From the Biology Department and Molecular Biology
Institute and the
Department of Chemistry, San Diego State
University, San Diego, California 92182 and the ¶ Department of
Biology, University of York, P.O. Box 373, York YO1 5YW, United Kingdom
Received for publication, September 13, 2000, and in revised form, December 6, 2000
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ABSTRACT |
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To investigate the molecular functions of the
regions encoded by alternative exons from the single
Drosophila myosin heavy chain gene, we made the first
kinetic measurements of two muscle myosin isoforms that differ in all
alternative regions. Myosin was purified from the indirect flight
muscles of wild-type and transgenic flies expressing a major embryonic
isoform. The in vitro actin sliding velocity on the flight
muscle isoform (6.4 µm·s The functional properties of muscle myosins have been investigated
in many systems. Developmental- and tissue-specific isoforms have distinct ATPase rates and velocities that are the primary determinants of muscle contractile properties (1). However, it is not
well understood how functional properties of myosin are determined by
variations in myosin heavy chain
(MHC)1 sequence and structure
(2, 3).
Genetic engineering, sequence comparisons, in vitro motility
assays, and crystallographic structures have facilitated recent investigations into MHC structural regions that might influence function properties (3). For example, the two flexible loops (25/50-kDa, loop 1; 50/20-kDa, loop 2) in the chicken skeletal and
Dictyostelium myosin heads, unresolved in the atomic
structures, have been examined as possible determinants of functional
variation between isoforms (4-6). Substituting different loop 1s with
varying charge and length affected ADP release rate, ATPase rate, and in vitro actin sliding velocity (4). However, these changed properties did not correlate well with the relative speed of the native
myosin from which the loop was derived, suggesting that additional
regions need to be altered in concert to give the unique kinetic and
velocity characteristics of a specific isoform.
Investigations using species-specific myosins differing only in one or
a few regions result in better insights. In phasic smooth muscle
myosin, seven additional amino acids near the nucleotide binding site
increase actin-activated ATPase rate and in vitro motility
velocities compared with the tonic smooth muscle isoform (7, 8).
Similarly, scallop catch and phasic muscle isoforms have different
motility and ATPase rates (9).
The Drosophila system has the potential to provide even
greater insights. Fifteen different MHC isoforms, arising from
alternative splicing of mRNA transcripts from the single
Mhc gene (10, 11), have been found so far in a wide variety
of muscle types, ranging from the supercontractile larval muscles to
the highly ordered, asynchronous indirect flight muscles (IFMs)
(12-15). Four of the five alternatively spliced exon sets occur in the
myosin head coding region and have been proposed to determine
functional differences between isoforms (Fig. 1) (16). The variety of
myosins normally expressed and the ease of creating transgenic flies
make the Drosophila Mhc gene a powerful system for studying
the relationships between myosin sequence and in vivo
functional variation (17). However, to take full advantage of this
system, it is necessary to develop in vitro assays to
determine the different properties of Drosophila myosins. As
the IFMs constitute a major part of the adult fly (approximately 20%
of body weight), myosin isolation from these muscles following
dissection is relatively straightforward compared with attempting
recovery from other muscle types.
Recently, a major embryonic isoform (Emb) was transgenically expressed
in a myosin null background (18) such that every muscle type expressed
only the Emb isoform. Although the transgenic flies were viable, they
exhibited a flightless phenotype and were severely impaired in jumping
and walking abilities. In addition, the IFMs showed severe myofibrillar
degeneration soon after the time at which flight normally commences.
Clearly, this embryonic isoform cannot substitute functionally for the
IFM isoform. The embryonic isoform differs from that of the IFM at all
alternatively spliced regions; therefore, one or more of the
alternative exons must be responsible for the functional differences
between these isoforms.
We report on the purification and functional characterization of
Drosophila IFM and modified embryonic (Emb18)
myosins. We present the first optical trap measurements of
Drosophila myosin step size, attachment lifetimes, in
vitro motility velocities, and isoform-specific ATPase
measurements. The results demonstrate that alternatively spliced exon
regions of MHC dramatically influence cross-bridge cycle kinetics and
set the stage for the evaluation of chimeric MHC constructs to pinpoint
functional variations to particular polypeptide sequences encoded by
the alternative exons.
Myosin Isolation--
Myosin was prepared from the
dorsolongitudinal IFMs (DLMs) of 100-120 wild-type (for muscle genes;
the flies carry the yellow body color and white eye mutations) or
transgenic Emb18 flies. Emb18 myosin (hereafter
referred to as Emb18) was expressed transgenically
following P element-mediated germline transformation of a modified
version of a major embryonic isoform cDNA (17). Emb18
was modified from a native embryonic myosin to have the shorter adult
tailpiece encoded by exon 18. The adult tailpiece enhances myosin
accumulation in the adult IFMs (17). By expressing Emb18 in
a Mhc10 background (null for myosin in flight
and jump muscle), the Emb18 isoform could be isolated from
the IFM without contamination by other myosin isoforms. However, it had
the IFM-specific essential myosin light chain isoform. Thus, the only
differences between the isolated myosin isoforms were located in head
regions encoded by exons 3, 7, 9, and 11 (Fig. 1), and the rod hinge
region, encoded by exon 15.
DLMs were dissected at 4 °C from split thoraces in York Modified
Glycerol (YMG: 20 mM potassium phosphate, pH 7.0, 2 mM MgCl2, 1 mM EGTA, 8 mM DTT, 2% (v/v) Triton X-100, and 50% (v/v) glycerol) (19) without Triton X-100, but with the addition of a protease inhibitor mixture (Complete, Roche Molecular Biochemicals). A protease
inhibitor mixture was also included in all subsequent myosin extraction
solutions. IFMs were suspended in YMG (with Triton X-100), incubated
for 30 min, centrifuged (8,500 × g), and washed in YMG
without Triton X-100 and glycerol. Myosin was extracted into three
volumes (55 µl) of 1.0 M KCl, 0.15 M
potassium phosphate, pH 6.8, 10 mM sodium pyrophosphate, 5 mM MgCl2, 0.5 mM EGTA, and 8 mM DTT for 10 min and centrifuged at 8,500 × g for 5 min. The proteins in this initial extraction are
shown in Fig. 2A (lane 2). The pellet
was sometimes used to make acetone powder for actin isolation (see
below). The extracted myosin was precipitated by decreasing KCl to 40 mM and incubating for 16 h (overnight). Following a
15-min centrifugation in a Beckman TL-100.3 rotor at 100,000 × g, the pellet (Fig. 2A, lane
3) was dissolved in an equal volume (18 µl) of Wash B (2.4 M KCl, 100 mM histidine, pH 6.8, 0.5 mM EGTA, 8 mM DTT) (20). Myosin that did not
dissociate from actin (shown in Fig. 2A, lane
6) was precipitated by slowly adding water until the KCl
concentration was decreased to 0.3 M and then centrifuged
at 60,000 × g for 30 min. The supernatant was removed
and further diluted until the KCl concentration reached 30 mM and centrifuged at 100,000 × g for 30 min. The final pellet was resuspended in 30 µl of myosin storage
buffer (0.5 M KCl, 20 mM MOPS, pH 7.0, 2 mM MgCl2, and 8 mM DTT).
Drosophila myosin preparations were quantified by their
absorbance at 280 nm using an absorbance coefficient of 0.53 cm Actin Isolation--
Actin was isolated from dissected DLMs as
described by Razzaq et al. (22). Purified F-actin was
centrifuged at 150,000 × g for 1.5 h, and
resuspended in actin buffer (25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl2, 1 mM EGTA, and 1 mM DTT) (23). Fig. 2C
shows the purity of the actin preparation. Actin was quantified by
subtracting absorbance at 310 nm from that at 290 nm and dividing by an
absorbance coefficient of 0.62 cm ATPase Assays--
The Ca-ATPase activity of myosin was measured
using a modification of the method described by Pullman (25). Activity
was determined in a 0.5-ml reaction mixture containing 10 mM imidazole, pH 6.0, 0.1 M KCl, 10 mM CaCl2, and 1 mM
[
For determination of the Km of myosin Ca-ATPase
activity, ATP was varied from 5 to 300 µM for IFM myosin
and from 2 to 40 µM for Emb18 myosin.
Specific radioactivity of [32P]ATP was increased to
5,000-8,000 cpm·nmol
Mg-ATPase activity was determined in 0.5 ml of solution containing 10 mM imidazole, pH 6.0, 20 mM KCl, 2 mM MgCl2, 0.1 mM CaCl2,
and 1 mM ATP with 2-5 µg of protein for 30 min at
22-23 °C, and processed as described above for Ca-ATPase.
Sliding Filament Assay--
F-actin in vitro motility
assays were conducted similarly to Kron et al. (23), except
for the following modifications. 1) The nitrocellulose-coated coverslip
of the flow cell was blocked by incubating with assay buffer plus BSA
(AB/BSA) prior to myosin addition at 0.5 mg·ml
In some experiments, smooth muscle tropomyosin (smTM) (chicken gizzard,
Sigma) was bound to Drosophila IFM actin by mixing at a 1:1
molar ratio. Centrifugation and one-dimensional SDS-PAGE electrophoresis of the pellet and supernatant confirmed the binding of
smTM to Drosophila F-actin (data not shown). smTM (100 nM) was included in all solutions added to the flow cell
after actin addition to prevent smTM dissociation from actin (28).
The assays were conducted at 22-23 °C, and filament movement was
recorded onto videotape. Video sequences were captured at 10 frames/s
with an Apple PowerMac computer. Actin filament velocity was calculated
using the Autotrack Macro (29) from the public domain NIH Image
program. A filament's velocity was determined only if it moved
smoothly for >2 s. At least 50 filaments from a flow cell with the
best quality of movement were averaged to determine the mean velocity
for each myosin preparation. Means for all preparations were then
averaged to determine mean myosin isoform velocity. Standard deviations
were calculated from the preparation averages. A Student's
t test was used for all statistical analysis, with
p values < 0.05 considered statistically significant.
Single Molecule Mechanical Experiments--
The single
actomyosin cross-bridge mechanical experiments were carried out using
an optical tweezers transducer in the "three-bead" configuration
(30-32). Two independently trapped latex beads, attached to the ends
of an actin filament, held the filament close to a glass bead coated
with a low surface density of Drosophila myosin molecules
(0.1 µg of protein applied to a 22 × 22-mm2
coverslip) allowing interactions between single myosin heads and the
actin filament. Beam steering of the traps and calibrations were
performed as described previously (31, 32). Actomyosin interactions
were measured at trap stiffnesses between 0.02 and 0.04 pN·nm
The size of the working strokes of the two Drosophila
myosins interacting with rabbit or Drosophila IFM actin were
determined by analyzing hundreds of displacement events (for details,
see Refs. 31 and 32). Histograms of the displacement events were fitted
with a Gaussian distribution whose midpoint was shifted from zero,
reflecting the size of the working stroke, and whose width was
determined by the trap stiffness (31).
Protein Isolation--
The Drosophila myosin isolation
method yielded an enzymatically active myosin capable of moving actin
filaments in a smooth and continuous manner. The IFM myosin yield was
300-400 µg (2.5-3.3 µg/fly) and was at least 90% pure, as judged
from the proportion of heavy chain (200 kDa) and light chains on
SDS-PAGE gels (Fig. 2B). Two light chains associate with
Drosophila MHC: the essential light chain and the regulatory
light chain, which appears as two bands due to multiple phosphorylated
versions (~14 isoelectric variants; Refs. 33 and 34). A negligible
amount of actin was present. The Emb18 myosin yield was 200 µg (
The yield of IFM actin was ~0.5 µg/fly, similar to that
reported by Razzaq et al. (22).
ATPase Activities of IFM and Emb18
Myosins--
Drosophila IFM myosin exhibited a high
Ca-ATPase activity, of 7.5-9 s
Mg-ATPase activity of the IFM isoform was only 1-2% of the Ca-ATPase
activity. The Ca-ATPase and Mg-ATPase activities of transgenic Emb18 myosin were 20-37% of those obtained with IFM
myosin (Table I, Fig. 3). Drosophila myosin K-ATPase (EDTA)
activity at 0.5 M KCl was slightly higher than the
Mg-ATPase rates (data not shown), and about 50% of the K-ATPase
activity reported by Takahashi and Maruyama (26) for whole thorax myosin.
Sliding Filament Assay--
The in vitro motility assay
required a few modifications from the standard rabbit heavy meromyosin
(HMM) assay (23) to obtain smooth and continuous movement of actin on
Drosophila myosin. Actin filaments diffused off the
myosin-coated surface after ATP addition unless 0.4% methylcellulose
was added to the AB/BSA/glucose/glucose oxidase/catalase and ATP
solutions. Actin interactions with myosin were enhanced when the
overall ionic strength was lowered to about 25 mM (no added
KCl). Higher ionic strengths decreased the number of filaments moving,
and some filaments would leave the myosin surface even in the presence
of methylcellulose. The best actin movement (greatest number of
filaments moving smoothly and continuously) occurred at ~0.5
mg·ml
Surprisingly, the binding of smTM to actin increased filament velocity
over Emb18 myosin almost 6-fold (Table II, Fig.
4A). In contrast, smTM had a slight inhibitory effect on IFM
myosin driven movement, reducing the velocity (Fig. 4B) and
decreasing the number of filaments that moved smoothly and uniformly.
Reduced movement quality was not observed with Emb18
myosin. Thus, with smTM bound to the Drosophila IFM actin,
the difference in sliding velocity between IFM and Emb18
myosins was only 1.4-fold.
Myosin Displacement Size--
The displacements produced by the
two Drosophila myosins in single molecule interactions with
an F-actin in the optical trap are shown in Fig. 5. A short section of
signal recording the position of a bead (one of a bead pair attached to
F-actin) is shown (Fig. 5A), which is similar to those
presented previously for other myosins (32, 37). There are periods of
reduced noise due to the increased stiffness when myosin, on the third
bead, binds to the actin filament. Signal noise is due to thermal
fluctuation of the bead held in the optical trap. The amplitude of the
thermal fluctuations was estimated from a running variance (lower
trace), which is used to objectively and automatically identify
individual myosin attachment events. A histogram (Fig. 5B,
gray) shows the distribution of bead positions during
periods when no myosin is attached. This noise can be fitted with a
Gaussian distribution whose S.D. is defined by the combined stiffness
of the optical tweezers (
Fig. 5C shows the distribution of displacement means (binned
into 2-nm bins) for each actin filament interacting with either Emb18 (gray) or IFM (black). We found
that data collected from different pedestals (third bead) with the same
F-actin filament had a large variation in mean displacement. Because of
this large variability between different estimates, we performed
additional control experiments. 1) Only Drosophila myosin
preparations that demonstrated good quality movement in the in
vitro motility assay were used for step size measurements. 2) Step
size of rabbit full-length myosin was also measured under the same
conditions (Fig. 5C). This gave a step size of 4.20 ± 0.93 nm (mean ± S.D. of 8 filament means, each with >25 events),
which is not significantly different from the values for the
Drosophila isoforms (p = 0.832, one-way
analysis of variance). 3) Neither using rabbit actin instead of IFM
actin nor adding tropomyosin affected the step size of either
Drosophila myosin.
The duration of myosin attachments to F-actin in the optical tweezers
traces provides a measure of the second order rate constant of A.M + ATP We have demonstrated that myosin can be isolated and purified from
Drosophila IFMs in sufficient quantities for optical
trapping, in vitro motility, and ATPase assays. More
significantly, we can isolate other myosin isoforms by expressing them
transgenically in the IFMs of mutant flies,
Mhc10, which do not express the native IFM
myosin. Myosin isoform differences do not appear to influence assembly
properties (17, 18). Even though embryonic isoforms cause myofibrillar
breakdown and a reduction in myosin content as the flies age (17, 18),
yields of Emb18 from young flies (1-2 days old) were no
less than 50% compared with wild type.
Actin Velocity in Vitro and in Vivo--
Both
Drosophila myosins move actin in vitro only at
high surface myosin concentrations, in the presence of methylcellulose and at low ionic strength. All these factors suggest that these myosins
have much lower actin affinities than rabbit skeletal muscle myosin.
Methylcellulose enhances solution viscosity, reducing diffusion of
actin filaments away from the surface and increasing the probability of
further interactions with myosin heads. It thus maintains movement at
very low myosin surface densities (27) or when actomyosin affinity is
reduced, either by various actin or myosin mutants (6, 38) or by higher
ionic strengths (39, 40). Indirect flight muscle myosin from
Lethocerus (waterbug) has a Km for actin,
measured from actin-activated ATPase studies, which is 6-15 times
greater than vertebrate fast muscle (41). Thus, it seems likely that
IFM myosins may generally have low actin affinities. Since
Emb18 has similar in vitro motility
requirements, the low actin affinity likely extends also to the
embryonic isoform, normally expressed in the supercontractile muscles
of the larva.
Our results show that Drosophila IFM myosin produces one of
the fastest in vitro actin sliding velocities reported for a
skeletal muscle isoform. It is faster than rabbit skeletal myosin (4-5 µm·s
It is clear from the Drosophila IFM myosin data, as
previously observed with rabbit skeletal myosin, that in
vitro velocity tends to underestimate the values of
Vmax achieved in vivo (47). This is
partly due to the random orientation of cross-bridge heads (48) and
because the filaments are not fully unloaded (e.g. binding
of other myosin heads) in vitro. Further, in
vitro velocities are usually higher with a full complement of thin
filament accessory proteins (28).
The mean velocity produced by the Emb18 myosin is 9-fold
slower than the IFM isoform and correlates with the slower contraction velocities of the larval body wall muscles. Emb myosin is normally expressed in the supercontractile dorsal oblique muscles that have long
sarcomeres and a high degree of shortening (49). The mechanical
properties of these muscles are unknown, but they are used to power
crawling at 1 mm·s
Although the functional measures of Emb18 correlate with
the mechanical properties of its native muscles, it should be noted that due to transgenic expression the Emb18 isoform has the
IFM-specific essential light chain. The Drosophila genome
contains single copies of both the regulatory and essential muscle
myosin light chain genes. The IFMs express a unique essential light
chain isoform that differs in 14 amino acids of the C terminus due to
alternative splicing of its transcript (50). As light chain isoforms
influence velocity (51), the properties of the endogenous embryonic
isoform may differ slightly from those measured for Emb18.
However, since the IFM and the transgenically expressed
Emb18 contain the same essential light chain, any
functional differences we observed must derive from the myosin heavy
chain sequence.
The binding of smooth muscle tropomyosin dramatically increases actin
velocity on Emb18 myosin but slightly decreased the sliding
rate on IFM myosin. The molecular basis for this potentiation, which
has also been found for actin activation of myosin ATPase (52, 53)
remains generally unexplained. The effect varies according to myosin
type and isoform. Smooth muscle tropomyosin increases actin sliding velocity ~10-fold with phosphorylated Limulus striated
muscle myosin (54) and 2-4-fold on smooth muscle myosin (55), but only
slightly increases velocities on rabbit skeletal HMM (56). On this
basis the Emb18 isoform acts like a smooth muscle myosin,
while the IFM myosin behaves more like rabbit skeletal myosin.
IFM Myosin ATPase Rates--
The fast nature of the IFM myosin
isoform was supported by ATPase measurements. The Ca-ATPase rate of the
IFM isoform was ~2-4-fold higher than rabbit skeletal muscle and
Lethocerus flight muscle myosins measured under similar
conditions (36, 57). The significance of the Km
difference for ATP of the Ca-ATPase activation for the two isoforms is
not immediately apparent, since ATP concentrations in vivo
are vastly in excess of 1 µM. However, it will be
interesting to determine which alternative exon influences this
property, even if it does not impact the fly physiologically.
White et al. (41) measured Mg-ATPase values of 0.01-0.05
s Single Molecule Mechanics--
There were no significant
differences in the overall mean displacements measured for rabbit, IFM,
and Emb18 full-length myosins. However, there was large
scatter in the individual estimates of mean step size obtained for each
myosin isoform. Separate estimates of the mean step size were obtained from each third bead tested (and therefore each individual myosin molecule). The individual binding events used to obtain each estimate had an amplitude distribution that was Gaussian with S.D. similar to
that of the free bead movement (as described by Molloy et
al. (31)). The new feature in the data that we report here is that the mean estimates (some obtained using the same actin filament but
testing a different myosin molecule of the same isoform) have a greater
standard error of the mean than expected on the basis of sampling
statistics. In other words, trials on the same myosin type gave results
that were significantly variable between myosin molecules tested. This
discovery implies that the individual myosin molecules being tested
behave differently in this assay. There are three possible
interpretations. 1) The myosin molecules are biochemically
heterogeneous as a result of differential post-translational modifications; 2) the Drosophila myosins occupy a wider
range of functionally active molecular orientations in the way they are
deposited on the third bead substrate, some of which restrict the
production of the full displacement; or 3) the myosin step size depends
upon the geometry of myosin binding to actin and the degrees of freedom
of movement within the myosin molecule. Any single myosin molecule
would give a movement proportional to the cosine of the angle that it
makes to the actin filament (as suggested by Molloy et al.
(Ref. 31) and as shown by Tanaka et al. (Ref. 48)). Since in
these assays the myosin head orientation is random with respect to the
F-actin axis, different flexibilities within the myosins might produce
step size distributions with different mean variance. On the basis of
our data set, it is not possible to discriminate among these models.
However, it does appear that, in these experiments, the insect myosin
step sizes give a broader distribution than rabbit myosin.
The Functional Role of MHC Alternative Exons--
At least one of
the five MHC regions encoded by alternative exons must be responsible
for the observed differences in actomyosin kinetics between the IFM and
Emb18 isoforms. The remainder of the molecule is identical
between these two isoforms as it arises from constitutive exons of the same gene, and because we engineered the adult tailpiece onto Emb18. Based on mapping the four motor domain alternative
exons (exons 3, 7, 9, and 11) onto the three-dimensional chicken
skeletal myosin head (16), we currently believe that exon 7 may be
responsible for the measured differences in ATPase rates as it encodes
part of the lip of the nucleotide site and is part of "switch one." Exons 11 and/or 3 could contribute to the differences in actin sliding
velocity, as they are located near the pivot point of the lever arm
(Fig. 1) and have been proposed
previously to affect step size (17). However, no difference in step
size was found in this study; therefore, neither exons 3a and 3b nor
exons 11e and 11c regulate step size. We cannot exclude the possibility that exon 11a, 11b, or 11d expressed in one or more of the other myosin
isoforms may affect step size. Exon 9 encodes the "rigid relay
loop" that may be involved in propagating signals from the nucleotide
site to the actin binding site and lever arm region (59). The final
alternative exon, 15, encodes the rod hinge region. Although it has
been shown that myosin S-1 head fragment is sufficient for in
vitro motility (31, 60), exon 15 could be involved in modulating
function in vivo (61).
None of the four alternative exon regions in the myosin head
corresponds to the loop 1 and loop 2 regions that have been
investigated as possible sources of functional variation between
myosins from different muscle types (4-9). The seven-amino acid
insert, which determines differences in ATPase rate and in
vitro motility velocity between the tonic and phasic smooth muscle
isoforms, is not within a homologous region encoded by a
Drosophila alternative exon (7, 8). Thus, multiple
mechanisms appear to have evolved to modify myosin function.
In summary, we have observed dramatic differences in the in
vitro motilities, the effects of smooth muscle tropomyosin on motility, Ca- and Mg-ATPases, and Km for ATP between the IFM and Emb18 myosin isoforms. These differences are
certainly sufficient to explain why the Emb18 isoform
cannot functionally substitute for the native isoforms in the flight
muscle. Most obviously, the Emb isoform is not fast enough to power
flight. Although the details of how these functional differences arise
(e.g. different ADP release rates) remain to be
investigated, we have shown that functional myosins can be expressed
and purified from the IFMs in sufficient quantities for biochemical
experiments. This will enable us to develop more sophisticated assays
to determine which steps in the cross-bridge cycle are different
between these two isoforms.
By exchanging alternative exons between the IFM and Emb18
isoforms, we can relate the functional differences described here to
specific structural regions of the myosin heavy chain. These alternative regions can then be examined in more detail using site-directed mutagenesis to determine specific amino acids or groups
of residues that are responsible for particular functional modifications. The advantage of the Drosophila Mhc system
for this analysis of myosin is that we can, in transgenic organisms, observe the effects from the level of myosin molecular properties, through muscle mechanics (20, 33) to whole animal locomotory performance (17) to gain a fully integrative understanding of the role
of the alternative exon regions.
1 at 22 °C) is
among the fastest reported for a type II myosin and was 9-fold faster
than with the embryonic isoform. With smooth muscle tropomyosin bound
to actin, the actin sliding velocity on the embryonic isoform increased
6-fold, whereas that on the flight muscle myosin slightly decreased. No
difference in the step sizes of Drosophila and rabbit
skeletal myosins were found using optical tweezers, suggesting that the
slower in vitro velocity with the embryonic isoform is due
to altered kinetics. Basal ATPase rates for flight muscle myosin are
higher than those of embryonic and rabbit myosin. These differences
explain why the embryonic myosin cannot functionally substitute
in vivo for the native flight muscle isoform, and
demonstrate that one or more of the five myosin heavy chain alternative
exons must influence Drosophila myosin kinetics.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 for 1 mg·ml
1
(21). The Bradford assay (Coomassie Plus-200, Pierce) and
semiquantitative SDS-PAGE gave similar results when rabbit myosin was
used as the protein standard.
1 for 1 mg·ml
1 (24). For the motility assay, actin
was labeled with rhodamine-phalloidin as described by Kron et
al. (23).
-32P]ATP (500-800
cpm·nmol
1) with 2-5 µg of protein. The
reaction was initiated by ATP addition. After incubating for 15 min at
22-23 °C, the reaction was stopped by addition of 0.1 ml of 1.8 M HClO4. Following centrifugation, aliquots of
the supernatant were added to 0.5 ml of 5% ammonium molybdate, 2 ml of
1.25 M HClO4, and 2.5 ml of isobutanol-benzene (1:1). After vigorous mixing and phase separation, 1 ml of the organic
phase containing 32Pi was assayed by Cerenkov
counting. We found that the Ca-ATPase activity at pH 6 was
approximately twice that obtained at pH 7.4 (data not shown), similar
to previous reports for whole thorax myosin (26). Thus, we measured
ATPase activity at pH 6.0 to maximize ATPase rates.
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Fig. 1.
MHC motor domain depicting the locations
encoded by alternative exons in Drosophila. The
regions encoded by alternative exons are superimposed, in color, on the
chicken molecule S-1 backbone depicted in yellow (62).
All four alternative exon regions are different between the two
isoforms, IFM/Emb18. Spheres represent positions
of nonconserved amino acid residues within these regions.
ELC, essential light chain; RLC, regulatory light
chain. The reactive thiol groups are depicted as large
yellow spheres. The location of the -phosphate group
of ATP is shown as a large red sphere (modified
from Ref. 16).
1. The reaction time
was reduced to 3 min to ensure that initial rates were determined.
1. 2) After myosin addition, dead
heads (myosin that binds irreversibly to actin) were blocked by adding
5 µM phalloidin-stabilized IFM actin followed by AB plus
ATP, 0.4% methylcellulose, and oxygen scavengers (27). After a 10-min
incubation, ATP was washed out of the flow cell by several additions of
AB/BSA. Labeled actin was added and the remainder of the protocol was
as described by Kron et al. (23). This method was very
effective, as continuous movement of actin filaments would last ~10
min in unblocked cells before the majority of filaments were arrested
by dead heads, while in cells employing dead head blocking, most
filaments were still moving after 30 min. Filament movement in
unblocked cells was used as an indicator of myosin quality. If
continuous movement by the majority of actin filaments did not last for
~10 min, the myosin preparation was not used for ATPase measurements.
3) The final activating motility solution contained 0.4%
methylcellulose and no KCl.
1 at 22-23 °C. The optical trap
assay buffer was 25 mM KCl, 4 mM MgCl2, 1 mM EGTA, 25 mM imidazolium
chloride, pH 7.4, 20 mM DTT and glucose/glucose
oxidase/catalase (0.2 mg·ml
1 glucose
oxidase, 0.05 mg·ml
1 catalase, and 3 mg·ml
1 glucose). It included an ATP
regeneration system (2 mM creatine phosphate, 0.1 mg·ml
1 creatine phosphokinase) and 5 µg·ml
1 rhodamine-phalloidin-labeled actin
(32). The ATP concentration was 3 µM for both step size
and attached lifetime measurements. The latter estimates the second
order rate constant of A·M + ATP
A + M·ATP and was determined
by fitting a least squares regression to a plot of frequency against
the logarithm of binned event durations (31).
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Fig. 2.
Myosin and actin purification from
Drosophila IFM. A, an 8%
polyacrylamide SDS gel depicting the purification of myosin.
Lane 2 is the initial myosin high salt extract
from the IFMs. Lane 3 is the pellet after
precipitation of the initial myosin high salt extract and resuspension
in Wash B. Lane 6 is the pellet (normally
discarded) after precipitation of myosin that did not dissociate from
actin. Lane 4 is the final purified IFM myosin
resuspended in myosin storage buffer, and lane 3 is purified Emb18. B, a 12% gel showing the
light chains associated with IFM MHC, in lane 1,
and Emb18 MHC in lane 2. There are
multiple phosphorylated versions of the RLC (~14 isoelectric
variants), which result in two bands, 30 and 33 kDa, on the gel (33).
C, lane 1 is IFM actin and arthrin,
and lane 2 is rabbit actin. Arthrin is the
ubiquinated form of actin found only in the IFM (63).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 µg/fly), and its purity was similar to the IFM isoform
(Fig. 2B). The purified Drosophila myosins were
kept in storage buffer at 5-10 mg·ml
1, and
were used the same day they were isolated, as ATPase activity declines
to 50-75% of the original activity within 2 days. The decline in
activity is similar to that reported for other myosin types, including
rat cardiac myosin (35).
1 (Table
I). These values are the same as for
phosphorylated myosin from whole Drosophila thoraces (36).
The Ca-ATPase assay conditions, selected to maximize ATPase rate, were
similar to those used by Takahashi et al. (36), except that
we used 10 mM calcium rather than 3 mM. The
Emb18 Ca-ATPase activity is similar to that reported for
whole larval myosin (26), which was 25% that of their adult myosin
mixture (whole thorax). The Km for ATP of the
Emb18 myosin Ca-ATPase activity was approximately one-fifth
that of the IFM myosin (Fig. 3).
ATPase activities
1 of myosin, which is at the high end
of values reported for other myosin types (0.1-0.5
mg·ml
1; Ref. 28). Movement was not achieved
below 0.3 mg/ml. Mean actin velocity on IFM myosin, 6.4 µm·s
1, was faster than that typically
found with rabbit myosin (4-5 µm·s
1;
Refs. 23 and 28). Mean actin velocity on IFM myosin was 9-fold faster
than with Emb18 myosin (Table
II). Although it was much slower, the
embryonic myosin still required methylcellulose to prevent actin
diffusion away from the myosin-coated surface, and moved actin best at
a myosin concentration of 0.5 mg·ml
1.
Motility characteristics
=
(kT/
trap)). A distribution of individual
mean displacements produced by Drosophila IFM myosin
attachments is shown (Fig. 5B, black). These
data are well fit by a Gaussian distribution of the thermal noise of
the unattached bead-actin-bead assembly, but stiffened in the
x axis by the size of the myosin working stroke. These are
representative data from all the interactions of an individual F-actin
bead pair. The shift in position of this distribution from the zero
position on the x axis estimates the average size of the
myosin displacements (37). For the Drosophila IFM myosin
this produces a displacement of 3.91 ± 2.36 nm (mean of 3547 attachment events obtained from 43 F-actin filaments on a total of 13 IFM myosin preparations; ± S.D. of 43 means). For Emb18
myosin we obtained a mean displacement of 4.38 ± 2.27 nm (1011 events involving 8 F-actin filaments and 4 myosin preparations; ± S.D.
of 8 means). These estimates are not significantly different (p = 0.605, Student's t test), but are
rather smaller than previously measured with rabbit HMM (5.5 nm) (37)
and S-1 (32).
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Fig. 3.
Double-reciprocal plot of myosin Ca-ATPase
activities and ATP concentrations. Each plot is from one IFM or
Emb18 myosin preparation. ATP concentration was
varied from 5 to 300 µM for IFM and from 2 to 40 µM for Emb18.
A + M·ATP. Since the rate of detachment is so fast once ATP
binds, the rate constant estimates the ATP affinity of nucleotide free
actomyosin. At 3 µM ATP, we obtained estimates for the
two Drosophila myosins (Table II), which are essentially the
same and similar to that obtained (31) for the heavy meromyosin fragment of rabbit myosin (0.8 × 106
M
1·s
1).
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Fig. 4.
In vitro actin sliding
velocities. A, actin velocity with IFM myosin was
slightly decreased when smTM was bound to actin. B, actin
velocity was much slower with Emb18 myosin, but was
dramatically increased with smTM bound to actin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1) under similar conditions and
temperature (27, 42). The high in vitro velocities produced
by the IFM myosin are consistent with its role in powering one of the
fastest contracting Drosophila skeletal muscle types.
Drosophila IFMs produce oscillatory contractions of ~200
Hz at 22 °C (43) and shorten by about 3.4% (44). The thick and thin
filaments of each set of IFMs, the opposing dorso-longitudinal and
dorso-ventral muscles, slide past each other for up to 2.5 ms during a
contraction. Starting with a sarcomere length at rest (45) of 3.4 µm,
the length change for each half sarcomere will be 58 nm and contraction
velocity will be around ~23 µm·s
1,
almost 4 times the speed measured in the motility assay. In comparison,
rabbit psoas muscle shortens about 7 µm·s
1 per half sarcomere at 22 °C (46,
47) or about 3-fold slower than Drosophila IFMs.
1,2
with individual contractions visible to the naked eye, and must have
much slower contraction velocities than IFMs. The actin velocity on
Emb18 myosin that we have measured more closely resembles
those described for phasic smooth muscle isoforms than those of
vertebrate slow skeletal muscle isoforms (4, 7, 8).
View larger version (32K):
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Fig. 5.
Step size determination for IFM and
Emb18 myosin. A, a raw data recording from
an optical trapping experiment is shown. A single actin filament was
held suspended between two 1-µm diameter plastic microspheres close
to a third, surface-fixed bead on which was deposited a low surface
density of IFM myosin (see text for details). The position of one of
the optically trapped beads was monitored with a four-quadrant
photodetector. The upper record shows how the
position of the optically trapped bead varies with time. Intervals of
high noise are interspersed with intervals of reduced noise. Noise
amplitude reflects the system stiffness and periods of low noise
correspond to myosin binding events. The lower
trace is the calculated S.D. of the data record. Events are
defined as periods during which the S.D. remained below the threshold
level (dotted line). The amplitude of separate
events is measured from the displacement from local mean position.
Panel B shows (gray) the distribution
of background thermal vibration of the bead (in the absence of myosin
attachments). The shape of this histogram is consistent with the
principle of equipartition of energy in which the Gaussian distribution
of x positions is given by A = A0exp( x2/kT) (where
is the combined optical trap stiffness of 0.038 pN·nm
1, see text). The black
histogram shows the distribution in amplitude of myosin
binding events measured from a much longer data recording than shown in
panel A (335 events). The mean position in this
data set is shifted by about 5 nm from zero. Panel
C shows how the estimates of mean position are distributed
between experimental runs. Individual estimates are highly variable
between runs, and even within experiments performed using the same
actin filament. This implies that behavior of the individual myosin
molecules is heterogeneous. The overall means determined from many
experimental runs measured for IFM (black),
Emb18 (gray), and rabbit myosins
(white) are not significantly different from each other (see
text for details).
1 for Lethocerus myosin
subfragment 1, S-1, at 20 °C, pH 7; this is about the same as our
Drosophila IFM myosin value (0.1 s
1). Drosophila IFM ATPase
activity correlates with IFMs being classified as fast muscles.
However, Lethocerus flight muscles, which are physiologically similar, only contract at 30 Hz, yet have nearly the
same myosin ATPase activities. Clearly, myosin ATPase activity does not
scale with contraction velocity within this muscle type. This
observation is very similar to the results of Molloy et al. (58), who showed that IFM fiber ATPase activity is constant across a
wide range of insect wing beat frequencies, with the exception of the wasp.
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ACKNOWLEDGEMENTS |
---|
We thank Floyd Sarsoza and Gracielle Manipon (supported by National Institutes of Health NIGMS MBRS Program Grant GM 58906), for help analyzing the motility data.
![]() |
FOOTNOTES |
---|
* This work was supported by postdoctoral fellowships from the National Institutes of Health and the American Heart Association Western affiliate (to D. M. S.); by a Biotechnology and Biologicol Sciences Research Council, UK, research grant (to J. C. S. and J. E. M., which supported M. L. B.); by NATO Collaborative Research Program Grant CRG940669 for travel funds, which were instrumental in enabling much of the collaborative work to be completed; and by National Institutes of Health Grant GM32443 (to S. I. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: E-mail: dswank@sciences.sdsu.edu.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M008379200
2 D. M. Swank, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: MHC, myosin heavy chain; IFM, indirect flight muscle; DLM, dorsal longitudinal muscle; Emb, embryonic myosin isoform; Emb18, embryonic isoform with the adult C terminus; smTM, smooth muscle tropomyosin; BSA, bovine serum albumin; DTT, dithiothreitol; YMG, York Modified Glycerol; MOPS, 4-morpholinopropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; AB, assay buffer; N, newton(s); HMM, heavy meromyosin.
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