Determining Proximities of Sarcomeric Proteins in Living Skeletal Muscle Cells Using FRET Imaging

ABSTRACT

The technique of fluorescence resonance energy transfer (FRET) has been used in this study to determine the proximities (efficiencies, i.e., E%) of interacting proteins in sarcomeres in living cultured embryonic chicken skeletal muscle cells to draw up a FRETsome of a sarcomere in mature myofibrils. During this study, two known Z‐band proteins, FATZ (also called myozenin), and telethonin, were found to be actin binding proteins. The Efficiency of the F‐actin and telethonin pair was measured to be 15%, while the F‐actin and FATZ pair was 18%. The known F‐actin and alpha–actinin interactions resulted in an efficiency of 13%. The new actin binding properties of FATZ and telethonin were confirmed by F‐actin pull down experiments. By placing a donor probe (CeFP) on one end of the FATZ molecule and the acceptor probe (YFP) on the other end of FATZ, we were able to determine that the two termini moved apart from each other as the dense bodies of the nascent myofibrils fused with one another to form the Z‐bands of mature myofibrils. Three other proteins of the dense bodies, i.e., myotilin, alpha‐actinin and ArgBP2 also underwent similar termini separation changes as the dense bodies transformed into Z‐bands of mature myofibrils. The highest efficiencies were measured for MyBP‐C interactions with myosin heavy chains (35%), myosin light chains (35%), and F‐actin (31%). The establishment of a sarcomere FRETsome of these interactions in control sarcomeres in mature myofibrils in living transfected skeletal muscles establishes a foundation on which the introduction of individual mutated sarcomeric proteins involved in different muscle diseases will have on their interactions with control partner sarcomeric proteins in mature myofibrils.