Download Ectopic expression of beta-cytoplasmic actin

3627
Journal of Cell Science 112, 3627-3639 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0612
Substitution of flight muscle-specific actin by human β-cytoplasmic actin in
the indirect flight muscle of Drosophila
Véronique Brault1, Mary C. Reedy2, Ursula Sauder3, Richard A. Kammerer4, Ueli Aebi1
and Cora-Ann Schoenenberger1,*
1M.E. Müller Institute, Biozentrum, University of Basel, CH-4056 Basel, Switzerland
2Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA
3Interdepartmental Electronmicroscopy, Biozentrum, University of Basel, CH-4056, Basel, Switzerland
4Department of Biophysical Chemistry, Biozentrum, University of Basel, CH-4056, Basel, Switzerland
*Author for correspondence (e-mail: [email protected])
Accepted 16 August; published on WWW 18 October 1999
SUMMARY
The human β-cytoplasmic actin differs by only 15 amino
acids from Act88F actin which is the only actin expressed
in the indirect flight muscle (IFM) of Drosophila
melanogaster. To test the structural and functional
significance of this difference, we ectopically expressed βcytoplasmic actin in the IFM of Drosophila that lack
endogenous Act88F. When expression of the heterologous
actin was regulated by ~1.5 kb of the 5′′ promoter region of
the Act88F gene, little β-cytoplasmic actin accumulated in
the IFM of the flightless transformants. Including Act88Fspecific 5′′ and 3′′ untranslated regions (UTRs) yielded
transformants that expressed wild-type amounts of β-
cytoplasmic actin. Despite the assembly of β-cytoplasmic
actin containing thin filaments to which endogenous
myosin crossbridges attached, sarcomere organization was
deficient, leaving the transformants flightless. Rather than
affecting primarily actin-myosin interactions, our findings
suggest that the β-cytoplasmic actin isoform is not
competent to interact with other actin-binding proteins in
the IFM that are involved in the organization of functional
myofibrils.
INTRODUCTION
studies of actin isoforms have been rather restricted. A further
drawback in analyzing the functional significance of the closely
related isoforms is the scarcity of specific antibodies that reliably
distinguish one isoform from another in mixtures of different
actins.
Expressing a particular actin isoform or mutant in vivo in
order to analyze subtle functional or structural differences is
complicated by the toxicity of excess amounts of actin, the
presence of more than one type of actin in a given cell, or the
frequently disruptive effects of deleting or mutating this essential
protein (Hennessey et al., 1992). Recently, substitution of whole
actin isoforms has been achieved in yeast and in mouse heart,
where the substituted actin isoform rescued lethality and/or
improved function (Karlsson et al., 1991; Kumar et al., 1997).
In contrast to other organisms in which detailed structural and
functional analysis of actin isoform substitution is difficult, the
IFMs of Drosophila provide an excellent system for exploring
actin isoform diversity. IFMs are dispensable for viability, so
disruption of IFM structure and function simply impedes flight
performance, providing an easy assay for functional change
(Sparrow et al., 1991a; Bernstein et al., 1993). Because the IFMs
are not required for viability, a stable strain that is completely
null for IFM-specific Act88F actin could be established (e.g.
KM88; Hiromi and Hotta, 1985; Mahaffey et al., 1985) which
Multiple isoforms of actin have been described in almost all
eukaryotic organisms (Gallwitz and Seidel, 1980; Fidel et al.,
1988; Hirono et al., 1987; for review see Rubenstein, 1990;
Herman, 1993). Humans have six actin isoforms, four of which
are muscle-specific, and two are found in the cytoplasm of
nonmuscle cells. Likewise, Drosophila melanogaster expresses
two nonmuscle and four muscle-specific actins in a temporally
and spatially regulated pattern (Fyrberg et al., 1983). For
instance, Act88F, is exclusively expressed in the indirect flight
muscle (IFM), and encodes all of the actin contained in the
myofibrils of these muscles (Ball et al., 1987). Among
mammals, specific isoforms are extremely conserved if not
identical, and Drosophila actins share 93 to 97% identical amino
acid residues with mammals. Although the high sequence
conservation raises the question as to whether there is a
functional significance of the multiple actin isoforms, the stageand tissue-specific expression pattern of different actins suggests
that isoforms have distinct functions (McKenna et al., 1985;
DeNofrio et al., 1989; Sawtell and Lessard, 1989; EppenbergerEberhardt et al., 1990; Peng and Fischman, 1991; Mounier et al.,
1997). To date, there is no expression system that produces large
quantities of pure, fully functional actin. Therefore, in vitro
Key words: Actin, β-Cytoplasmic actin, Isoform, Myofibril, Thin
filament
3628 V. Brault and others
allows expression of another isoform against an IFM
background free of wild-type Act88F actin. In addition, IFMs
display a high degree of structural order (Reedy and Beall,
1993), thereby providing a sensitive experimental system for
examining even subtle structural as well as functional changes
resulting from the substitution of one isoform for another.
Recently, Fyrberg and co-workers have used the IFM to test the
consequences of isoform-specific amino acid replacements in
Act88F actin (Reedy et al., 1991; Fyrberg et al., 1998). When a
single isoform-specific amino acid of Act88F was substituted
with a residue corresponding to another Drosophila actin
isoform, the exchange affected myofibrillar function only in one
out of ten cases. Flies transformed with chimeric genes
containing multiple replacements showed flight impairment, and
replacement of all IFM-specific residues with amino acids
corresponding to Drosophila nonmuscle Act42A actin, produced
flightlessness and disorganized myofibrils.
One might expect that cytoplasmic actin isoforms, which
function in a very different environment from the highly ordered
IFMs, differ most from muscle actin isoforms. However, human
β-cytoplasmic actin diverges from the Drosophila IFM-specific
isoform by only 15 residues. Insect muscle actins form a distinct
family of related proteins characterized by about 10 amino acids
which appear to distinguish them from the vertebrate
cytoplasmic actins (Mounier et al., 1992; see also Fig. 8). These
residues may be critical in tailoring actin to perform its isoformspecific function in a muscle environment.
Here we report the ectopic expression of an entire human βcytoplasmic actin isoform in a Drosophila muscle environment
free of any other actin. Consistent with the assumption of
functional diversity among actin isoforms, we find that human
β-cytoplasmic actin does not fully compensate for the
endogenous Act88F isoform, even when present in amounts
similar to Act88F in wild-type Drosophila.
MATERIALS AND METHODS
Construction of plasmids
A PstI-EcoRI fragment comprising the Act88F gene including the 5′
UTR with the first intron (Okamoto et al., 1986), the 3′ UTR, and
approximately 1.5 kb regulatory sequences upstream of the
transcription initiation site was excised from the P[ry+;CSB] plasmid
(Hiromi et al., 1986) and cloned into the pW8 Drosophila
transformation vector which contains the selectable white (w) marker
gene (Klemenz et al., 1987). The resulting parent pW8Act88F plasmid
is shown in Fig. 1A.
pW8(5′Act88F)β-cyto construct
A PstI-HincII fragment containing ~1.5 kb promoter region of the
Act88F gene including the transcription initiation site was excised
from the P[ry+;CSB] plasmid and ligated into pW8 at the PstI and
StuI sites yielding an intermediate pW8(5′Act88F) construct. The fulllength cDNA encoding human β-cytoplasmic actin (Gunning et al.,
1983) was inserted as a blunt end-BamHI fragment into the
pW8(5′Act88F) transformation vector downstream of the Act88F
transcription initiation site (Okamoto et al., 1986) using the HpaI site
adjacent to the former HincII site at the 5′ and the BamHI site at the
3′ end. The resulting pW8(5′Act88F)β-cyto transformation
construction is shown in Fig. 1B.
pW8Act88F(−3′UTR) construct
The KpnI-EcoRI fragment which contains the Act88F 3′UTR was
replaced in the parent pW8Act88F (Fig. 1A) construct by a
corresponding fragment lacking the 3′UTR. This fragment was
obtained by PCR using a sense primer 5′-CGGCGGTACCACCATGTACCCTGG-3′ spanning the upstream KpnI site in Act88F and
an antisense primer 5′-CCGGAATTCTTAAAAGCATTTGCGGTG3′, which inserts an EcoRI site 3′ of the TAA stop codon. The
final pW8Act88F(−3′UTR) transformation construct is shown in Fig.
1C.
pW8(−5′UTR)Act88F construct
The parent pW8Act88F was digested with BamHI/EcoRI and
the purified Act88F gene fragment subcloned in pUC19 cloning
vector (Amersham Pharmacia Biotech). The same BamHI-EcoRI
Act88F fragment was used as template in order to introduce a
new BamHI site 25 nucleotides upstream of the Act88F translation
start site by PCR. The sense primer 5′-CGGGATCCCAGATAAACAACTGCCAAGATG-3′ and the antisense primer 5′CGCACGGTGTGGGAGACACCAT-3′, which spans the DraIII site
in the Act88F coding sequence, were used. The amplified BamHIDraIII fragment was cloned into the pUC19Act88F construct in place
of the endogenous BamHI-DraIII fragment. Subsequently, a BamHIEcoRI fragment of this construct was ligated into pW8(5′Act88F) at
the respective sites yielding the pW8(−5′UTR)Act88F transformation
construct which lacks the 5′UTR (Fig. 1D).
pW8(StuI)Act88F(EcoRI) construct
To replace the Act88F coding sequence with the human β-cytoplasmic
actin cDNA, two unique restriction sites were generated at opposite
ends of the Act88F coding sequence in pW8Act88F by the following
procedure. First, the EcoRI site at the 3′ end of pW8Act88F was
eliminated by DNA fill-in synthesis. A new EcoRI site was obtained
3′ of the TAA stop codon by inserting a corresponding linker (5′GAATTCGCCCGCCGCGAAAGCTCTTCAAG-3′) between the
stop codon and the SapI site in the 3′UTR of Act88F. At the 5′
end, changing the original nucleotide sequence at positions −11 to
−9 upstream of the ATG translation start site from CAA to
GGC was achieved by site-directed mutagenesis by PCR using 5′CCTTCCAGATAAAGGCCTGCCAAGATGTGTG-3′ as sense, and
5′-CACACATCTTGGCAGGCCTTTATCTGGAAGG-3′ as antisense
primer. Mutating the sequence yielded a unique StuI site without
changing the splice acceptor sequence. The resulting
pW8(StuI)Act88F(EcoRI) construct (Fig. 1E) served as a control to
verify wild-type levels of Act88F actin expression.
pW8(StuI)β-cyto(EcoRI) construct
The human β-cytoplasmic actin cDNA was modified by PCR in order
to add a StuI site at its N terminus and an EcoRI site at its C terminus.
The sense primer (5′-GAAGGCCTGCCAAGATGGATGATGATATCGCCGC-3′) was designed to obtain a StuI site followed by 5′GCCAAG-3′ which corresponds to the sequence immediately 5′ of
the start ATG in Act88F. The 3′ antisense primer (5′-CGGAATTCC
TAGAAGCATTTGCGGTGGA 3′) was designed to introduce an
EcoRI site 3′ of the TAG stop codon. Finally, the endogenous StuIEcoRI Act88F fragment of pW8(StuI)Act88F(EcoRI) (Fig. 1C) was
replaced with the StuI-EcoRI β-cytoplasmic actin PCR product,
yielding the pW8(StuI)β-cyto(EcoRI) transformation construct
(Fig. 1F).
All constructs were confirmed by DNA sequencing.
Germ line transformation
Germ line transformation was carried out essentially as described by
Rubin and Spradling (1982) using the helper P element plasmid
pπ25.7∆2-3 wc. The recipient strain for all constructs was the KM88
null mutant (w; Act88F KM88) (Hiromi and Hotta, 1985; Okamoto et
al., 1986).
The posterior ends of homozygous KM88 embryos were injected
with 100 ng/µl of helper plasmid and 100-300 ng/µl of pW8
Ectopic expression of β-cytoplasmic actin 3629
transformation constructs. Individual adult Go flies were back-crossed
to KM88 flies and the progeny was scored for red eyes. For each
construct independent homozygous lines were established using
balancer chromosomes (Lindsley and Zimm, 1992).
Electron microscopy
IFMs were prepared for transmission electron microscopy according
to the method of Reedy and Beall (1993) with minor modifications.
The part containing the dorsal longitudinal IFMs was dissected from
thoraces of 24- to 48-hour-old females and directly immersed in a
freshly prepared fixative consisting of 3% glutaraldehyde and 0.2%
tannic acid in MOPS buffered Drosophila Ringer’s solution (Fyrberg
et al., 1990) without phosphate (pH 6.8) for 2 hours at room
temperature. After primary fixation, the dorsal thoraces were rinsed
in MOPS-buffered Drosophila Ringer’s solution, in 100 mM
phosphate buffer (pH 6.0) and then immersed for 1 hour in ice-cold
secondary fixative consisting of 1% osmium tetroxide in 100 mM
phosphate buffer and 10 mM MgCl2 (pH 6.0). After washing, thoraces
were block-stained in aqueous 2% uranyl acetate for 1 hour at 4°C.
Dehydrated specimens were embedded in Epon. In some cases,
thoraces were split down the midline and the resulting hemithoraces
embedded in Araldite 506, allowing staining of the sections in
KMnO4. For glycerination of IFMs in situ, hemithoraces were used.
IFMs were glycerinated according to the method of Reedy et al.
(1989) and subsequently fixed and stained as described above.
Electron micrographs were recorded on Kodak (Rochester, NY)
SO163 film using a LEO 910 (LEO, Oberkochen, Germany) or a
Philips EM 420 transmission electron microscope operated at 80 kV.
SDS-PAGE and immunoblot analysis of IFM protein extracts
Dorsal longitudinal flight muscles were dissected from thorax halves
according to the method of Brault et al. (1999), transferred to SDSPAGE sample loading buffer (2.3% SDS, 62.5 mM Tris-HCl, pH 7.0,
15% glycerol, 2.5% β-mercaptoethanol, 0.05% bromophenol blue),
and boiled for 5 minutes. Protein extracts corresponding to the IFMs
of half a thorax were separated on 11.5% SDS-polyacrylamide gels
Pst I
A
BamH I ATG
TIS
Pst I
HincII
Stop
TIS
Pst I
C
D
pW8Act88F
Stop BamH I
pW8(5′Act88F)β-cyto
β-cytoplasmic
actin cDNA
BamH I ATG
BamH I/ATG
Regulation of transgene expression by only the 5′′
promoter region of Act88F yields low levels of
human β-cytoplasmic actin in the IFM
In the initial pW8(5′Act88F)β-cyto transformation construct
(Fig. 1B), expression of the heterologous human β-cytoplasmic
actin in the IFM of transformed Drosophila is regulated by
approximately 1.5 kb Act88F promoter region including the
Act88F transcription initiation site. Transformants established
with the pW8(5′Act88F)β-cyto construct carried the insertion
on the third chromosome and were unable to fly.
To examine the cause of the flightless phenotype at the
Sap I
DraIII
Kpn I
Stop/EcoR I
pW8Act88F(-3′UTR)
5′UTR
Pst I
RESULTS
EcoR I
3′UTR
HincI HpaI
I
ATG
B
Kpn I Stop/EcoR I EcoR I
pW8(-5′UTR)Act88F
3′UTR
HincII
Pst I
E
Stu I
ATG Kpn I Stop/EcoR I EcoR I
3′UTR
5′UTR
Pst I
F
Kpn I
5′UTR
(Laemmli, 1970). Gels were electroblotted onto an Immobilon
polyvinylidene difluoride membrane (Millipore, Bedford, MA). Blots
were rinsed with PBS and transiently stained with Coomassie Brilliant
Blue to confirm that protein extractions, gel loadings and
electrophoretic transfer were comparable for each lane. After
complete destaining with methanol, blots were washed in PBS and
0.1% Tween-20 (PBS-T), blocked in 5% milk powder in PBS-T, and
incubated for 2 hours at room temperature with either a mouse mAb
recognizing different actin isoforms (Amersham Pharmacia Biotech;
diluted 1:7,500 in PBS-T), or with a mouse mAb which specifically
recognizes β-cytoplasmic actin (Sigma; diluted 1:2,500 in PBS-T).
Blots were washed for 15 minutes each with 5% milk powder in PBST, in PBS-T, and 1% blocking solution (Roche Molecular
Biochemicals) in PBS-T, followed by a 2 hour incubation with a
1:5,000 dilution of a goat anti-mouse IgG alkaline phosphatase
conjugated secondary antibody (Sigma). Blots were washed 3 times
for 15 minutes with PBS-T, once with 100 mM Tris (pH 9.5), 100
mM NaCl2, and 5 mM MgCl2, and then developed with Western Blue
stabilized substrate for alkaline phosphatase (Promega).
Stu I
5′UTR
ATG
pW8(StuI)Act88F(EcoRI)
Stop/EcoR IEcoR I
β-cytoplasmic
actin cDNA
3′UTR
pW8(StuI)β-cyto(EcoRI)
Fig. 1. Act88F and β-cytoplasmic actin constructs for
Drosophila transformation. (A) The endogenous
Act88F gene inserted into the pW8 vector
(pW8Act88F) was used to generate all subsequent
constructs. (B) In the pW8(5′Act88F)β-cyto actin
construct, expression of β-cytoplasmic actin is
regulated by ~1.5 kb of the 5′ promoter region from
the Act88F gene including the autologous
transcription initiation site (TIS). (C) In
pW8Act88F(−3′UTR), the 3′ untranslated region
downstream of the TAA stop codon was deleted. (D)
In pW8(−5′UTR)Act88F, the Act88F gene including
18 nucleotides upstream of the translation start codon
and the Act88F-specific 3′UTR was linked to the
Act88F regulatory region at the HincII site 5′
of the transcription initiation site. (E) In
pW8(StuI)Act88F(EcoRI), a StuI and an EcoRI
restriction site flank the Act88F coding sequence at
the 5′ and 3′ end, respectively. (F) In pW8(StuI)βcyto(EcoRI), the Act88F actin coding sequence was
replaced with the cDNA encoding human βcytoplasmic actin. Solid black boxes represent
translated regions of the Act88F gene, whereas
untranslated sequences are shown as white boxes with
stippled regions representing intron sequences. The βcytoplasmic actin cDNA is shown as a shaded box.
TIS, Act88F transciption initiation site.
3630 V. Brault and others
Ectopic expression of β-cytoplasmic actin 3631
Fig. 2. Ultrastructure of IFM from initial pW8(5′Act88F)β-cyto
transformants compared to pW8(StuI)Act88F(EcoRI) control IFM.
(A) In longitudinal sections of IFM from transformants expressing
low amounts of β-cytoplasmic actin, sarcomeric organisation and Zdiscs are absent. M-line-like structures (arrowhead and inset) indicate
some degree of thick filament register. (B) Cross-sections of IFM
from pW8(5′Act88F)β-cyto transformants suggest that thin filaments
are virtually absent. Accordingly, there is no ordered lattice of thin
and thick myofilaments. (C) Control transformants display regular
sarcomeres marked by Z-discs (Z) and M-lines (M). (D) Myofibrils
of control transformants have a wild-type appearance. The higher
magnification (inset) reveals the almost crystalline hexagonal array
of myofilaments. Bars: (A) 500 nm (inset, 500 nm); (B) 500 nm
(inset, 100 nm); (C) 500 nm (inset, 200 nm); (D) 500 nm (inset, 100
nm).
ultrastructural level, the thoraces of young adult transformants
were fixed in situ and processed for transmission electron
microscopy (TEM). Longitudinal IFM sections from the
pW8(5′Act88F)β-cyto transformant reveal only some
imperfectly aligned skeins of thick filaments (Fig. 2A). In
essence, sarcomeric organisation including Z-discs is
completely missing and thin filaments appear to be absent.
Occasionally, indications of an M-line-like structure
(arrowhead) can be discerned. In contrast, control
transformants expressing wild-type amounts of Act88F actin
Fig. 3. The 5′ promoter region of Act88F yields low levels of βcytoplasmic actin in the IFM of transformants. (A) Immunoblot
revealing the accumulation of β-cytoplasmic actin in thoracic
extracts from pW8(5′Act88F)β-cyto transformants. (B) Immunoblot
with a mAb that recognizes different actin isoforms. The amounts of
β-cytoplasmic actin in the IFM of the pW8(5′Act88F)β-cyto
transformant are significantly lower than the amounts of Act88F
actin in wild-type flies and in pW8(StuI)Act88F(EcoRI) control
transformants. Normal amounts of Act88F are present in
pW8Act88F(−3′UTR) transformants, whereas truncation of Act88F
at the 5′ end abrogates actin accumulation in pW8(−5′UTR)Act88F.
The additional ~55 kDa band corresponding to arthrin is present only
in wild-type flies and in transformants accumulating wild-type levels
of Act88F actin.
display the typical, highly ordered sarcomeres (Fig. 2C). A
cross-section of the IFM from pW8(5′Act88F)β-cyto
transformants (Fig. 2B) reveals the absence of round, highly
organised myofibrils which are characteristic of wild-type IFM
and the control transformants (Fig. 2D). In contrast to the
almost crystalline double-hexagonal pattern of control
myofibrils (Fig. 2D, inset), cross-sections of pW8(5′Act88F)βcyto transformant IFM (Fig. 2B, inset) display randomly
distributed myosin thick filaments with no apparent hexagonal
array of actin containing thin filaments surrounding them.
To examine the expression of β-cytoplasmic actin in the IFM
of the pW8(5′Act88F)β-cyto transformants, an immunoblot of
IFM protein extracts from the transformant, KM88, and wildtype flies was probed with a mAb that specifically recognizes
the β-cytoplasmic actin isoform (Gimona et al., 1994). The anti
β-actin antibody detected a single 43-kDa band in IFM extracts
from pW8(5′Act88F)β-cyto transformants (Fig. 3A). As
expected, the β-actin band is absent in IFM extracts from
KM88 and wild-type flies. To compare the amount of βcytoplasmic actin that accumulated in the IFM of
pW8(5′Act88F)β-cyto transformants to the amount of
endogenous Act88F in wild-type IFM, we probed a
corresponding blot with a mAb that equally recognizes a
number of different actin isoforms (Fig. 3B). This anti-actin
antibody revealed that β-cytoplasmic actin in the IFM of
pW8(5′Act88F)β-cyto transformants accumulates at a much
lower level than the endogenous Act88F actin in wild-type
flies. The faint ~55-kDa band which is detected by the antiactin antibody in all lines accumulating wild-type levels of
Act88F actin most likely represents arthrin (i.e. ubiquitinated
actin; Bullard et al., 1985; Ball et al., 1987). As anticipated,
actin was virtually undetectable in IFM extracts from KM88
flies.
The 5′′ untranslated region of the Act88F gene is
important for proper β-cytoplasmic actin
accumulation
The coding sequence of Act88F is flanked both at the 5′ and
3′ end by UTRs (Fyrberg et al., 1981; Sanchez et al., 1983;
Geyer and Fyrberg, 1986; Okamoto et al., 1986). The 5′UTR
contains an intron which is 552 bp in length and spans the
region from −568 to −17 upstream of the translation start site
(Okamoto et al., 1986). In contrast to the low levels of βcytoplasmic actin in the initial pW8(5′Act88F)β-cyto
transformants, transgenic Act88F was expressed at wild-type
levels in control transformants that were established with the
parent pW8Act88F construct (Fig. 1A), which contains an
intact Act88F gene including the homologous 5′UTR and
3′UTRs (data not shown).
To examine the role of the 5′UTR and 3′UTR in the
expression of IFM actin, we established transformants with the
pW8Act88F(−3′UTR) (Fig. 1C) in which the 3′UTR had been
removed from the otherwise authentic Act88F gene, and with
the pW8(−5′ UTR)Act88F construct missing most of the
5′UTR (from −535 to −20) (Fig. 1D). Transgenic lines
homozygous for the 3′UTR-truncated Act88F gene accumulate
wild-type amounts of Act88F in their IFMs (Fig. 3B).
Moreover, the ultrastructural IFM morphology was
indistinguishable from that of wild-type flies and the
transformants were able to fly normally (data not shown). In
contrast, transformants containing a 5′ truncated Act88F did
3632 V. Brault and others
Fig. 4. Regions flanking the Act88F coding
sequence confer wild-type levels of βcytoplasmic actin accumulation upon the IFM in
transgenic flies. (A) Immuno-blotting IFM
protein extracts with an antibody specific for βcytoplasmic actin reveals that the flies
transformed with the construct containing the
authentic Act88F UTRs accumulate significantly
more β-cytoplasmic actin than flies that express
the β-cytoplasmic actin under the 5′ promoter
region of Act88F gene without the corresponding
UTRs. Ubiquitinated β-cytoplasmic actin is
detected only in transformants where the Act88F
UTRs were included in the construct. (B) If intact
Act88F UTRs flank the β-cytoplasmic actin,
transformants accumulate wild-type levels of βcytoplasmic actin in the IFM.
not accumulate any detectable amount of Act88F actin or
arthrin (Fig. 3B) and were unable to fly. This finding indicates
that the 5′UTR of the Act88F gene including the intronic
sequence is critical for the expression of normal levels of actin
and that its absence in the previous pW8(5′Act88F)β-cyto
construct probably reduced actin accumulation and thus,
affected the IFM structure yielding a flightless phenotype.
In the light of these findings, we designed a new control
transformation vector, pW8(StuI)Act88F(EcoRI), in which the
Act88F UTRs were preserved (Fig. 1E). Generation of a
StuI restriction site at position −12 did not affect the splice
acceptor site and left the sequence directly upstream of the
ATG intact. Control transformants established with the
pW8(StuI)Act88F(EcoRI) construct accumulate wild-type
level of Act88F actin and their ultrastructural IFM morphology
(Fig. 2C and D) corresponds to that of wild-type flies, thus
confirming that the sequence modification had no adverse
affect. The unique restriction sites allowed us to exclusively
exchange the Act88F coding sequence with the coding
sequence of β-cytoplasmic actin, while retaining the
untranslated sequences of the endogenous Act88F gene.
Transformants expressing wild-type amounts of βcytoplasmic actin in the IFM
The accumulation of β-cytoplasmic actin protein in six
individual transformed lines established with the pW8(StuI)βcyto(EcoRI) construct (Fig. 1F), which for convenience are
called β-cyto transformants, is documented in Fig. 4A. In all
six transformants, the anti-β antibody recognizes a prominent
band with an apparent molecular mass of ~43 kDa which
corresponds to the size of actin. The level of expression does
not significantly vary between the different lines. In
comparison, the amount of β-cytoplasmic actin present in IFM
extracts of the initial pW8(5′Act88F)β-cyto transformant
which lacks the flanking Act88F UTRs (lane on the right) was
strikingly lower. Moreover, the detection of a ~55 kDa arthrin
band in β-cyto transformants supports the
notion that ubiquitination is not isoformspecific but rather a feature of the IFM (Ball
et al., 1987).
To directly compare the amount of βcytoplasmic actin present in the β-cyto
transformants with endogenous Act88F in
wild-type IFM, an equivalent blot was probed
with an anti-actin mAb that does not discriminate different
isoforms (Fig. 4B). This immunoblot revealed that the amount
of β-cytoplasmic actin in β-cyto transformants is similar to that
of Act88F actin in wild-type flies. Consistent with the result
from the immunoblot using the β-cytoplasmic actin-specifc
antibody (Fig. 4A), the actin level in the IFM of the initial
pW8(5′Act88F)β-cyto transformant was at least 10 times lower
than in the β-cyto transformants or in wild-type flies. In some
instances, the anti-actin antibody also detected a faint 43 kDa
band in control extracts from the KM88 null mutant. Although
the band could correspond to endogenous cytoplasmic actin
present in the IFM, we believe it rather represents other
Drosophila actin isoforms from surrounding muscle or nonmuscle tissue, especially since in the absence of a discernible
myofibrillar structure in KM88, it is extremely difficult to
exclusively dissect IFM. Similar to the anti-β mAb (Fig. 4A),
the anti-actin mAb also detected the 55 kDa arthrin band in
wild-type flies and in the β-cyto transformants.
By flanking the heterologous human β-cytoplasmic actin
with the 5′ and 3′ UTRs of the Act88F gene in the
transformation vector, we achieved ectopic expression of βcytoplasmic actin in the IFM that is regulated in a manner
similar to endogenous Act88F in wild-type flies.
Myofibrillar structure of β-cytoplasmic
transformants
As illustrated in Fig. 5, the IFM of β-cyto transformants
contain thin as well as thick filaments, which are bundled
together into myofibrillar-like structures. However, these are
not marked by regular sarcomeric repeats (Z-discs and Mlines) and frequently split or taper. In contrast to both wildtype and KM88 IFM, the β-cyto myofibrils very rarely display
M-lines, which may reflect the lack of thick filament register.
At higher magnification, ultra-thin sections of β-cyto
transformants clearly reveal that the thin filaments interdigitate
with thick filaments in an orderly fashion (see small arrows,
Ectopic expression of β-cytoplasmic actin 3633
Fig. 5. Thin filaments containing β-cytoplasmic actin assemble into myofibril-like bundles. (A) Longitudinal sections reveal rudiments of
sarcomeric organisation. Large arrows point to two Z-disc-like structures that are formed by alignment of Z-bodies (zb). Note that both Z-disclike structures are by-passed by out-of-register filaments, lying close to the arrowheads. Smaller arrows point to isolated Z-bodies.
Mitochondria (MI); bar, 500 nm. (B) Higher magnification of aligned Z-bodies (zb) bracketing rudimentary sarcomere. Arrowhead points to
discontinuity. Larger arrow points to an isolated Z-body showing that thin filaments are frequently not linked end-to-end and do not emerge
from both sides of Z-bodies. Only a few thick filaments are in-register at Z-bodies. Small arrows point along corridors in which thin filaments
can be seen. Bar, 500 nm. (C) Discontinuities between Z-bodies (arrowheads) and lack of thick and thin filament register. Bar, 250 nm.
(D) Myosin crossbridges forming angled chevrons along the β-cytoplasmic actin containing thin filaments. Bar, 150 nm. (E) The polarity of
rigor chevrons (indicated by arrowheads) is uniform along individual thin filaments. Chevrons on adjacent thin filaments may point in opposite
directions. Bar, 200 nm.
3634 V. Brault and others
Fig. 5B). Examination of longitudinal sections in rigor (Fig. 5D
and E) document that crossbridges bind to β-cytoplasmic actin
thin filaments, forming ~45° angled chevrons, which are
comparable to those found in wild-type IFM (Reedy and Reedy,
1985). Moreover, the polarity of the chevrons is uniform along
the entire length of a particular thin filament (Fig. 5D).
However, along an adjacent thin filament (arrowheads in Fig.
5E), chevrons may be oriented in the opposite direction. This
finding indicates that the laterally aligned thin filaments do not
have uniform polarity, possibly because they are not correctly
anchored in Z-discs. Consistent with this notion, the myofibrils
of β-cyto transformants display amorphous, dense ‘Z-bodies’
(zb, Fig. 5A,B and C) instead of Z-discs. In some areas, Zbodies are not integrated into myofibrils and occur as isolated
dense bodies with tangles of short thin filaments emerging from
them (medium size arrows in Fig. 5A and B). Some other Zbodies are narrow and extend axially over two or three hundred
nanometers (Fig. 5C), while other, less extended Z-bodies, are
so well aligned laterally that they appear to be a Z-disc (zb, Fig.
5A). However, unlike with a normal Z-disc, only a few thin
filaments appear achored into each Z-body, which is separated
from the neighboring one by a gap (Fig. 5C, arrowheads). In a
few cases, axially repeated Z-disc-like structures delineate a
rudimentary sarcomere (Fig. 5A and B; delineated by two large
arrows in Fig. 5A). Measurements of rudimentary sarcomeres
reveal their length to be most often ~2 µm, although examples
as short as 1.6 µm could also be found. These significantly
shorter sarcomere-like structures reflect the shorter lengths of
the thin and thick filaments.
Cross-sections (Fig. 6) reveal that β-cyto myofibrils have no
regular boundary. Central areas exhibiting hexagonal packing
of thin and thick filaments are seen (marked ‘A’ in Fig. 6A),
surrounded by a disorganized packing of thin and thick
filaments. Areas with ordered β-cyto myofibrils exhibit thin and
thick filaments that are laterally well aligned, in which case the
hollow profiles of the thick filaments in the A-band (marked ‘A’
in Fig. 6B) can be distinguished from the solid profiles that
characterize the bare zone of the thick filament (marked ‘M’ in
Fig. 6B). This observation indicates that the apparent lack of an
M-line is not due to misassembly of the midregion of the thick
filaments, and supports the idea that filament misregister is
responsible for the lack of a distinct M-line. Dense Z-body
formations of various sizes appear in scattered areas of the βcyto IFM (‘Zb’ in Fig. 6A), consistent with the longitudinal
views.
At the myotendon junction (MTJ), myofibrils are attached
to the cuticle via epithelial tendon cells. Substitution of
Act88F actin for β-cytoplasmic actin has a strong impact on
this junction. In wild-type flies, each myofibril is anchored to
the muscle cell membrane by a modified terminal Z-disc (MTZ) (double-headed arrow in Fig. 7A), which is extended into
a meshwork of actin filaments and dense material. A dense
zigzag junction (‘*’ in Fig. 7A) is formed between the muscle
membrane at the MT-Z and the apposed membrane of the
epithelial tendon cell. Microtubule bundles (arrowheads in
Fig. 7) extend from the tendon cell membrane opposite the
MT-Z to the tendon cell membrane apposed to the cuticle,
where dense tonofibrils (t) penetrate the cuticle and anchor the
Fig. 6. Cross-sections of β-cyto transformant IFMs. (A) Myofibrils of β-cyto transformants are irregular in size and shape. Even the best
ordered areas, such as the extensive region of hexagonal overlap seen in one A-band (A), are surrounded by out-of-register filaments. (Zb), Zbodies; scale bar, 500 nm. (B) Region of hexagonal overlap; in A, thick filaments are hollow, whereas the narrow, irregularly shaped region ‘M’
displays solid thick filament profiles that characterize the bare zone of the thick filament. Bar, 100 nm.
Ectopic expression of β-cytoplasmic actin 3635
Fig. 7. Longitudinal sections through the myotendon junction (MTJ) of wild type,
KM88, and the β-cyto transformant IFM. (A) At the wild-type MTJ, each myofibril is
linked to the cuticle (C) via an epithelial tendon cell. Myofibrils (mf) maintain
sarcomere organization right up to the modified terminal Z-disc, a dense meshwork of
actin filaments linking each myofibril to the muscle membrane (large double-headed
arrow). The muscle membrane forms the characteristic dense zig-zag junction
(asterisks) with the tendon cell membrane. Bundles of microtubules (arrowheads) link
to tonofibrils (t) that extend into the cuticle (C). (B) At the MTJ of the β-cyto
transformant, myofibrils (mf) terminate near the muscle membrane (arrows) and no
modified terminal Z-disc is formed. However, dense junctional plaques between the
membranes of the muscle and tendon cells are formed (asterisks), although not
necessarily opposite the terminus of a myofibril (longest arrow, bottom left). The
microtubule bundles (arrowheads) are fairly dense in this example and link to the
tonofibrils (t) which connect to the cuticle (C). (C) In KM88, irregular myofibrillar
bundles (mf) simply terminate near the muscle membrane and no modified terminal Zdisc is formed (vacant location indicated by double headed arrow). The membranes of
the muscle (upper asterisk) and tendon cell (lower asterisk) are widely separated. In
this example, the microtubule bundles in the tendon cell, and a junction between tendon
cells, are quite dense, and at low magnification, may give the misimpression that the
dense membrane junction is present. The tonofibrils (t) connect to the cuticle (C) in the
normal manner. Bars, 1 µm.
muscle in it (see Reedy and Beall, 1993, for a more complete
description).
In the β-cyto transformants (Fig. 7B), MT-Zs are absent,
leaving the myofibrils unconnected to the tendon cell and thus,
to the cuticle. However, the transformants exhibit dense
junctions between the membranes of the muscle and tendon
cells (arrows in Fig. 7B), and the tendon cell morphology is
nearly normal. This is an important detail, because in the
Act88F actin null IFM (Fig. 7C), both the MT-Z and the dense
3636 V. Brault and others
junctions between the tendon and muscle cell membranes are
missing (double-headed arrow in Fig. 7C). The membrane
surrounding the muscle cells in the Act88F actin null strain
balloons and blebs, thereby creating vacuoles between the
muscle and tendon cells (‘*’ in Fig. 7C), whereas the tendon
cell morphology remains almost normal.
DISCUSSION
Sequences that influence the accumulation of actin
in the IFM
Our Act88F control transformants establish which sequences
of the Act88F gene affect actin protein accumulation in the
IFM. These experiments demonstrate that the Act88F 5′UTR
including its intronic sequence is essential for the accumulation
of wild-type amounts of actin protein in the IFM. In our initial
pW8(5′Act88F)β-cyto transformants, expression of βcytoplasmic actin cDNA was regulated by the 5′ Act88F
promoter region because this region has been reported to
contain the sequences required for wild-type mRNA
accumulation in the IFM (Geyer and Fyrberg, 1986). However,
compared to the amounts of Act88F actin in the IFM of wildtype flies and pW8Act88F control transformants, the
pW8(5′Act88F)β-cyto transformants accumulated very little βcytoplasmic actin, and their IFM ultrastructure resembled that
of the KM88 null mutant.
Recently, Fyrberg et al. (1998) suggested that both the
intronic sequence in the 5′UTR and the intron within codon
307 of the protein-coding region (Fig. 1A) of the Act88F gene
were required for orderly temporal and spatial actin
expression. They based their conclusion primarily on
transformants expressing a chimeric actin gene, which after
the first 84 Act88F-specific amino acids encodes human αactin and thus lacks the downstream intron interrupting the
coding region in theAct88F gene. Since our corresponding
pW8(StuI)β-cyto(EcoRI) transformants also did not include
the downstream intron and still showed accumulation of wildtype amounts of β-cytoplasmic actin, we conclude that the
deficient expression might be related to the α-actin isoform
rather than the absence of the intron residing within the coding
region of Act88F.
By flanking the β-cytoplasmic actin cDNA with the Act88F
UTRs in the pW8(StuI)β-cyto(EcoRI) construct, we achieved
accumulation of wild-type levels of heterologous actin in the
IFM of β-cyto transformants. There is evidence that the amount
of Act88F actin plays a critical role in the proper structure and
function of the IFM (Mahaffey et al., 1985). Although
immunoblot analysis with an anti-actin antibody that does not
distinguish between Act88F and β-cytoplasmic actin suggests
that the latter accumulates at wild-type level, we cannot
exclude that there are minor differences which imbalance the
critical stoichiometry of thin and thick filaments (Beall et al.,
1989). However, if any minor reduction in the amount of actin
occurs, it has not impeded the ability of the β-cytoplasmic actin
to polymerize and form thin filaments in the IFM of the β-cyto
transformants.
Isoform-specific amino acid differences
The amino acid differences between IFM-specific Act88F
actin, human β-cytoplasmic actin, and rabbit skeletal muscle
actin are listed in Fig. 8A. N-terminally processed Act88F
differs by 15 amino acids from mammalian β-cytoplasmic
actin which, in turn, differs by 25 amino acids from
mammalian skeletal muscle actin. The majority of the
replacements are conservative. Residues 232, 278, and 368
(boxed in Fig. 8A) of Act88F, which belong to the small
group of muscle-specific residues in Drosophila (bold in Fig.
8A; Mounier et al., 1992), are replaced by polar amino acids
in β-cytoplasmic actin. To visualize the approximate position
of the differences with respect to the intersubunit contacts
and the surface of the F-actin filament, we have mapped the
residues that are different in β-cytoplasmic actin onto the
ribbon of a trimer representing Act88F (Fig. 8B). Based on
this stereo representation, differences in residues 325 and 169
possibly affect the intersubunit contacts along the two longpitch helical strands. The most likely candidates involved in
intersubunit contacts between the two long-pitch helical
strands appear to be residues 169 and 76. Slight changes in
geometry and/or conformation of the filament due to
isoform-specific differences could in turn interfere with the
proper interaction of F-actin with actin-binding proteins.
Amino acids that are exposed on the surface of the filament
according to the Holmes-Lorenz model of the F-actin
filament (Holmes et al., 1990; Lorenz et al., 1993) are
indicated in orange in Fig. 8. These residues, i.e. also
residues 232 and 368, are more likely to be involved in
interactions with actin-binding proteins. For example, it has
been proposed that the α-helix extending from residue
Asp222 to Ser233 could be part of a contact site between
actin and tropomyosin (Kabsch and Vandekerckhove, 1992),
and that Asn225 which is specific for muscle actins in
mammals directly interacts with tropomyosin (Mounier and
Sparrow, 1997). However, both Act88F and β-cytoplasmic
actin have Gln, which has a slightly longer side chain, at
position 225 and yet they interact with tropomyosin. In their
recent publication, Fyrberg et al. (1998) state that exchanging
singly residues 232, 278, or 368 of Act88F with the residue
corresponding to a Drosophila cytoplasmic actin had no
effect on IFM structure and function. This suggests that these
muscle-specific residues do not confer a unique function to
their respective isoforms.
It is noteworthy that the N terminus where several isoformspecific residues are clustered and which protrudes from the
filament surface at the largest radius, is directly involved in
myosin binding (Rayment et al., 1993). However, the different
amino acids at the N terminus of β-cytoplasmic actin did not
prevent rigor crossbridge formation of IFM myosin with thin
filaments built of β-cytoplasmic actin (Fig. 5D and E).
In those three positions (i.e. 76, 153, and 279) where the
amino acids in Act88F actin are identical to the mammalian
muscle-specific residues, the replacements in β-cyto
transformants are rather conservative: IrV; LrM; and YrF.
None of these residues have been directly implicated in
interactions with actin-binding proteins, albeit their location
on the surface of the F-actin filament would favor such
interactions. Interestingly, the substitution of Ile at position
76 with Val, which is a residue of similar stereochemistry,
produced mild perturbations in the myofibrillar structure of
transformants (Fyrberg et al., 1998), in particular, the Z-disc
morphology is affected (M. Reedy, unpublished observation).
As discussed above, this effect could possibly be mediated
Ectopic expression of β-cytoplasmic actin 3637
by modifications of intersubunit contacts in the F-actin
filament.
Although comparison of the primary sequence does not
reveal an obvious basis for the structural and functional
differences between the IFM expressing either Act88F or βcytoplasmic actin, one has to keep in mind that the respective
actin conformation in vivo may vary from the one predicted by
the atomic structure which has been derived from actin
complexed with actin monomer-binding proteins (Kabsch et
al., 1990; McLaughlin et al., 1993; Schutt et al., 1993).
Moreover, not much is known about how isoform differences
affect F-actin filament geometry which, in turn, appears to be
an important parameter in regulating the interactions of actin
with actin-binding proteins (McGough et al., 1997).
Structural and functional properties of a nonmuscle
actin in a muscle environment
The detection of arthrin in the β-cyto transformants documents
that ubiquitination is not restricted to IFM-specific Act88F.
Consistently, ubiquitinated actin was detected in transformants
expressing chimeric Act88F transgenes (Fyrberg et al., 1998).
Our findings also agree with other evidence that ubiquitination
and filament formation are closely correlated: (i) ubiquitination
lags several hours behind Act88F expression, paralleling
myofibril formation (Ball et al., 1987), and (ii) in transgenic
flies that express tagged variants of Act88F actin,
ubiquitination of tagged actin only occurred in transformants
that were able to form thin filaments (Brault et al., 1999).
However, arthrin was not detected in transformants expressing
Fig. 8. Amino acid
replacements between different
actin isoforms. In A, the
position of a given amino acid
is indicated by its residue
number. White numbers
indicate that we predict the
residue at this position to be
presumably exposed on the
filament surface based on the
Holmes-Lorenz F-actin
filament model (Holmes et
al.,1990; Lorenz et al., 1993).
A shaded box indicates a
partial surface exposure,
whereas black numbers are
positions of predominantly
internal residues. Insect
muscle-specific residues are
given in bold. Asterisks
identify mammalian musclespecific residues. The arrows
point to amino acids that differ
between Act88F and human βcytoplasmic actin. Orange
indicates that we predict
surface exposure for βcytoplasmic specific residues,
yellow indicates β-cytoplasmic
specific substitutions that are
likely to be buried in the
filament. The isoform-specific
replacement that had an effect
on IFM ultrastructure (Reedy et
al., 1991, Fyrberg et al., 1998)
is marked by an arrowhead.
(B) Stereo image of a ribbon
representation of an F-actin
trimer (Lorenz et al., 1993),
aligned and transparently
overlaid with an averaged and
refined 3-D helical
reconstruction of a rabbit
skeletal muscle F-actin filament
(Steinmetz et al., 1997). The 3-D reconstruction is surface-rendered to include 100% of the nominal molecular volume. The Act88F ribbon
representation of two neighbouring monomers along one long-pitch helical strand are shown in blue, whereas the ribbon representation of a
monomer on the opposite strand is depicted in turquoise. Residues that are different in β-cytoplasmic actin are indicated in orange if they
appear to be exposed on the filament surface. Residues that are presumably at least partially buried are indicated in yellow. The molecular
modeling work (courtesy of D. Stoffler, M.E. Müller Institute) was performed using Insight II (Biosym/Molecular Dynamics, Inc. San Diego).
3638 V. Brault and others
a chimeric Act88F-α-actin (Fyrberg et al., 1998), suggesting
that not all isoforms are invariably ubiquitinated.
Despite the competence to assemble β-cytoplasmic actin
containing thin filaments, with which the endogenous myosin
thick filaments can interact via typical rigor crossbridge
formation, there are only rudimentary indications of
sarcomeric organization in the IFMs of β-cyto transformants.
For one, they do not assemble normal Z-discs. Aberrant Zdiscs have been reported for a number of Drosophila
myofibrillar protein mutants (Reedy et al., 1989; Fyrberg et al.,
1990; Sparrow et al., 1991b, 1992; Miller et al., 1993). For
example, transheterozygous tropomyosin mutants contained
sarcomeres with multiple, tandemly arrayed Z-discs that often
did not span the entire width of the fibril (Kreuz et al., 1996).
A transformant with Drosophila Act42A-specific replacements
in the Act88F gene was reported by Fyrberg and co-workers
(1998) which, although not examined in detail, appears to have
disrupted Z-discs.
Because of the absence of proper thin filament anchoring
in Z-discs, actomyosin interactions may move thin and thick
filaments out of register. The lack of registration, in turn, may
prevent interactions necessary for forming mature Z-discs
and M-lines. Moreover, the mis-register or absence of regular
Z-discs may also affect thin and thick filament length
regulation, as suggested by the shorter filament lengths
inferred from the sarcomere-like structures in β-cyto
transformants versus the ~3.2 µm sarcomere length in wildtype flies. Most of the rudimentary sarcomere repeats in the
β-cyto transformants indicate a sarcomere length of ~2 µm or
less. This means that not only the thin filaments but also the
thick filaments are significantly shorter compared to wildtype myofilaments. In flight muscle assembly during
pupation, thick and thin filament lengths are closely coregulated: thin filaments grow from ~1 µm to 1.5 µm whereas
thick filaments start at 1.6 µm and grow to 3 µm. Thin and
thick filaments add the same amount of length simultaneously
at each stage of sarcomere assembly (Reedy and Beall, 1993).
The EMs do not clearly show the ends of actin filaments at
mid-sarcomere in the IFM of β-cyto transformants, so they
may be too long (~2 µm) or too short (1 µm) or of variable
length. An alternative explanation for the shorter filaments in
the β-cyto transformants is that insufficient amounts of actin
during pupation cause thin filaments to become shorter,
thereby secondarily affecting thick filament length. Slight
imbalances in the relative amounts of actin and myosin
caused both thin and thick filaments to assemble to shorter
lengths (Beall et al., 1989). However, quantitative analysis of
northern blots from mRNA from late pupae indicated that the
levels of respective mRNA in β-cyto transformants and wildtype were comparable (data not shown), supporting our
observation that the amount of actin protein present in the
IFM was also adequate.
Another explanation for aberrant Z-discs and filament
lengths is that β-cytoplasmic actin cannot interact properly
with α-actinin and/or actin capping or severing proteins during
sarcomere assembly (for review see Littlefield and Fowler,
1998), so length regulation of thin and thick filaments is
disturbed. Consistent with this notion, more extreme
tropomoyosin mutants also display shorter sarcomere lengths
(Kreuz et al., 1996). Conversely, it is possible that βcytoplasmic actin cannot appropriately interact with the
resident tropomyosin and/or troponin, thereby giving rise to
deficient sarcomeres.
Taken together, these results establish that β-cytoplasmic
actin expressed at wild-type levels partially substitutes for
muscle-specific Act88F in the IFM and that intrinsic
parameters of actin function (e.g. polymerization and
interaction with myosin) are retained in β-cytoplasmic actin,
even when it is expressed in the IFM environment. However,
the failure of β-cytoplasmic actin to fully substitute for the
endogenous Act88F isoform in the IFM exemplifies the
functional diversity of actin isoforms. We suggest that the
functional variation primarily depends on modulation of the
interaction of each isoform with particular actin-binding
proteins.
We are indebted to the members of Professor Walter J. Gehring’s
laboratory (Biozentrum, Basel) for the use of their equipment and for
their advice on all work involving flies. The cDNA for human βcytoplasmic actin was kindly provided by Prof. P. Gunning (The New
Children’s Hospital, University of Sydney). We thank Dr D. Stoffler
(M.E. Müller Institute, University of Basel) for preparing Fig. 8B.
This work was supported by the Swiss National Science Foundation,
the Canton Basel-Stadt, the M.E. Müller Foundation of Switzerland,
and an NIH grant to M.K.R.
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