Unravelling the structure of striated muscle M-band by electron tomography
Author(s)
Salim, Oday
Type
Thesis
Abstract
The sarcomeric M-band in vertebrate striated muscle is thought to maintain the
uniform hexagonal lattice of the thick filaments by crosslinking neighbouring thick filaments
in the middle of the A-band. It is also thought to maintain the fine axial alignment of the
thick filaments within the A-band. The M-band has been implicated with fundamental
signalling complexes that control sarcomeric protein turnover during development. There
are two classes of M-band: 3-line M-band, which has a prominent central M1 stripe; and 2-
line M-band that lacks the central M1 stripe. The aim of this study is to examine the
ultrastructure of the different M-band types using electron microscopy and tomography.
The 3-line M-band was examined thoroughly using electron microscopy and
tomography of longitudinal and transverse sections. Profile plot analysis of longitudinal
section micrographs and tomograms showed that the peaks of the M-band were found to
be associated with protrusions on the thick filaments, rather than the M-bridges. Thus the
M-bridges only contribute to the background density of the profile (envelope function)
rather than the peaks. Analysis of transverse section tomograms unravelled new structural
details of the M-band, which allowed us to model the possible pathways of M-band
proteins. Projection of our model into longitudinal section view shows that the M-band
appearance is a projection effect of the underlying 3D structure. Examination of the thick
filament backbone structure showed that it is rotating at the bare region and the M-band,
which suggested that the thick filament may have torsional flexibility.
The path of titin in the A-band is thought to run along the thick filament in the
crossbridge region, but it is not known whether titin is dislodged from the thick filament at
the M-region or not. We find evidence (from the 3-line M-band specimen) that titin could
be dislodged from the thick filament at the periphery of the M-region. We speculate that
titin may exist as a doublet along the crossbridge segment and splits into single filaments
after dislodging at the M-region.
For the 2-line M-band study, freshly dissected bony fish heart muscle was prepared
for plastic embedding. In longitudinal sections, the M-band did not show clear stripes but
the average profile plot showed clear M-band peaks that lack the central M1 peak.
Transverse sections of the M-band did not show clear hexagonal network of M-bridges. In
addition, thick filaments orientations and the thick filament lattice were less ordered than in
the 3-line M-band muscle. Modelling a thick filament from the raw tomograms shows a
backbone structure similar to the 3-line M-band; a novel M-band model was produced,
where the M-bridges originating at M4 (and M4’) do not cross the central M1 line but are
oriented outwards towards the bare region. We hypothesise that M-protein is responsible
for the crystalline appearance of the M-band and the thick filaments in 3-line M-bands.
The work carried out in this PhD investigation will help our understanding of the
structural role of the M-band in different muscles. It gives the most detailed view to date of
the M-band in fast, slow and cardiac muscle. It will help our understanding of the structural
changes in the M-band during activation that may initiate signalling events.
uniform hexagonal lattice of the thick filaments by crosslinking neighbouring thick filaments
in the middle of the A-band. It is also thought to maintain the fine axial alignment of the
thick filaments within the A-band. The M-band has been implicated with fundamental
signalling complexes that control sarcomeric protein turnover during development. There
are two classes of M-band: 3-line M-band, which has a prominent central M1 stripe; and 2-
line M-band that lacks the central M1 stripe. The aim of this study is to examine the
ultrastructure of the different M-band types using electron microscopy and tomography.
The 3-line M-band was examined thoroughly using electron microscopy and
tomography of longitudinal and transverse sections. Profile plot analysis of longitudinal
section micrographs and tomograms showed that the peaks of the M-band were found to
be associated with protrusions on the thick filaments, rather than the M-bridges. Thus the
M-bridges only contribute to the background density of the profile (envelope function)
rather than the peaks. Analysis of transverse section tomograms unravelled new structural
details of the M-band, which allowed us to model the possible pathways of M-band
proteins. Projection of our model into longitudinal section view shows that the M-band
appearance is a projection effect of the underlying 3D structure. Examination of the thick
filament backbone structure showed that it is rotating at the bare region and the M-band,
which suggested that the thick filament may have torsional flexibility.
The path of titin in the A-band is thought to run along the thick filament in the
crossbridge region, but it is not known whether titin is dislodged from the thick filament at
the M-region or not. We find evidence (from the 3-line M-band specimen) that titin could
be dislodged from the thick filament at the periphery of the M-region. We speculate that
titin may exist as a doublet along the crossbridge segment and splits into single filaments
after dislodging at the M-region.
For the 2-line M-band study, freshly dissected bony fish heart muscle was prepared
for plastic embedding. In longitudinal sections, the M-band did not show clear stripes but
the average profile plot showed clear M-band peaks that lack the central M1 peak.
Transverse sections of the M-band did not show clear hexagonal network of M-bridges. In
addition, thick filaments orientations and the thick filament lattice were less ordered than in
the 3-line M-band muscle. Modelling a thick filament from the raw tomograms shows a
backbone structure similar to the 3-line M-band; a novel M-band model was produced,
where the M-bridges originating at M4 (and M4’) do not cross the central M1 line but are
oriented outwards towards the bare region. We hypothesise that M-protein is responsible
for the crystalline appearance of the M-band and the thick filaments in 3-line M-bands.
The work carried out in this PhD investigation will help our understanding of the
structural role of the M-band in different muscles. It gives the most detailed view to date of
the M-band in fast, slow and cardiac muscle. It will help our understanding of the structural
changes in the M-band during activation that may initiate signalling events.
Date Issued
2011-09
Date Awarded
2012-03
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Luther, Pradeep
Nagase, Hideaki
Sponsor
Kennedy Institute of Rheumatology
Publisher Department
Medicine: Kennedy Institute of Rheumatology
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)