Strain-sensitivity of muscle fibre cross-bridges investigated by flourescence life-time imaging microscopy

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

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Technical Summary

We will take advantage of a novel methodological approach, Fluorescence Life-Time Imaging
Microscopy (FLIM), to investigate enzyme kinetics in organised cellular systems. This is probably the first application of this type, and the spatial averaging opportunities provided by the use of striated muscle cells should allow unparalleled spatial and time resolution. The project will pioneer the use of time-resolved FLIM in biomedical research. We aim to demonstrate that muscle force modifies directly the behaviour of the enzymatic core of molecular motors, thus explaining how muscle strain affects the catalytic activity of muscle proteins to maintain their optimal performance and efficiency. The work is important because it will enable better understanding of the fundamental aspects of energy conversion in muscle, and will demonstrate the interaction between externally applied forces and the behaviour of an enzyme’s catalytic site.
We shall draw on FLIM expertise from Physics at Imperial and fluorophore synthesis from NIMR.
Fluorescence life-time (FL) provides information about the environment of a fluorophore in a cell,
with the advantage, over imaging of fluorescence intensity, of independence from fluorophore
concentration. Preliminary results using fluorescently labelled analogues of ATP in isolated muscle fibres are exciting. With DEACpATP (3’-O-{N-[3-(7-Diethyl-aminocoumarin-3-carboxamido)-
propyl]carbamoyl}ATP), we have shown: 1. that FL of DEACpATP bound at the active site is
longer in the actin-myosin overlap region of the A-band compared to that in the non-overlap
region, suggesting that FL of actomyosin-DEACpADP (AM-DEACpADP) is different from that of
myosin-DEACpADP; 2. that the spatial distribution of FL changes with sarcomere length,
consistent with the change in overlap; 3. that multi-exponential fitting of the fluorescence decay
allow the measurement of the proportion of myosin cross-bridges bound or not to actin; 4. that
averaging of signals from many sarcomeres, and across the width of the muscle fibres greatly
improve the signal quality, spatial resolution and likely to improve time resolution.
The project consists in: 1. developing the above technology to detect strain-induced changes in
the FL of AM-DEACpADP. FL images will be acquired in phase with many cycles of length
oscillations to accumulate photons, reduce noise and improve FL measurements. The experiments will detect strain dependence of AM-DEACpADP cross-bridges; 2. characterising FL measurements of fluorescently labelled myosin light chains (LC1) introduced in the muscle fibres. This has the advantage of allowing experiments in fully contracting fibres at physiological concentrations of ATP. Experiments will determine FL changes upon strain changes in contracting muscle fibres.
 
Description BBSRC Project Grant (Muscle cross-bridge mechanism investigated by fluorescence lifetime imaging microscopy of myosin essential light chain)
Amount £544,441 (GBP)
Funding ID BB/E021573/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2007 
End 06/2010
 
Description BBSRC Project Grant (Novel approaches to elucidate the molecular basis of muscle contraction: FRET-FLIM imaging applied to single muscle fibres)
Amount £371,000 (GBP)
Funding ID BB/I019448/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2011 
End 07/2014
 
Description BHF Project grant: Molecular Mechanisms of contractile dysfunction in cardiac muscle hypertrophy and failure
Amount £636,000 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2008 
End 08/2011
 
Description Newton International Fellowship
Amount £98,000 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2009 
End 01/2011
 
Description Wellcome Trust-NIH studentship
Amount £90,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2010 
End 09/2014