The aim of the project is to understand, at the molecular level, how muscle thin filaments are regulated. Thin filament regulation by calcium and stretch will be studied in the flight muscle of Drosophila. The structure of the thin filament, the proteins and their interactions and the physiological properties of fibres and filaments will be investigated in wild type and mutant flies. The mechanisms controlling expression of troponin genes and possible functions of troponin isoforms will be investigated.
Isolated proteins. - Flight muscle will be isolated from Drosophila by density gradient centrifugation and proteins separated by column chromatography. When larger amounts of proteins are needed they will be isolated from Lethocerus.
Arthrin. - The lysine in actin to which ubiquitin is covalently bound will be determined. CNBr peptides of arthrin will be separated by HPLC and tested for reaction with anti-ubiquitin. Peptides containing ubiquitin will be sequenced. The function of arthrin will be determined by site directed mutagenesis of the actin gene to prevent ubiquitination.
Troponin H.- The function of TnH in stretch activation of insect
flight muscle will be investigated.The position of different parts of the C-
terminal half of the molecule relative to tropomyosin, TnT and TnC in the thin
filament will be determined. Epitopes will be mapped in the sequence by
screening a Drosophila cDNA library with existing monoclonal
antibodies. The position of epitopes in the thin filament and myofibril will
be determined by labelling with gold-conjugated antibody for electron
microscopy.
Contacts of TnH with thick filament proteins will be investigated.
Myofibrils will be chemically crosslinked and the proteins separated on gels.
Species containing TnH and the proteins to which TnH is linked, will be
identified with antibodies. If TnH binds directly to myosin, the position of
the binding site in the myosin molecule will be determined. Binding of TnH to
myosin S1 and myosin rod will be investigated by immunoblotting and by
sedimenting thin filaments in the presence of the myosin fragments with and
without ATP (to eliminate S1-actin interaction). If TnH contacts the thick
filament via another protein (e.g. flightin), the site of interaction of this
protein with myosin will be determined by immunoblotting with myosin
fragments.
The sites of calcium binding and phosphorylation in TnH will be
determined by peptide mapping and the stoichiometry and affinity of calcium
binding will be measured. The effect of these modifications on association of
TnH with thick filament proteins and on the regulatory function of the thin
filament will be investigated.
Troponin T.- The regulation of TnT by calcium binding and phosphorylation will be studied. Different muscle specific isoforms will be isolated and the stoichiometry and affinity of calcium binding measured. This study will be on the polypeptide isolated from muscles and on full length and truncated polypeptides synthesised in E.coli transformed with recombinant plasmid vectors carrying total and partial cDNA sequences. If native and recombinant TnTs have different properties, suggesting that post- translational modifications are involved in calcium binding, then the recombinant peptides will be synthesised in the insect baculovirus system. The sequence domains responsible for calcium binding will be identified using truncated expressed polypeptides and the chemical nature of any post- translational modification will be investigated by comparing full length polypeptides from the three sources. The sites of phosphorylation will be identified by phosphorylating in vivo and sequencing phosphorylated peptides; phosphatase inhibitors may be used.
Stoichiometry.- A model structure of the thin filament will be produced based on the position of the proteins and their stoichiometry. Ratios of in vivo radiolabelled methionone incorporated into proteins will be measured on 2- dimensional gels and stoichiometries estimated from the known amino acid sequences.
The function of isoforms of regulatory proteins and of domains within
the proteins will be investigated by standard techniques of molecular
genetics. The control of transcription will be investigated for TnT and TnI
initially and this may be extended to TnH and TnC.
The promotor region and other regulatory regions of the TnT gene will be
cloned by screening a genomic library with probes from the 5' exon of the TnT
gene. The promotor region will be sequenced and tested for in vivo
expression using reporter genes. Different constructs will be prepared using P
element vectors and injected into the germ line of Drosophila embryos.
The transcriptional regulation of TnT isoforms in different muscle types will
be determined. An attempt will be made to rescue the phenotypes of TnT mutants
in order to find the function of different domains of the protein. The
function of phosphorylation in TnT will be investigated by altering amino
acids in the phosphorylated sites in vitro. The in vivo
transformation assay will be used to test the effect of eliminating regulatory
phosphorylated sites in flies with different genetic backgrounds.
Functional interdependence between TnI and other muscle proteins will be
looked for by isolating suppressors of TnI mutations. Suppressor mutations
that have been isolated already are mutations in the loci of the myosin heavy
chain gene, actin 88F or tropomyosin genes. These mutant genes will be
sequenced in order to identify the amino acids involved in possible
interactions between TnI and other muscle proteins in vivo.
The mechanisms controlling the expression of the TnI gene will be
investigated. The gene produces ten isoforms and is close to another gene with
which it seems to share regulatory mechanisms, possibly through a
bidirectional promotor. The product of the gene adjacent to the TnI gene will
be identified and the common regulatory mechanisms will be elucidated. The
differential expression of a family of TnI exons will be studied by
identifying the RNA binding proteins that control the alternative splicing. To
that end, mutations that suppress a splicing defective mutation in the TnI
gene will be produced.
The function of TnH in flight muscle will be determined by deleting part
of the TnH molecule. Flies will be transformed with anti-sense constructs of
TnH specific exons and the effect on muscle structure and mechanics will be
found. In this experiment the TnH specific exons will be put under the control
of the flight muscle-specific Act88F promotor.
The sequence of a TnH isoform which has sequence in common with TnC will
be determined using existing cDNA clones. Domains of this protein will be
expressed in E.coli and possible calcium sensitive binding to thick
filament proteins will be studied by the methods to be used with native TnH.
The chromosomal location of the gene will be determined by in situ
hybridization.
The function of arthrin will be determined by site directed mutagenesis
of the cloned Act88F gene to remove the lysine to which ubiquitin is
conjugated. The gene will be replaced in the genome of a null strain for the
Act88F gene by P-element transfection. The effects on muscle structure
and mechanics will be found.
A model for the arrangement of proteins on the Drosophila thin
filament will be derived from information obtained in this study. The
structure of native thin filaments will be compared with that of filaments
reconstituted from isolated components. Filaments with mutant proteins will
also be examined. These will either be reconstituted using one or more mutant
proteins or they will be obtained from flies in which proteins are mutated, in
some cases by transfection (see above). Native thin filaments will be prepared
from a myosin nul mutant, Ifm(2)2 and some mutations produced by
transfection will be in the myosin nul flies.
Thin filaments will be embedded in amorphous ice and examined by low
dose electron microscopy. Ordered two dimensional arrays will be made in
lipid monolayers and the images will be analysed using image enhancement
techniques. Conditions under which TnH projections can be seen by cryo-
electron microscopy will be investigated. Thin filaments in the myofibrillar
lattice will be examined in thin cryo-sections of fibres. Dynamic studies of
actin-myosin interactions will be made using fast freezing techniques to study
the effect of mutant troponins on the structure of the actin-myosin S1
complex. Interaction of TnH with the rod fragment myosin will be tested by
attempting to decorate thin filaments with myosin rod.
The relative positions of troponin components and the position of
different parts of extended proteins, TnT amd TnH, will be determined by
labelling thin filaments with antibodies to epitopes of known sequence. Fab
fragments conjugated with 3nm gold will be used and binding patterns will be
analysed statistically to resolve epitopes separated by as little as 7nm.
Labelled filaments will be negatively stained. The shape of TnH will be
compared with that of tropomyosin using shadowing techniques. The overall
myofibrillar structure of troponin mutants will be examined in sectioned
fibres embedded in Lowicryl.
The effect of mutations in the troponin genes on the function of flight muscle fibres and isolated thin filaments will be studied.
Fibre mechanics. - Mechanics of skinned flight muscle fibres will be studied when the structure of the fibres in mutants appears to be normal by electron microscopy. Methods developed in York for mechanical measurements on Drosophila fibres will be used. The effects of stretch and calcium concentration on the ATPase activity of wild type and mutant Drosophila fibres will be measured.
Thin filament function. - Optical tweezers will be used to
measure the force produced by single thin filaments and single myosin
molecules. This method will be used for mutants in which filaments are not
assembled in a regular structure. Experiments will be done on native or
reconstituted filaments with troponin components that are altered or
omitted.
The in vitro motility assay will be used to test regulation of
the movement of thin filaments over immobilized myosin. The effect of calcium
on Vmax for the movement of filaments with mutant troponins will be
measured.
There are five laboratories in the network and each will contribute equally to the proposed study. The Co-ordinator (COO) and four Associated Contractors (AC) in different laboratories will each have a Post Doctoral student who will undertake a particular aspect of the work outlined in the Work Programme. It will be the responsibility of the COO and ACs to supervise the Post Doctoral students and to ensure that the work in the proposal is carried out. The Post Doctoral students will visit other laboratories in the network and it will be the responsibility of the host COO and ACs to train them in the techniques used in their laboratories. The COO will be responsible for the management of the project. She will organize meetings of the participants and will be the intermediary between the the ACs and the Commission.
Laboratories specialising in genetics, biochemistry, mechanics or structure will collaborate to study Drosophila thin filaments by all these techniques.