Molecular Genetic Analysis of Muscle Gene and
Protein Function During Drosophila Development
We use the fruit fly, Drosophila melanogaster,
to explore gene expression and protein function in muscle cells. Defects
in contractile proteins cause human cardiac and skeletal muscle disease.
Therefore, it is useful to pursue an understanding of contractile protein
function in an organism that is readily manipulated by genetic and transgenic
means. Our integrative approach allows us to study muscle from the molecular
level (using biochemical and biophysical assays), through the cellular
level (using microscopy and fiber mechanical assays) to the level of whole
organism muscle function.
The molecular motor of muscle is myosin heavy chain,
a protein that interacts with actin to cause muscle contraction. We determined
that Drosophila possesses a single myosin heavy chain gene, and
that multiple forms of the myosin protein are produced by the process of
alternative RNA splicing. Specific regions of the myosin RNA are included
in specific muscle types to yield myosin protein isoforms. The regions
that differ among the isoforms must be critical for the myosin-mediated
ultrastructural and physiological differences among muscle types. In collaboration
with Dr. Ron Milligan (the Scripps Research Institute), we determined the
location of the isoform differences on the three dimensional map of the
myosin molecule. Using these data, we developed hypotheses regarding how
these differences can affect myosin function.
Tissue-specific accumulation
of alternative forms of myosin heavy chain RNA in Drosophila embryos (from
Zhang and Bernstein, 2001)
Stereo image of the myosin head with
locations of domains encoded by alternative exons highlighted (from Kronert
et al., 1999)
To experimentally test our hypotheses on myosin isoform
function, we developed transgenic strains of Drosophila that express
the wrong isoforms in particular muscle types. As an initial approach,
we showed that the normal myosin gene can rescue muscle defects associated
with myosin null mutations. Interestingly, additional copies of the myosin
gene result in defective muscles as well, due to an overabundance of myosin-containing
thick filaments. We next succeeded in expressing the embryonic form of
myosin in adult flight muscle. To our surprise, the adult muscle assembles
normally using the embryonic protein, indicating that structural properties
of the muscle are not affected by the isoform of myosin present. However,
the flight muscles do not function when the embryonic myosin substitutes
for the normal form, indicating that myosin isoforms are functionally different.
As a result of our efforts, we created a fly line
that expresses a single myosin isoform in all its muscle types. We have
produced additional lines that express other myosin isoforms. Currently,
we are isolating single isoforms of the myosin protein from these lines
to determine the biochemical and biophysical properties that are encoded
by particular variable regions of the protein, e.g. actin binding
ability, ATPase kinetics, in vitro thin filament motility, in
vitro force generation. Transient kinetic studies are performed in
collaboration with Dr. Michael Geeves (University of Kent at Canterbury)
while single myosin molecule assays are performed in collaboration with
Dr. Justin Molloy and Dr. John Sparrow (University of York). Muscles with
altered myosin isoforms are studied in collaboration with Dr. David Maughan
(University of Vermont) to assess how mechanical properties are affected
by the alterations. Overall our approach should lead to a complete understanding
of the in vitro and in vivo properties imparted by particular
regions of the myosin molecule
We are pursuing similar functional studies on alternative
forms of the thick filament protein paramyosin. We discovered that the
gene encoding this alpha-helical protein also produces a novel muscle protein,
miniparamyosin. By mutational analysis, isoform substitution, biochemical
studies and the use of the yeast two-hybrid interaction assay, we intend
to define the role of paramyosin and miniparamyosin in determining muscle
structure and function.
We are also studying Drosophila
UNC-45, a molecular chaperone/co-chaperone that aids
in folding of muscle myosin heavy chain and possibly
other muscle or non-muscle proteins. We are pursuing
a detailed structure/function analysis of this protein
in vivo and in vitro as well determining mechanisms
whereby defects in protein folding can be ameliorated
via genetic and transgenic suppression. Our research
will lead to insights as to how mutations that result
in production of abnormally folded contractile proteins
cause phenotypic defects and how these may be ameliorated.
Since several neuromuscular diseases arise from aberrant
protein folding and accumulation of misfolded protein
aggregates, our work will contribute to understanding
the disease process and may yield insight into therapeutic
modalities.
We are studying elements responsible for tissue-specific
transcription of contractile protein genes during
muscle development. We attach a putative transcriptional
promoter region to a "reporter gene" that is then
returned to the Drosophila genome. The transgenic
lines readily indicate the location of trangene expression
upon histochemical treatment. We then produce deletions
of the suspected transcriptional elements in the construct
and make additional transgenic lines to find which
muscle-specific transcriptional elements we have deleted.
We are using both genetic and biochemical approaches
to discover the trans-acting factors that activate
contractile protein genes by binding to the transcriptional
elements. We are also interested in the regulation
of post-transcriptional contractile protein gene expression,
i.e. the elements governing alternative RNA
splicing. Using an in vitro splicing system,
production of transgenic lines containing in vitro
manipulated genes, as well as Drosophila genetics,
we are identifying cis-acting signals and trans-acting
factors responsible for the tissue-specific regulation
of RNA splicing. Our studies on the molecular biology
of contractile protein genes yield insight into how
gene expression is regulated during organismal development.
Representative Publications
Hodges, D., R.M. Cripps, M. O'Connor, and S.I. Bernstein.
The role of evolutionarily-conserved sequences in alternative splicing
at the 3' end of Drosophila melanogaster myosin heavy chain RNA.
Genetics 151: 263-276 (1999).
Kronert, W.A., A. Acebes, A. Ferrús and S.I.
Bernstein. Specific myosin heavy chain mutations suppress troponin I defects
in Drosophila muscles. J. Cell Biol. 144: 989-1000 (1999).
Cripps, R.M., J.A. Suggs and S.I. Bernstein. Assembly
of thick filaments and myofibrils occurs in the absence of the myosin head.
EMBO J. 18: 1793-1804 (1999).
Swank, D.M., L. Wells, W.A. Kronert, G.E. Morrill
and S.I. Bernstein. (2000) Determining structure/function relationships
for sarcomeric myosin heavy chain by genetic and transgenic manipulation
of Drosophila. Microsc. Res. Tech. (special issue: The Biology of Myosin).
50: 430-442.
Zhang, S. and S.I. Bernstein. (2001). Spatially
and temporally regulated expression of myosin heavy chain alternative exons
during embryogenesis of Drosophila. Mech. Dev. 101: 35-39.
Arredondo, J.J., R.M. Ferreres, M. Maroto, R.M. Cripps,
R. Marco, S. I. Bernstein and M. Cervera. (2001) Control of
Drosophila paramyosin/miniparamyosin gene expression: differential regulatory
mechanisms for muscle-specific transcription. J. Biol. Chem. 276: 8278-8287.
Swank, D.M., M.L. Bartoo, A.F. Knowles, C. Iliffe,
S.I. Bernstein, J.E. Molloy and J.C. Sparrow. (2001) Alternative
exon-encoded regions of Drosophila myosin heavy chain modulate ATPase rates
and actin sliding velocity. J. Biol. Chem. 276: 15117-15124.
Arredondo, J. J., M. Mardahl-Dumesnil, R.M. Cripps,
M. Cervera and S.I. Bernstein. (2001) Overexpression of minipara-myosin
causes dysfunction and myofibril degeneration in the indirect flight muscles
of Drosophila melanogaster. J. Muscle Res. Cell Motil. 22: 287-299.
Swank, D. M., A. F. Knowles, J. A. Suggs, F. Sarsoza, A.
Lee, D. W. Maughan and S. I. Bernstein. (2002) The
myosin converter domain modulates muscle performance.
Nature Cell Biol. 4: 312-317.
Littlefield, K. P., D. M. Swank, B. M. Sanchez, A.
F. Knowles, D. M. Warshaw and S. I. Bernstein.
(2003) The converter domain modulates the kinetic
properties of Drosophila myosin. Am. J. Physiol.
Cell Physiol. 284: C1031-C1038.
Swank, D. M., A. F. Knowles, W. A. Kronert, J. A.
Suggs, G. Morrill, M. Nikkhoy, G. G. Manipon, and
S. I. Bernstein. (2003) Variable N-terminal regions
of muscle myosin heavy chain modulate ATPase rate
and actin sliding velocity. J. Biol. Chem. 278: 17475-17482.
Yu, Q. and S. I. Bernstein. (2003) UCS proteins:
managing the myosin motor. Curr. Biol. 13: R525-R527.
Miller, B.M., M. Nyitrai, S. I. Bernstein, and M.
A. Geeves. (2003) Kinetic analysis of Drosophila muscle
myosin isoforms suggests a novel mode of mechanochemical
coupling. J. Biol. Chem. 278: 50293-50300.
Swank, D.M., W.A. Kronert, S.I. Bernstein and D.W.
Maughan. (2004) Alternative N-terminal regions of
Drosophila myosin heavy chain tune cross-bridge kinetics
for optimal muscle power output. Biophys. J. 87: 1805-1814.
Hao, Y., S. I. Bernstein and G.H. Pollack. (2004)
Passive stiffness of Drosophila IFM myofibrils: A
novel, high accuracy measurement method. J. Muscle
Res. Cell Motil. 25: 359-366.
Liu, H., M. S. Miller, D. M. Swank, W. A. Kronert,
D. W. Maughan, and S. I. Bernstein. (2005) Paramyosin
phosphorylation site disruption affects indirect flight
muscle stiffness and power generation in Drosophila
melanogaster. Proc. Natl. Acad. Sci. U.S.A. 102:
10522-10527.
Miller, B. M., S. Zhang, J. A. Suggs, D. M. Swank,
K. P. Littlefield, A. F. Knowles and S.I. Bernstein.
(2005) An alternative domain near the nucleotide-binding
site of Drosophila muscle myosin affects
ATPase kinetics. J. Mol. Biol. 353: 14-25.
Swank, D. M., J. Braddock, W. Brown, H. Lesage, S.
I. Bernstein and D. W. Maughan (2006) An alternative
domain near the ATP binding pocket of Drosophila
myosin affects muscle fiber kinetics. Biophys. J.
90; 2427-2435.
Melkani, G. C., A. Cammarato and S. I. Bernstein
(2006) alphaB-Crystallin maintains skeletal muscle
myosin enzymatic activity and prevents its aggregation
under heat-shock stress. J. Mol. Biol. 358: 635-645.
Miller, B. M. and S. I. Bernstein. (2006) Myosin.
In Nature's Versatile Engine: Insect Flight
Muscle Inside and Out. (J. Vigoreaux, ed.). Landes
Biosciences, Georgetown TX. 62-75.
Hao, Y., M. S. Miller, D. M. Swank, H. Liu, S. I.
Bernstein, D. W. Maughan and G. H. Pollack (2006)
Passive stiffness in Drosophila indirect
flight muscle reduced by disrupting paramyosin phosphorylation,
but not by embryonic myosin S2 hinge substitution.
Biophys. J. 91: 4500-4506.
Hess, N. K., P. A. Singer, K. Trinh, M. Nikkhoy and
S. I. Bernstein (2007) Transcriptional regulation
of the Drosophila melanogaster muscle myosin
heavy-chain gene. Gene Expr. Patterns 7: 413-422.
Suggs, J. A., A. Cammarato, W. A. Kronert, M. Nikkhoy,
C. M. Dambacher, A. Megighian and S. I. Bernstein.
(2007) Alternative S2 hinge regions of the myosin
rod differentially affect muscle function, myofibril
dimensions and myosin tail length. J. Mol. Biol. 367:
1312-1329.
Miller, B. M., M. J., Bloemink, M. Nyitrai, S. I.
Bernstein and M. A. Geeves. (2007) A variable domain
near the ATP binding site in Drosophila muscle
myosin is part of the communication pathway between
the nucleotide and actin-binding sites. J. Mol. Biol.
368:1051-1066.
|