Muscle contraction is a highly fine-tuned process that requires the precise and
timely construction of large protein sub-assemblies to form sarcomeres, the
individual contractile units. Mutations in many of the genes encoding constituent
proteins of this macromolecular machine result in defective functioning of the
muscle tissue, and in humans, often lead to myopathic conditions like cardiomyopathies and muscular dystrophies, which affect a considerable number of people the world over. As more information regarding causative mutations becomes available, it becomes imperative to explore mechanisms of muscle
development, maintenance and pathology.
In striated muscles, contraction is regulated by the thin filament-specific
tropomyosin (Tm) – troponin (Tn) complex (Ca2+-binding troponin-C, inhibitory
troponin-I and tropomyosin-binding troponin-T). These troponin subunits are
present in 1:1:1 ratio on thin filaments, with 1 Tm-Tn complex present on every
7th actin molecule. This stoichiometry is tightly regulated, and disturbances have
been associated with functional defects. Each of these proteins has multiple
isoforms, whose expression is controlled both spatially and temporally. The expression of specific combination of isoforms confers specific contractile properties to each muscle subtype.
Drosophila melanogaster has been a preferred model of choice to study various
aspects of muscle development for decades. In this study, the Indirect Flight
Muscles (IFMs) of Drosophila have been used to investigate developmental and
functional roles of two temporally regulated isoforms of a vital structural and
regulatory component of the sarcomere – Troponin T (TnT). On a larger scale,
whole genome expression profiles of mutants that are null for major myofbrillar
proteins have also been discussed. IFMs serve as an excellent model system to
address these questions, owing to the extreme ease of genetic manipulability in
this system, and high degree of homology between mammalian and Dipteran
cytoskeletal proteins.
Chapter 1 covers basics of muscle biology, and the role of TnT in muscle contraction. Phenomena responsible for generating diversity in genes encoding muscle proteins – alternative splicing and isoform switching – have also been discussed. These mechanisms are highly conserved, as are patterns of TnT splicing and isoform expression across phyla. Mutations leading to altered splicing
patterns lead to myopathic conditions, and the importance of model systems to
study muscle biology has been emphasized. The advantages of studying Drosophila IFMs and a comprehensive overview of IFM development has been covered. The resources and experimental tools used have been described in
Chapter 2.
Two isoforms of TnT are alternatively spliced in the Drosophila thorax – one containing alternative exon 10a (expressed in adult IFMs and jump muscle); and one containing alternative exon 10b (expressed in pupae and newly eclosed flies).
These exons are spliced in a mutually exclusive manner, and defects in splicing
have been reported to cause uncontrolled, auto-destructive contractions. In Chapter 3, a splice mutant of TnT, up1, has been discussed, with respect to its developmental profile. Transgenic rescue experiments with two separate isoforms demonstrate rescue at the structural as well as functional level.
Transgenic over-expression, however, leads to functional abnormalities, highlighting the importance of stoichiometry in multi-protein complexes. In Chapter 4, molecular signals that bring about the developmentally regulated TnT isoform switch are discussed. A splicing factor, Muscleblind, has been
transgenically knocked down in normal and mutant IFMs to study effects on muscle function.
Chapter 5 looks at whole genome transcriptional alterations in muscles null for either actin or myosin. All significant expression changes have been classified into
categories based on different biological processes, and an attempt to differentiate
generic muscle responses from filament-specific responses has been made.
In conclusion, the studies have highlighted the importance of TnT isoform switching, and that extended expression of a pupal stage-specific isoform can partially compensate for loss of the adult isoform. Also, in the absence of major myofibrillar proteins, stress response pathways like heat shock response and protein degradation pathways are activated, along with a subset of metabolic
responses that are unique to the thin or thick filament systems.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.iisc.ernet.in:2005/3601 |
| Date | January 2015 |
| Creators | Madan, Aditi |
| Contributors | Nongthomba, Upendra |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Relation | G27158 |
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