly desirable.
Research into muscle function has spanned from the
molecular to the organismal, resulting in a broad body of
knowledge about the properties of this remarkable tissue. It is
Department of Biology, University of Vermont, Burlington, VT 05405
USA. E-mail: [email protected]
BioEssays 23:1047±1063, ß 2001 John Wiley & Sons, Inc.
often difficult to consolidate information gained from disparate
disciplines, either because the research is conducted on
different experimental specimens (organism or muscle type)
or because we lack the conceptual tools to properly transpose
the findings of one discipline to another.
This essay reviews the historical tradition in Drosophila IFM
research and discusses how this experimental model can be
exploited to provide an integrated view of muscle function.
Genetics plays a pivotal role in affording an integrative
approach and enabling the validation of functional assays
across levels of biological organization. Genetics also
provides the bridge between traditional, small-scale research
and new, large-scale approaches. Examples of mutations in
myofibrillar proteins are presented to illustrate the multiple
faces of protein function and how genetics provides information about proteins as individuals and as pegs in the cellular
proteome. An understanding of the broad properties and
multifarious functions of myofibrillar proteins is seen as an
important step towards a systems-wide understanding of
muscle function.
Historical synopsis
It has been almost 100 years since Drosophila melanogaster
entered genetics research. The fly has made outstanding
contributions to our knowledge of genes and genomes, from
the first genetic linkage map to the annotated sequence of the
entire genome.(1) Fly genetics has played a primary role in the
functional annotation of genes and in deciphering gene
networks that control embryogenesis, development and
differentiation.
For the past six decades, insect IFM has been a staple of
research on muscle function. The pioneering work of Pringle
and colleagues with Lethocerus IFM led to the discovery of
stretch activation(2) (Fig. 1). Research on Lethocerus IFM also
uncovered the ATP-dependent changes in crossbridge angle
that contributed to the formulation of the ``swinging crossbridge'' model of muscle contraction.(3) The high structural
order of insect IFM has lent itself well to X-ray diffraction
studies, electron microscopy and three-dimensional reconstruction, the results of which continue to provide a privileged
view of the fine details of actomyosin interaction.(3)
Drosophila IFM shares many of the structural features of its
Lethocerus counterpart and, in addition, is amenable to
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Figure 1. The indirect flight muscles of Drosophila and the mechanics of insect flight. Side view of the thorax showing the IFM consists
of two sets of antagonistic muscles that are oriented nearly perpendicular to each other, the dorsal longitudinal muscles (DLM, left to
right) and the dorsal ventral muscles (DVM, top to bottom). Both sets of muscles are anchored at the cuticle by epithelial tendon cells and
move the wings indirectly through deformation of the thoracic cuticle. Contraction of the DVM raise the wings and stretches the DLM; in
turn, contraction of the DLM lower the wings and also stretches the DVM. The stretch (L, length change) results in a concomitant
increase in force (F) that rapidly decays but rises again to a high level after a short delay. This ``stretch activation'' response underlies the
ability of the IFM to perform oscillatory work and produce the high power necessary for flight. For clarity, other muscles of the thorax are
not shown.
genetic manipulation. The properties and features that make
fly IFM an excellent experimental model are well documented.(4) It is remarkable that the IFM has served to study
biological phenomena ranging from the gene (transcriptional
regulation) to the population (fitness traits), at practically every
level of biological organization (Table 1). More significantly,
genetics has afforded the integration of molecular structure
and cellular function with muscle performance and animal
locomotion.(5) In addition to vertical integration across levels of
biological organization, studies of Drosophila flight aerodynamics afford a horizontal integration across physiological
systems that contribute to locomotion.(6)
Genetics provides a useful experimental approach for
uncovering protein function. As with a pickax, genetics can be
utilized in two different ways. The pick end of the tool is used to
``dig out'' individual amino acids that dictate specific properties
of the protein (herein referred to as molecular function). The
broader, chisel-edge is used to ``scrape away'' the protein's
association with other proteins and its contribution to cellular
processes (herein referred to as cellular function).
Genetics and the molecular function
of muscle proteins
Identification of myofibrillar protein genes
Table 2 lists all known IFM myofibrillar proteins and summarizes how each gene or protein was first identified. Recent
analysis of the complete Drosophila genome with gene
prediction software has led to the identification of a potentially
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new myofibrillar gene, stretchin-mlck, that encodes several
conceptual proteins, including a large 926 kDa titin-like
kinase.(7) Thus, an important application of genome data is
to identify new myofibrillar genes. This is particularly important
in light of the fact that additional genes whose mutations give
muscle phenotypes or that are expressed in muscle (as
determined by enhancer trap lines) remain to be identified.(4)
Some of these genes may encode novel myofibrillar proteins
while others are likely to encode proteins involved in muscle
development, muscle attachment, and/or neuromuscular
interaction.(8)
Molecular studies that identified myofibrillar protein genes
were, in many instances, preceded by genetic studies of IFM
function. The convergence of these two approaches has
played a major role in the functional annotation of these genes.
Early genetic studies
A number of genetic loci involved in muscle development and
contractile function were first identified through mutant
screens designed to isolate flightless flies (Fig. 2A).(4) These
screens offered the advantage that flightless flies could be
easily identified among their flighted cohorts so large numbers
of flies were tested for autosomal dominant and/or sex-linked
flightless mutations.(9±11) Furthermore, several mutant strains
recovered exhibited additional phenotypes such as abnormal
wing position or indented thoracic cuticle indicative of a
defective IFM. Later studies revealed that many of these
loci encode the major myofibrillar proteins actin (Act88F),
myosin heavy chain (Mhc), tropomyosin (Tm), troponin I
Review articles
Table 1. Genetic and molecular genetic manipulations of Drosophila IFM afford an experimental model for broad
ranging biological questions
Level
G
Gene
RNA
Protein
E
N
E
T
I
C
Myo®lament
Myo®bril
Fiber
IFM
Fly
S
Population
Properties
Transcriptional regulation
Alternative splicing
Abundance, modi®cation
Biochemical & biomechanical
Protein binding
Proteome
Polymerization
Assembly
Morphology & biomechanics
Biomechanics
Morphology
Length oscillations in vivo
Vertical ¯ight (free)
Aerodynamics (tethered)
Myo®lament spacing & ordering
Fitness
Free ¯ight performance
(wings up A/heldup (wupA/hdp)) and troponin T (upheld/
indented (up/int)), among others. However, a handful of the
mutant genes had more unexpected functions. For example,
erect wing encodes a DNA-binding protein and flapwing
encodes a protein phosphatase.(8) Other studies revealed that
mutations that affect muscle innervation can also result in
dominant flightlessness.(4) Results from these early genetic
studies suggested that only a limited number of myofibrillar
genes are haploinsufficient for flight and that the majority of
mutations interfere with the expression of multiple proteins
within the IFM.
Saturation mutagenesis
To establish if additional autosomal genes can be mutated to
produce a dominant flightless phenotype, Cripps et al.
screened over 40,000 flies and obtained 22 new mutants that
mapped to three loci.(12) Most of the mutants recovered were
Mhc or Act88F alleles. However, a third dominant flightless
and recessive lethal loci, named lethal(3)Laker was also
uncovered. An and Mogami conducted a massive screen
(&120,000 flies) on the third chromosome from which they
recovered ten Act88F alleles and thirteen other dominant
flightless mutations.(11) The availability of genome sequence
should speed up the identification of the gene or genes
affected by these mutations, especially since their genetic
position is already known.(11) In summary, the saturation
studies, together with those described in the previous section,
demonstrate that sarcomeric protein genes can be identified
from relatively simple, high throughput genetic screens.
However, the number of loci identified from these screens
Assays
Examples
Transgenic expression of marker gene
Transgenic expression of minigene/RT-PCR
2DE
Stop ¯ow kinetics, in vitro motility, optical trap
Filament sedimentation, solid phase binding
Mass spectrometry
Centrifugation
In vivo length measurements
Transmission electron microscope
Atomic force microscope
Force transducer rig
Scanning electron microscope
Light microscope
High speed video microscopy
Flight box
Flight arena (respirometry/kinematics chamber)
X-ray diffraction
Wind tunnel
Three-dimensional motion analysis
69
70
9
32,66
71
67
26
34
61,72
73
36
12
15
74
35
36
37
75
appears to be limited to those encoding abundant sarcomeric
proteins.
In addition to the discovery of lethal(3)Laker, the study of
Cripps et al was significant for two other reasons. First, they
identified two Mhc alleles, Mhc13 and Mhc19, whose phenotype
differed from all other mutants recovered. These MHC
mutations appear to have little or no effect on IFM development but resulted in severe and irreversible shortening of
the muscle fibers in the adult. Later studies showed that these
mutations harbor single amino acid changes within the coiled
coil region and probably affect the interaction of flightin with
thick filaments, an interaction that appears to be critical for
muscle cell integrity.(13,14) Second, their study concluded that
``not all major muscle protein genes appear to mutate readily to
give dominant flightlessness and other techniques must be
applied in order to identify additional genes and mutations
within them.'' A subsequent study provided the first systematic
screen for autosomal (second chromosome) recessive muscle mutations in the IFM (Fig. 2C).(15) The study relied on direct
examination of IFM fiber morphology using polarized light
microscopy and resulted in the identification of eight new
complementation groups that result in IFM defects. Five of the
eight complementation groups were mapped genetically but
none of the genes have been identified.
Reverse genetics
A second strategy for obtaining recessive flightless mutations
relies on hemizygosity for a chromosomal deletion or
deficiency. Once a cloned gene or cDNA is in hand, the
cytological position of the gene can be identified by in situ
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Table 2. Myofibrillar proteins of Drosophila IFM
Structure
Thick
®laments
Protein1
MHC (36B)
Cross hybridization
DMLC2 (99DE)
Flightin (76DE)
Paramyosin (66D)
Abundantly expressed RNA
during adult muscle
development, in vitro
translation
Abundantly expressed RNA
during adult muscle
development, in vitro
translation
Antibody
Antibody
Mini-paramyosin (66D)
Cross hybridization
Actin (88F)
Arthrin (88F)
Tropomyosin (88F)
Cross hybridization
Mutation, antibody
Chromosome walking
Troponin T (12A)
Antibody
Troponin I (16F)
Abundantly expressed RNA
during IFM development,
mapping to cytological
region of known mutant
PCR/cross-hybridization
ELC1/3 (98B)
Thin ®laments
Troponin C (73F, 41C)
Z bands
Connecting
®laments
Other
1
2
Identification2
Troponin H (88F)
Antibody, chromosome
walking
GST-2 (53F)
a-Actinin (2C)
Mass spectrometry
Antibody
Z(210) (Ð)
Antibody
Kettin (62C)
Antibody
Z(400/600) (Ð)
Projectin (102C/D)
Antibody
Antibody, PCR
Titin (62C)
Antibody, Mutation
Stretchin-MLCK (52DE?)
PCR, database mining
ADP/ATP translocase
(9EF)
Mass spectrometry
Comments
References
Alternative RNA splicing generates > 14 isoforms.
Embryonic isoform competent for assembly but
not function of IFM
46 aa N-terminal extension; multiple phosphovariants
4, 33
Alternative RNA splicing generates IFM-speci®c
isoform which differs in C-terminal 12 aa
IFM-speci®c protein; multiple phosphovariants
Low levels in IFM, distributed throughout thick
®lament. Multiple phosphovariants
Unique N-term 114 residues not found in other
paramyosins. Localized at the M line and both
ends of thick ®laments. Multiple phosphovariants,
IFM-speci®c 62kD isoform
Act88F gene expressed in IFM only
IFM-speci®c ubiquinated Act88F actin
C-term 27 residues encoded by IFM-speci®c exon;
switching of this region with non-IFM sequence
does not affect function
Long (136aa) C-terminal extension with
polyglutamic tail; 17 aa insertion; phosphorylated,
binds calcium
Alternative IFM-speci®c exon encodes N-terminal
Ala-Pro rich 61aa sequence
Encoded by three genes; the most distantly related
gene is adult-speci®c; IFM expression has not
been established
IFM-speci®c Tropomyosin fusion with 200 aa
hydrophobic, Pro-rich C-term extension;
two spliced variants
Not expressed in non-IFM muscles
Muscle and non-muscle isoforms generated
by alternative RNA splicing from
same gene
Expression limited to IFM and large cells of TDT.
Renamed zetalin
Elongated protein with 35 Ig domains extends from
Z band center to I band
Most likely Kettin
Mutation that deletes kinase domain affects
crossbridge kinetics and stretch activation
Lacks characteristic domains found in A band
and M line region of vertebrate Titin
Seven proteins encoded by three transcription
units. One unit encodes a standard MLCK.
Tissue distribution not yet established
but one form, A(225) identi®ed
in IFM A bands
Abundant in IFM myo®brils; unclear whether
it represents mitochondrial contamination
or a novel isoform
cytological position of gene indicated in parenthesis; (±) no published reference.
Indicates how the Drosophila gene or protein was first identified.
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4
4
76
4, 77
4, 77
4
4
4
4, 78
4
79
80, 59
60
65
65
71
81
65
65, 82, 43
83, 84, 85
7
67
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hybridization to larval polytene chromosomes. Mutagenesis
screens can then be designed to ``target'' the cytogenetic
region of interest. For example, Warmke et al. used a synthetic
deficiency of the myosin regulatory light chain (Dmlc2 ) gene to
screen for recessive lethal mutations.(16) The screen identified
44 mutations that mapped to 23 complementation groups but
none corresponded to Dmlc2. However, a null allele of Dmlc2,
E38 (renamed Dmlc2 E38) was obtained in a similar screen
conducted independently by John Merriam.(16) Interestingly,
the screen by Warmke et al. generated four alleles of a
noncomplementing group that maps outside the region
uncovered by the deficiency and exhibits dominant lethal
synergism with Dmlc2 E38. This group, named l(3)nc99Eb, was
later shown to be within the Tm2 gene.(17) This study
epitomizes both the strength and weaknesses of the genetic
approach. On one hand, they failed to obtain mutant alleles of
Dmlc2 in two separate screens designed to recover dominant
flightless and recessive lethal mutations, respectively, despite
the fact that Dmlc2 E38 shows both characteristics. On the other
hand, they uncovered an unexpected functional interaction
Figure 2. Genetic strategies for isolation of mutations that affect
IFM. A: The traditional approach for obtaining dominant IFM
mutants is to subject male flies to a mutagen (irradiation or feeding
a chemical, most commonly ethyl-methane sulfonate (EMS)), mate
mutagenized males to normal females, and select flightless
progeny that fall to and are captured at the bottom of a flight
testing column.(9) Recessive mutations on the X chromosome are
obtained via a similar screen that selects only males, which are
hemizygous for the sex chromosome.(10) B: (top) Many flightless
mutants also exhibit an abnormal wing position phenotype which
can be selected for without the need for a flight test. (Bottom) Once
a muscle mutant is available, intragenic or intergenic modifier
mutations can be obtained by screening for revertants of the wing
phenotype.(41,47) C: Mutagenized flies can be scored directly for
morphological muscle defects using polarized light to visualize the
IFM after chemical clarification of the cuticle.(15) D: Once the
chromosomal position of a gene is established by in situ
hybridization, it is possible to select for deletions or deficiencies
of that region by screening for the absence of a visible marker (e.g.,
eye color) encoded by a transposable element inserted nearby.
Southern blots of genomic DNA and in situ hybridization to polytene
chromosomes are performed to ascertain the region of interest has
been deleted in lines that fail to express the visible marker.(18) The
vast collection of single transposable element insertion lines makes
this approach nearly universally applicable. The deficiency also
serves to identify null mutations in recessive genes. De novo
mutagenized males are mated to females carrying the deficiency
chromosome and the progeny screened for the absence of the
gene product.(14) This approach permits the isolation of null
mutations without the need to score for a specific muscle
phenotype. E: Transgenic manipulation of Drosophila has enabled
molecular genetic approaches to study protein function in vivo.
Mutations engineered into cloned genes are introduced into the
germline via P element mediated transformation. Expression of the
transformed gene can be restricted to the IFM with the use of
the tissue-specific Act88F promoter.
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between Dmlc2 and Tm2 that may reveal important features of
the muscle regulatory system.
Knowing the cytological location of a gene also allows the
isolation of mutations without having to select for a particular
functional phenotype. One example is the approach taken to
isolate a null mutation for flightin that did not rely on a muscle
phenotype. Instead, a protein dot±blot approach was used to
detect the absence of the protein (Fig. 2D).(14,18) This
approach offers the advantage of high throughput, but it is
useful only for identifying null mutations. More recently,
another phenotype-independent, high throughput approach
based on denaturing HPLC has been developed to identify
missense mutations in any Drosophila gene.(19)
Molecular genetics dissection of protein function
Molecular genetics has served to expand the repertoire of
mutants obtained from the aforementioned studies. The
availability of IFM-specific null mutants has enabled transgenic approaches to study protein structure±function in vivo
without the preoccupation that mutations may affect viability
(Fig. 2E). This has been the case for MHC (Ifm(2)2, alias
Mhc7), ACT88F(KM88), and TM2 (Ifm(3)Tm23).(4) Null alleles
and genetic deficiencies also permit the study of dosage
requirements for IFM function and these have been shown to
differ among contractile protein genes. Thus, while the
expression of MHC, DMLC2, TM2, and ACT88F actin have
been shown to be sensitive to dosage effects, flightin, kettin,
a-actinin, TnT and TnH are dosage insensitive.(4,17,18) Interestingly, Mhc haploids are flightless but the presence of three
genes appears to have little effect upon IFM function.(20,21) If
gene dosage is increased to four, however, defects in IFM
structure appear and flight performance is compromised. In
the case of Act88F, which is also haploinsufficient for flight, the
presence of two extra gene copies has little effect upon flight
performance.
The Act88F gene is not essential for viability of the fly and
has allowed the only genetic study of a muscle actin gene.(22)
These genetic studies have provided information about actin
polymerization and myofibril assembly, actomyosin kinetics
and stretch activation, and torsional flexibility of crossbridges.(22±25) Missense alleles obtained from flightless
screens point to obvious residues that are critical for proper
actin function in the IFM. Nevertheless, this type of screen has
the disadvantage that the selection is based on an extreme
phenotype (lack of flight) and mutations that result in subtle but
important functional changes remain undetected. Drummond
et al. overcame this screening bias by performing random
mutagenesis on a cloned C-terminal fragment of the Act88F
gene and introducing the mutated genes back into flies to study
their effect in vivo.(26) All seven missense mutations generated
in their study were antimorphic for flight but showed varying
effects on IFM myofibril structure and flight performance.
Generally, the alleles obtained from flightless screens tend to
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have more pronounced effects on muscle structure than the
alleles obtained from random in vitro mutagenesis, no doubt a
reflection of the strong selection bias used for the in vivo
screens.(22) Interestingly, only one mutant obtained from the
random in vitro approach corresponds to a mutated residue
(G366S) obtained from large-scale in vivo screens for
dominant flightless mutants.(9,11,27,28) The combination of in
vivo and in vitro mutagenesis yields non-overlapping information about the role of specific amino acids in dictating actin
function in vivo.
Actin mutant alleles that affect flight performance have
been scrutinized to disclose the role of individual residues on
protein function. Early studies of mutant alleles focused on in
vivo effects on myofibril assembly, morphology, and accumulation of myofibrillar proteins.(9) These studies revealed the
large scope of changes that occur as a consequence of each
mutation. Later studies examined more specific actin properties such as stability, conformation and polymerization.(26,29)
In a wide-ranging study, Sparrow et al. documented the effects
of Act88F E93K (an actin mutant in which glutamic acid 93 is
replaced by lysine) on IFM ultrastructure, myofibrillar protein
accumulation, actomyosin interaction, and mechanical properties of skinned fibers.(30) Myofibrils from Act88F E93K mutant
IFM are severely disorganized and have no discernable Z
bands or sarcomeres. As a result, the fibers produce very little
active tension and cannot be stretch activated. The lack of Z
bands results from the absence of a-actinin, and perhaps
reduced amounts of high molecular weight Z band proteins.
Based on these results, the authors proposed that Act88F E93K
mutation may affect the binding of actin to a protein, possibly aactinin, critical for Z band assembly and/or stability.
The development of macroscale (from whole flies) and
microscale (from dissected IFM) purification protocols for
Act88F actin has permitted the application of biochemical
assays to study the function of mutant actins in vitro.(24,31,32)
For example, Bing et al. conducted in vitro motility assays to
determine that the charge reversal in Act88F E93Kcauses a
shift in the position of tropomyosin along the actin filament helix
towards the ``off'' state.(31) Further studies of Act88F E93Kactin
using stop-flow solution kinetics and single molecule mechanics (optical trap) showed that, while the mutation has no
effect on force transmission, it does affect the ability of actin to
bind rabbit skeletal S1 myosin.(32) The reduced affinity for
myosin suggests that actin residue 93 forms part of a
secondary myosin-binding site. In summary, the combination
of molecular assays has provided a detailed description of how
a single amino acid change affects the biochemical properties
of actin (Fig. 3, top).
It is important to correlate the functional properties of the
mutant protein obtained from in vitro studies with measured
fiber parameters in situ to gain insight on the in vivo molecular
mechanism of function. Unfortunately, Act88F E93K affects the
accumulation of several proteins in addition to actin and
Review articles
causes major structural defects in IFM myofibrils. As a result,
it is not possible to ascertain the direct effect of the mutant actin
on IFM biomechanical properties. It is very likely, however, that
the widespread disruption in sarcomere assembly caused by
Act88FE93Kis not a consequence of its affect on tropomyosin
position or myosin binding (Fig. 3, middle). Instead, residue 93
may form part of a binding domain between Z band actin and
another Z band protein, possibly a-actinin. Thus the same
amino acid appears to play distinct functional roles for actins
that differ in their sarcomeric distribution.
The functional properties of MHC also have been investigated intensively by targeted mutagenesis.(33) MHC is
encoded by a single gene that undergoes alternative RNA
splicing to generate isoforms whose expression is developmentally and/or spatially restricted.(4,33) To understand how
different MHC isoforms confer fiber-type-specific properties,
Bernstein's group has generated transgenic flies to express
incorrect isoforms in the IFM. In one study, flies expressing an
embryonic MHC containing five alternative exons not normally
expressed in IFM, were able to assemble a normal IFM, which
later became dystrophic.(33) Transgenic studies also have
shown that myosin molecules lacking the head region can
assemble into thick filaments in vivo but the resulting
sarcomeres and myofibrils have varying lengths and irregular
dimensions.(34)
In summary, in vivo and in vitro mutagenesis has been
employed to study the function of contractile proteins. While
the majority of the studies have examined the mutation effect
upon in vivo muscle structure, more recent work has focused
on the biochemical and biophysical properties of mutant
proteins. However, this work has been limited largely to actin
and MHC. Standardized purification protocols and in vitro
Figure 3. Multiple dimensions of protein function are revealed by
the combination of genetics, biochemistry, and proteomics. (Top)
Traditional genetics elucidate the role of particular amino acids in
dictating the molecular function of a protein (e.g., enzymatic
activity, binding properties). Each coordinate (e.g., m1a1) represents a ``pixel'' of functional information obtained from performing a
biochemical assay on a mutant protein. Points along the y-axis
(e.g., m0a1, m0a2) represent assays performed on the normal (nonmutant) protein. Points along x axis (e.g., m2a0, m3a0) represent
different mutants. Molecular genetics provides additional pixels by
allowing systematic dissection of the protein sequence. (Middle)
Examination of each mutant effect on cell ultrastructure provides
insight about the role of the protein in myofibril assembly and
stability. In some cases in vitro biochemical studies reveal mutant
characteristics that are incongruous with cellular phenotype (e.g.,
m1) while in other cases in vitro and ultrastructural data are
mutually supportive and portray a unified perspective on protein
function (e.g., m2). (Bottom) Proteomics provides an additional
dimension to the study of muscle mutants. By enhancing the view
of the mutant effect on the protein panorama of the cell, proteomics
consolidates molecular and cellular function and enables interpretation of mutant effects within the context of the entire cell.
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biochemical assays need to be developed for the other
contractile proteins. Biochemical studies of low abundance
contractile proteins and/or proteins that do not express well in
recombinant systems remains a challenge.
Molecular genetics and integrative
approaches to protein function
The aforementioned studies on Act88F E93K illustrate a
problem common in studies of contractile protein function that
many of these proteins serve essential roles during myofibril
assembly. If the mutation in question interferes with normal
assembly, its effect on muscle function cannot be ascertained.
For this reason, alleles that have little or no effect on myofibrillar assembly are highly desirable. Three such mutations
were engineered by Tohtong et al. who replaced two myosin
light-chain kinase (MLCK)-phosphorylated serines in DMLC2
with alanines, singly and in combination.(35) The double
mutant gene, Dmlc2 S66A,S67A, is able to rescue the recessive
lethality engendered by Dmlc2 E38 and has no discernible
effect on IFM myofibril assembly ultrastructure. However,
flight is impaired and mechanical studies on skinned fibers
showed a dramatic decrease in oscillatory work production
(i.e., stretch activation).(35,36) Time-resolved X-ray diffraction
of IFM in living flies revealed that a reduction in the number of
power-generating cross-bridges is the most likely explanation
for the drop in oscillatory work.(37)
Dickinson et al. performed an integrative functional
analysis on all three Dmlc2 phosphorylation site mutants and
were able to trace the power deficiencies from the molecular to
the organismal level.(5,36) Their study revealed how specific
amino acid changes affect the in vivo function of the flight
system. In the case of the double mutant Dmlc2 S66A,S67A,
mechanical and metabolic power outputs were significantly
decreased, as was wing beat frequency. Interestingly, double
mutants were able to compensate for the drop in wing beat
frequency by increasing stroke amplitude while hovering,
presumably through the action of the synchronous steering
muscles.(36)
A multifaceted approach similar to that of Dickinson et al.
was taken to study the role of the N-terminal 46 amino acid
extension of DMLC2.(38) Transgenic flies expressing a
truncated DMLC2 (DMLC2 D2±46) and no full-length DMLC2
are viable, are able to walk and jump, have normal IFM
ultrastructure, and can fly, albeit with diminished performance.
Under conditions that stimulated maximum flight performance
in tethered flies, DMLC2 D2±46 flies were capable of generating
flight forces comparable to body weight, or &35% lower than
those produced by wild-type flies. Skinned fiber mechanics
experiments revealed that the elastic modulus (the in-phase
component of the fiber's dynamic stiffness) is significantly
reduced. These studies suggest that the N-terminal extension
of DMLC2 contributes to the elastic properties of the IFM,
possibly by forming a parallel link between the thick filament
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and the thin filament, and that this structure is required for
maximal mechanical efficiency of the IFM.(38) In summary,
genetic manipulations in Drosophila IFM afford an integrated
approach to muscle protein function that spans from the
molecular to the organismal level.
Genetics and the cellular function
of muscle protein
The myofibril is a complex organelle where each protein is
likely to interact with several other proteins. The interaction
between two proteins may be influenced by other proteins that
interact separately with either partner. Several hundred or
more protein contacts underlie a functional myofibril; proper
performance of the contractile machine is dependent upon
correct assembly and stability of the myofibril. Only a small
number of ``functional interactions'' between proteins have
been identified from biochemical studies of partially reconstituted systems (e.g., actin-S1, troponin/tropomyosin). The
difficulty in ascertaining the precise nature of protein contacts
from biochemical studies is even more pronounced for
proteins that interact transiently during assembly or during
the contractile cycle. Genetics, and more recently Functional
Genomics, offer complementary approaches to the study of
protein function by revealing how a protein fits into the cellular
web of interacting proteins, and how these interactions define
biological processes and functions. Unlike conventional
biochemical approaches, the study of protein function by
genetics is not dependent on protein abundance but rather on
functional relevance. Hence genetics can reveal functional
features of protein complexes that are not easily accessible
through biochemistry. Two genetic strategies have been
undertaken to examine protein±protein interactions in IFM:
(i) genetic interactions among extant mutants and (ii) genetic
screens for dominant modifiers of extant mutants.
Genetic interactions from pairwise
combinations of known mutants
Two fly strains carrying mutations in two separate genes can
be mated to generate progeny that are heterozygous for the
two mutations (double heterozygotes). A genetic interaction is
evident if the phenotype of the progeny is unlike that of either
parent (e.g., flightless progeny from two flighted parents) or if
the progeny exhibits an otherwise recessive phenotype. There
are several circumstances that can give rise to a genetic
interaction. One common situation is when mutations in two
genes affect binding between their encoded proteins. Genetic
interactions also result even if the two mutant proteins are not
in direct physical contact but function in the same or in parallel
pathways, or are components of a large multiprotein complex.
Such is the case in muscle cells, where proper assembly and
structural stability of the myofibril is essential for function.
Given the range of underlying molecular causes, interpretation
of genetic interactions is not straightforward but is potentially
Review articles
very important and informative. Fortunately, muscle research
provides a panorama of ultrastructural, cellular, biochemical,
and biomechanical information that enlightens the interpretation of genetic studies.
To date, the most exhaustive analysis of muscle mutant
interactions was conducted by Homyk and Emerson in which
they examined alleles for several myofibrillar protein genes
including Act88F, Mhc, TnI, TnT, Tm2 and a-actinin.(20) The
study uncovered two general types of interactions among the
muscle mutants: combinations that resulted in synthetic
lethality and combinations that resulted in flight impairment
and/or wing posture defects. Lethality results from defects in
the larval musculature while flight impairment and abnormal
wing posture result mostly from defects in IFM. The inability of
either muscle type to perform their functions could stem from
abnormal development giving rise to a defective muscle
lattice, or from a mechanical or biochemical defect which may
prevent contractile force generation or propagation from a
normally assembled muscle myofibril.
The most extensive set of interactions among non-Mhc
genes was observed between hdp and up/int mutants. These
loci were later shown to encode TnI and TnT, respectively. The
genetic interaction between TnI and TnT mutants is not
surprising given these two proteins function within the same
complex and it is well established that they interact physically.(39) One example is the interaction between int 3 and
hdp 2. Both mutant alleles are recessive for flight and show
normal wing posture as heterozygotes, but the double
heterozygote hdp 2‡/‡ int 3 is flightless and shows abnormal
wing posture. hdp2 is a missense mutation in TnI that results in
substitution of alanine 116 for valine.(40) This mutation is
located in a region that in vertebrate TnI has been shown to
interact with TnC.(41) int 3 is a TnT missense mutation that
results in lysine substituting glutamate 100, a residue that is
not part of the TnI-binding site.(39) The genetic interaction
between hdp 2 and int 3 does not stem from improper binding
between TnI and TnT, assuming that the organization of the fly
troponin complex is similar to the vertebrate troponin complex.
This conclusion is also supported by the fact that the hdp
interaction with up/int is not allele specific: several int alleles
interact with several hdp alleles.(20)
Since the mutations do not affect the contact sites between
TnI and TnT, what then is the basis for the genetic interaction
and why are double heterozygous mutants flightless? One
possible explanation is that each mutation has a destabilizing
effect on the troponin complex, either affecting the regulatory
function of troponin or stability of the whole thin filament. In
homozygotes, int 3 does not appear to affect myofibril
assembly while hdp 2 gives rise to minor sarcomeric irregularities.(40,42) Nevertheless, in both mutant homozygotes myofibrils degenerate by eclosion, most likely as a result of stresses
produced by contractile activity, and thin filaments are no
longer visible in hdp 2 adult IFM.(10,40,42) Future experiments
should address the underlying causes of muscle degeneration. For example, the effect of each mutation on Ca2‡
regulation and IFM contractile properties can be ascertained
from mechanical experiments of skinned fibers from very
young int 3 and hdp 2 homozygote adults (i.e., before the onset
of muscle breakdown), as well as from hdp 2 ‡/‡ int 3 double
heterozygotes. These studies should be complemented by
studies in vitro that examine the properties of mutant troponin
complexes (containing hdp2 TnI, int3 TnT, or both) in regulating
myosin ATPase activity. Finally, it is important also to examine
muscle structure and function in the single mutant heterozygotes because early studies relied on flight tests as the sole
criteria of IFM function. More recent studies have revealed
that, while some mutant heterozygotes may perform well on
the flight test, their IFM exhibit ultrastructural and/or contractile
defects.(18,43) In summary, an understanding of the changes in
molecular properties caused by hdp 2 and int 3 will provide the
foundation by which to interpret the genetic interaction; this, in
turn, will serve to unravel the mechanistic and functional
principles of the troponin complex.
Homyk and Emerson also uncovered genetic interactions
that do not conform to current structural or biochemical models
of muscle function. One example is the strong interaction
between hdp2and the recessive flightless a-actinin alleles fliA3
and fliA4. fliA4 is a hypomorphic allele that results in greatly
reduced expression of both nonmuscle and muscle isoforms.(44) As a result, muscle insertions into the cuticle are
defective but, surprisingly, sarcomere structure is only
marginally affected. Like hdp 2/‡, fliA4 heterozygotes can fly
suggesting that one copy of the wild-type allele can compensate for the reduced production of the mutant allele. As
mentioned above, however, the flight test is not a rigorous
assay for muscle function and it is possible that underlying
structural and contractile defects may have gone undetected
in fliA4 and hdp2 heterozygotes. Regardless, double heterozygous fliA4 ‡/‡ hdp2 are flightless, and we must ask why?
A direct interaction between a-actinin and TnI has not been
described in any system and therefore it is not considered a
likely explanation for the phenotype. Furthermore, TnI was not
detected in a yeast two-hybrid screen for a-actinin-binding
proteins.(45) Nevertheless, both proteins physically interact
with actin and the flightless phenotype most likely arises from
separate, but additive effects of the two mutations on thin
filament stability. Alternatively, the two mutations could act
autonomously: hdp 2 affect the ability of the myofibril to
produce force and fliA4 affect the ability of the myofibril to
sustain force. Thus, if hdp2 TnI inhibits actomyosin interaction
poorly or not at all, unregulated contractile force may reach
levels that are unsustainable by a fliA4-structurally compromised lattice.
In addition to TnT and a-actinin alleles, hdp 2 also interacts
with several Mhc alleles.(20) This genetic promiscuity is a
double-edge sword. On the one hand, hdp 2 will serve to
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uncover important features of myofibrillar architecture and
mechanics; on the other hand, this mutant serves as a warning
that more rigorous biochemical and biomechanical tests are
needed before the nature of its multiple genetic interactions
can be fully ascertained.
A less direct approach to identify genetic interactions that
may shed new light on muscle function is the use of
chromosomal ``deficiency kits'', collections of mutants in which
each fly strain carries an unique deficiency. The kits provide
contiguous coverage of most of the euchromatic portion of the
fly genome. Crosses of deficiency strains to a mutant of
interest helps to identify potential interacting genes. This
approach offers the advantage that a large portion of the
genome can be screened relatively quickly for interacting
genes. Because of the possibility of non-specific and multiple
locus interactions, this approach serves only as a first cue to
identify potential candidate genes, which then must be
subjected to further scrutiny. In addition, the large-scale
Drosophila (P element) gene disruption project currently
underway promises to deliver a mutant for every gene.(46)
This collection, together with deficiency kits, will facilitate
genome-wide scanning of genetic interactions.
Genetic interactions from screens
for modifier mutations
Genetic screens for dominant modifiers of gain-of-function
mutations have been used extensively in Drosophila to identify
novel components of cell signaling pathways. In particular, the
compound eye has served as an excellent system to dissect
the ras signaling pathway genetically since, like the IFM, this
organ is dispensable for viability and fertility of laboratory
strains. Until recently, however, modifier screens similar to
those deployed to study eye function had not been conducted
for IFM mutants.
In a screen designed to identify proteins that interact with
TnI, Prado et al. mutagenized hdp 2 males and isolated
mutations that suppressed the wings up phenotype
(Fig. 2B).(47) They obtained six suppressor lines, four on the
Mhc gene (see below),(41) one on Tm2,(48) and an intragenic
mutation (called D3) that results in a leucine-to-phenylalanine
substitution at position 188.(47) This residue borders the
putative actin-binding site in TnI, but it is not known if
the mutation affects the affinity of TnI for actin. Nevertheless,
D3 (A116V‡L188F) flies have normal wing position, show
substantial improvements in IFM ultrastructure, and have
partly regained flight ability.
The four suppressors that map to Mhc alter residues within
the myosin head.(41) Two of the suppressors are at the actinbinding loop, the third is near the ATP entry and the ATPbinding site, and the fourth affects the region at the lip of the
nucleotide-binding pocket.(41) Unlike D3, none of the Mhc
suppressors restores flight. All four suppressors markedly
improve IFM ultrastructure, but there are persistent abnorm-
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alities in fiber morphology and sarcomere architecture.
Because the biomechanical properties of hdp2 fibers have
not been studied, we do not know to what extent, if any, the
Mhc suppressor mutations improve functional performance of
hdp 2 IFM.
What is the mechanism by which mutations in the myosin
head are able to compensate for defects engendered by a
mutant TnI? In order to answer this question one most
consider whether the Mhc mutations can suppress other TnI
alleles and whether other Mhc mutations are able to suppress
the hdp2 phenotype. Kronert et al. tested both of these
possibilities and found that the suppression uncovered in their
screen is allele specific.(41) Based on these results, the authors
espoused the hypothesis that the suppressors may identify
specific molecular interactions between MHC and TnI. This
putative interaction could potentially couple kinetic and
regulatory processes, perhaps as an added strategy for rapid
activation/deactivation given the scarcity of sarcoplasmic
reticulum in the IFM. However, it is unlikely that all four suppressors act by the same mechanism and physical interaction
between the myosin residues affected in two suppressors,
D41 and D45, and TnI appears unlikely.(41) It is possible that
D41 and D45 suppress TnI-induced degeneration indirectly,
by curtailing the ability of the myosin motor to produce force. At
any rate, the studies of Prado et al. and Kronert et al. have
validated the in vivo approach of using mutations in one gene
to identify interacting genes. The real challenge remains in
elucidating the molecular basis of the interactions and
distinguishing between specific protein interactions versus
general effects. An understanding of how each suppressor
works will require not only genetic tools but also biochemical,
biomechanical and ultrastructural methods. The reward, a
comprehensive appreciation of how molecular components of
muscle work as a unit, is very much worth the effort.
Unexpected genetic interactions
Drosophila muscles are equipped with all the molecular
components for dual thin filament and thick filament regulation
of contraction. There is very little understanding of how the
action of these separate regulatory systems is coordinated to
achieve unified control, however, since very few studies have
addressed this issue. Mutants discovered by Warmke et al.
may be useful in the study of dual regulation of contraction.(16)
These authors uncovered a lethal interaction between
Dmlc2 E38 and several Tm2 alleles. Two of the alleles, J8
and L2, are essentially nulls (they encode very short Tm2
polypeptides)(17) suggesting that lethality results from the
additive haploinsufficiency of two essential larval muscle
genes that act in tandem to regulate muscle contraction.
The third allele, S2, encodes a single amino acid substitution
(aspartic acid 121 to asparagine) and it is not clear why this
mutant is lethal in heterozygous combination with Dmlc2 E38.
It is important to point out that Kreuz et al. were unable
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to reproduce the lethal effect between Dmlc2 E38 and Tm2
mutants.(17)
Functional genomics
Functional Genomics designates a variety of experimental and
computational approaches that aim to assign function to each
and every gene of an organism. The first step in this approach,
gene annotation, involves finding the genes within the genome
sequence and assigning function based on sequence similarities to known genes. An intense ``annotation jamboree'' was
successful in identifying 13,601 Drosophila genes and assigning a putative function to &50% of these genes.(49) Validation
of the assigned functions, and establishing the function of the
remaining genes whose sequences do not match known
sequences in the database, will require other experimental
approaches.
A new method for assigning gene function, known as ``guilt
by association'', is based on microarray data from global gene
expression experiments. An uncharacterized open reading
frame can be assigned a tentative function if its expression
pattern mimics that of a gene with known function.(50) Other,
more conventional techniques for identifying protein±protein
interactions, such as the yeast two-hybrid system, have been
customized for high throughput screening.(51) Pooling data
from high-throughput screens with that from published
interactions has resulted in the generation of a global network
of protein interactions in yeast.(52) The function of newly
discovered or uncharacterized proteins can be inferred from
their interactions with proteins of known function. The
combination of RNA and protein profiling, together with other
functional genomics methods, is capable of providing a
roaming view of cellular protein networks.(53) In the postgenome era, traditional genetics will serve an increasingly
important role in the validation of protein networks derived
from functional genomics approaches.
Proteomics
An increasingly popular approach for assigning gene function
is proteomics , whose initial aim is to provide a comprehensive
catalogue of all proteins in a cell, tissue, or organism.
Proteomics also endeavors to provide an in-depth description
of protein function that includes knowledge of protein
modifications, protein localization, protein interactions, and
changes throughout development. The goal is to furnish an
inclusive view of the cellular function of each protein. The
panorama provided by proteomics would show how each
protein fits naturally within the functional landscape of the cell
and can serve to unify all aspects of protein function (Fig. 3,
bottom).
The traditional platform in proteomics is to separate
proteins by two dimensional gels (2DE) followed by mass
spectrometry to identify proteins and their modifications
(Fig. 4). While useful, a gel-based approach suffers from
several handicaps, namely the exclusion of some proteins
(e.g., hydrophobic proteins) and the inability to reliably detect
low abundance proteins.(54) One way to overcome the
limitation in visualizing rare proteins is to pre-fractionate the
sample before gel loading, thereby enriching for low abundance proteins. Pre-fractionation offers the additional advantage that it provides information about some property of the
protein. Multidimensional chromatography (e.g., ion exchange
and reverse phase)(55) affords separation based on the
protein's intrinsic properties. A second approach is to fractionate proteins according to their subcellular distribution (Fig. 4).
IFM proteomics in the pre-genomics era
IFM proteomics dates back to the early 1980s when
investigators began to assemble a 2DE database from normal
and mutant flies.(9,56) Coomassie blue-stained gels of whole
thorax homogenates revealed 186 protein spots; of these 20
spots were also detected in a myofibrillar fraction, and 12 of
these protein spots appear to be IFM-specific.(56) To date, only
three of the 20 myofibrillar proteins (spots 98, 99, and 184)
have not been identified. Furthermore, additional myofibrillar
proteins have been since identified (Table 2), including high
mass proteins (e.g., titin, projectin, and kettin) and proteins
that do not resolve well in standard 2DE protocols (e.g., miniPm, TnT).
The examination of mutants by 2DE was an attempt at
establishing gene networks based on the presence or absence
of a given protein in different mutant IFM. The premise of this
approach is that, if two proteins function together, the absence
of one protein would affect the accumulation of the other.
Mogami and Hotta examined the IFM proteins of 57 newly
isolated dominant flightless mutants and found that, in 26 lines,
more than one protein spot was absent or reduced, as a result
of the mutation.(9) Analysis of the protein profile of the 26 lines
revealed that the presence or accumulation of some protein
spots is strictly dependent on the presence of other spots
(Fig. 5). They proposed that this dependency reflects
functional and/or architectural relationships among the proteins. In addition, they used this information to predict the
primary site of the mutation.
The gene defects of several of the mutant strains generated
by Mogami and Hotta are now known. A re-examination of the
results from the early studies in light of the new information
validates the gel comparison approach as a means of
identifying functional protein relationships. Nevertheless the
2DE approach, limited by the resolution and range of the gel
system and the sensitivity of the detection method, lacks the
capacity to reveal the full spectrum of protein changes that
may occur as a result of a mutation.
Figure 5 summarizes the interrelationships among several
protein spots deduced from 2DE analysis of IFM mutants.(9)
Except for spot 184, the identity of all the other spots are
known. Their results correctly predicted that spots 158 and
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Figure 4. Building a functional map of IFM proteins through the study of mutant proteomes. A: Approach pioneered by Mogami and
Hotta for examining mutant effect on IFM function. Myofibrillar proteins from wild-type IFM (left panel) and mutant IFM (right panel) are
separated by 2DE and their profiles compared.(9) Many IFM mutations affect the expression of multiple proteins and, in some cases, the
presence of a protein is completely prevented (red circles). However, the range and resolution of the gel, and the sensitivity of
the detection method (e.g., coomassie blue) limits the number of proteins that can be detected. B: A proteomics approach enhances the
study of mutant effect on IFM function. A dissected IFM fiber is split and transferred sequentially through three different solutions that
allow fractionation of proteins by their subcellular distribution: drop 1, soluble (cytosolic) proteins; drop 2, detergent-extractable
(membrane and organelle) proteins; drop 3, insoluble (cytomatrix) proteins.(64) The pre-fractionation reduces the complexity of
the sample and enriches low abundance proteins, which are now detected by 2DE (blue squares). Individual protein spots (red circle)
are cut out from the gel, digested with trypsin, and the resulting peptides analyzed by mass spectrometry.(67) Alternatively, 2DE is
bypassed altogether and proteins fractionated by HPLC or multidimensional chromatography before or after trypsin digestion.(55)
Proteins are identified by using peptide fingerprints (MS) and/or partial sequence of representative peptides (MS-MS) to scan a virtual
translated Drosophila Genome. The protein profile of each mutant, along with relevant information from other studies (e.g., genetic
interactions, in vitro assays) is used to assemble an IFM mutant database and the formulation of functional protein networks.
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Figure 5. Interpretation of functional gene networks deduced from 2DE analyses of IFM mutants (flow diagram from
Mogami and Hotta).(9) Arrows point to spot numbers that are
reduced or missing in all mutants where the spot from which
the arrow originates is also reduced or missing. Numbers in
boxes connected by doubleheaded arrows indicate spots that
always decrease together. Note that reduction in actin (spot
101) always results in reduction of thin filament proteins
(arthrin, Tm2, and TnH) and flightin, but do not affect DMLC2
or ELC 1/3. Flightin is the only myofibrillar protein whose
accumulation is affected in all homozygous mutants that were
studied. Furthermore, flightin expression is affected by
practically all Act88F alleles that have been examined and
by some TnT and Tm2 alleles.(9,11,58,68) Flightin is a thick
filament associated protein(14) but these results suggest that
flightin interacts with actin or some other protein of the thin
filament. These results validate the mutant proteome comparative approach to identify functionally significant protein
interactions.
159, two isovariants of flightin, are strictly dependent on each
other (double arrow in Fig. 5). Furthermore, flightin is absent in
the mutant Mhc7, which is also missing spot 138 (DMLC2) and
spot 185 (ELC1/3), the latter one hypothesized to be the
primary site of the mutation.(9) A later study of Mhc7 revealed
that this mutant is an IFM-specific null allele of MHC that fails to
assemble thick filaments.(57) More than ten protein spots
(DMLC2, ELC1/3, and flightin among them) were shown to be
missing in Mhc7 IFM.(57) All the missing proteins are presumed
to be thick filament components since thin filaments and Z
bands appear marginally affected in Mhc7.(57)
Mogami and Hotta also examined several Act88F alleles,
including Ifm(3)2 (renamed Act88F R28C) and Ifm(3)7
(Act88FW356@-2), which affect the accumulation of known thin
filament proteins, namely Tm2, arthrin, and TnH (Fig. 5).
However, no changes were noted in other thin filament
proteins such as GST-2 (spot 137), TnI or TnT. The latter
two probably did not resolve well in their gel system, making
their presence more difficult to ascertain. Interestingly, some
TnT alleles also affect the accumulation of actin or other
proteins.(42,58)
At least three important lessons are derived from the
studies that analyzed muscle protein accumulation in IFM
mutants. First, the total analysis of protein accumulation in
mutants espoused by Mogami and Hotta is an extremely
valuable approach for deciphering functional protein networks.
The limitations encountered in their study are easily overcome
using higher resolution 2DE systems and more sensitive
stains, or replacing 2DE altogether with other means of protein
separation (e.g., HPLC or multidimensional LC; Fig. 4).(55)
Second, the strict interrelationship between one isoform of
TnH (spot 34) and spot 184 is intriguing and deserves further
consideration. TnH co-purifies with the Troponin complex in
Lethocerus(59) and binds GST-2 in Drosophila IFM,(60) but an
interaction with protein 184 has not been uncovered by
biochemical studies. A later study showed that the accumulation of spot 184 is affected by many Act88F alleles suggesting
that this protein may be a thin filament component.(11) In
addition, the interrelationship between TnH and spot 184 holds
for 14 of 15 Act88F alleles.(11) Since both TnH and spot 184 are
IFM-specific,(56) this putative interaction may play a defining
role in stretch activation. So what is spot 184? One possibility is
that this protein is another member of the troponin complex,
perhaps TnC. In fact, if one queries the genome using the
known properties of spot 184 (pI, molecular weight, cytoskeletal protein), the two most likely candidates that emerge are
TnC and a calmodulin-related protein.
Third, some Act88F alleles affect accumulation of thick
filament proteins, namely flightin and DMLC2. The effect is
particularly pronounced in several antimorphic alleles in which
DMLC2 is not detected at all.(11) These results are consistent
with the proposal that DMLC2 interacts with actin, perhaps
through its extended N terminus.(38) More importantly, however, these results indicate that the assembly of functional thin
and thick myofilaments may not be as independent of each
other as is often assumed.(61)
It is clear from the aforementioned studies that most mutant
genes affect more than their encoded products, they also
affect the expression of multiple other proteins some of which
are likely to interact physically with the product of the mutant
gene. These studies certainly represent ``the tip of the iceberg''
since they were based on visual inspection of 2DE gels of
limited range and resolution. An enhanced view of the
proteome would expose the full range of each mutation effect
and should prove particularly beneficial in cases where the
observed phenotype cannot be explained logically. A study of
three single amino acid mutations in the MHC rod illustrates
the validity of a proteomics-type approach, even if it is limited in
scope. Three Mhc alleles (Mhc6, Mhc13, and Mhc19 ) were
shown to have a pronounced effect on IFM structure and
function and little or no effect on other muscles despite the fact
that the mutations are located in an MHC constitutive exon.(13)
Two-dimensional gel analysis revealed that a reduction in
flightin accumulation was a common secondary manifestation
of the three mutants. Furthermore, the severity of the muscle
defect worsened in direct proportion to the reduction in flightin
abundance,Mhc13 in which little or no flightin is present, has a
more severe muscle defect than Mhc6, in which some
unphosphorylated flightin is present.(13) More recently Reedy
et al. described the isolation of fln0, a flightin null mutant whose
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phenotype is remarkably like that of Mhc13 despite the fact that
MHC is unaffected in fln0.(14) The convergent phenotypes
suggest that the mutations in two separate genes may affect a
common aspect of muscle structure and/or function. Together,
the studies of Kronert et al. and Reedy et al. provide
persuasive evidence that the phenotypic manifestations of
Mhc rod alleles result from their effect on flightin abundance,
rather than to some intrinsic effect on the myosin molecule.
This situation is intriguingly similar to that described for
vertebrate b cardiac MHC and myosin-binding protein C
(MyBP-C). On the one hand, mutations in the S2 region of b
myosin that cause familial hypertrophic cardiomyopathy
(FHC) have been shown to affect MyBP-C binding but have
no effect on myosin coiled-coil structure or stability.(62) On
the other hand, more than 30 mutations in cardiac MyBP-C
have been found in FHC patients and most of the mutations
result in truncated MyBP-C that lack the myosin-binding
domain.(63) These examples in flies and humans illustrate how
mutations in one gene (myosin) may engender muscle defects
by interfering with the activity of a separate gene product
(MyBP-C or flightin). These studies also highlight the need to
look beyond the primary site of the genetic defect in order to
obtain an accurate portrait of the molecular consequences and
pathobiological manifestations of each mutation. In summary,
the analysis of mutant IFM proteomes offers new perspectives
on the cellular function of muscle proteins and provides fresh
insight into the correlation of genotype and phenotype. This
view may be particularly beneficial for the study of human
genetic diseases, such as FHC, in which knowledge of the
molecular defect has not translated into an understanding of
disease pathogenesis.
The assignment of cellular function also requires knowledge of the protein's subcellular distribution. Warmke et al.
pioneered subcellular fractionation of IFM fibers in microliter
volumes to obtain distinct cytosolic, organelle, and cytomatrix
fractions (Fig. 4).(64) This approach, as well as large-scale
fractionation,(65) permits the isolation of a nearly intact
myofibril and allows myofibrillar protein composition to be
directly correlated with mechanical performance.(64) Furthermore, the myofibrillar fraction can be subsequently separated
into thick filament, thin filament or Z disk subfractions.(34,65) In
a recent study, Reedy et al. combined genetics and subcellular
fractionation to demonstrate that flightin remains associated
with thick filaments composed of headless myosin, suggesting
that flightin interacts primarily with the thick filament shaft.(14)
Summary and prospective
The function of the myofibril, like the function of other complex
subcellular machines, is predicated on the proper assembly
and maintenance of a supramolecular structure. Innumerable
protein contacts underlie a functional myofibril. The three
cases highlighted here (Act88F E93K, hdp2, and Mhc13/fln0)
emphasize the high degree of interdependency among
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myofibrillar proteins and demonstrate the multiple layers of
function that are served by protein constituents of a mechanical ensemble. The fundamental complexity of the myofibril is
evident in the varied functional expression of Act88F E93K, the
genetic promiscuity of hdp 2, and the congruent phenotypic
manifestations of Mhc13 and fln0. These studies serve as a
humbling warning that no single experimental approach is a
panacea for understanding muscle function. Fortunately,
Drosophila IFM is well suited for the multifaceted experimental
assault that will be needed to generate a comprehensive
description of how muscle works. The long tradition in crossdisciplinary research, together with the adeptness of IFM to be
queried from a top-down or bottom-up approach, provide a
solid foundation from which to embark on a systems approach
to function (Fig. 6). The knowledge gained will impact our
understanding of insect flight and of muscle function in
general.
Figure 6. A systems view of the myofibril. The striated
muscle myofibril is a stable supramolecular ensemble made
of a finite number of parts (proteins) organized in a spatially
precise lattice. As such, it presents an outstanding opportunity
for understanding how protein interactions define a complex
biological system. The myofibril functions to produce and
sustain mechanical forces in a regulated manner. Power
(work per unit time) takes into account force and velocity of
contraction. In the view presented here, the properties of the
myofibril are ascribed to separate but interdependent subsystems. Many fundamental questions in muscle biology can
be reduced to an understanding of how the subsystems
communicate to operate as one. The challenge remains to
identify how each myofibrillar protein contributes to each
subsystem.
Review articles
We have made great strides towards our goal of understanding IFM function but many important challenges remain.
Critical questions about the functional defects of many extant
mutants have not been addressed, and the full potential of
most of these mutants has not been unleashed. Large-scale
interaction screens and modifier screens will provide much
needed information about biologically significant protein
interactions. The results of these studies will provide the
impetus for the formulation of increasingly inclusive models of
myofibril form and function. Existing methodologies, ranging
from optical trapping of single molecules(66) to X-ray diffraction
of IFM in live flies,(37) will provide ample and comprehensive
means for validation of genetic data. The marriage of genetics
and proteomics is enthralling and has tremendous potential for
unveiling intimate details of contractile protein function,
including unexpected relationships between proteins. Bioinformatics will play an increasingly important role in enhancing
our ability to assimilate and integrate information from multidisciplinary investigations.
Years from now historians of science may view the current
genomics revolution as the beginning of the end of reductionism. The ability of ``Big science'' to generate information has
rapidly outpaced our ability to comprehend it. The situation
is not unlike that faced by early 19th century western
naturalists, who had gained little understanding about
nature despite large compilations of geological and biological
phenomena. The publication of The Origin of Species in
1859 provided the contextual framework by which a lot of
the information began to make sense. Whether we will
need a similar intellectual breakthrough in order to make
sense of genome-wide data remains to be seen. What is
evident is that time-tested genetic approaches will continue to
reap important rewards in the post-genome era of large-scale
biology.
Acknowledgments
I am grateful to David Maughan and Gretchen Ayer for critical
comments on the manuscript, and to members of the UVM
Drosophila group for many stimulating discussions. I would
also like to acknowledge the reviewers for their insightful
comments and suggestions. I want to thank Karen Moore for
help with the graphics, to David Maughan for providing
Figure 1, and the National Science Foundation for their
support.
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