The Structural and Biomechanical Properties of Insect Thick Filaments Expressing Flightin and Cardiac Myosin Binding Protein-C Lynda Menard 1 , Lori Nyland 2 , Jim Vigoreaux 1 Department of Biology 1 and Molecular Physiology and Biophysics 2 , University of Vermont, Burlington, Vermont Cardiac myosin binding protein-c (cMyBP-C) of mammalian cardiac muscle and flightin (FLN) of invertebrate indirect flight muscle (IFM) have been shown to contribute to thick filament stiffness, as determined by calculations of persistence length (PL), an index of flexural rigidity [1, 2] in their corresponding muscle systems. FLN and cMyBP-C in vitro bind to a common site in the coiled-coil region of myosin II, and both proteins are known to be regulated by phosphorylation [3, 4]. To test the hypothesis that FLN and cMyBP-C are functionally homologous, we have determined the extent to which cMyBP-C can rescue the phenotypes manifested in the Drosophila FLN knockout strain fln 0 . Structural characteristics of flight muscle sarcomeres were analyzed by transmission electron microscopy (TEM) and the contour and end- to-end length of isolated, hydrated native thick filaments was measured by atomic force microscopy (AFM). Experiments were carried out on four D. melanogaster mutant and transgenic strains: (i) FLN knockout strain (fln 0 ), (ii) a knockout rescued transgenic strain (fln 0 ;fln + ), (iii) a transgenic cMyBP-C strain without FLN expression (fln 0 ;cMyBPC + ), and (iv) a transgenic strain with FLN expression alongside cMyBP-C expression (fln + ;cMyBPC + ). In preparation for TEM, thoraces from newly eclosed (<1 hour) D. melanogaster were bisected, fixed, dehydrated, infiltrated, embedded, sectioned and imaged by TEM [5]. The length of sarcomeres from 4-5 flies for each Drosophila strain was measured using ImageJ. AFM data of isolated thick filaments were evaluated using the parameters and programs described by [6]. Statistical analysis was done using JMP 9 software. The TEM results confirmed both the sarcomere length measurements and level of structural order previously seen for fln 0 and fln 0 ;fln + , while revealing shorter sarcomeres in the transgenic lines involving cMyBP-C alone (Fig. 1, Table. 1). When cMyBP-C is expressed alongside FLN, sarcomere length is slightly but significantly longer than sarcomere length in the control fln 0 ;fln + . These results support the idea of cMyBP-C binding to myosin in thick filaments of D. melanogaster and influencing the length of the filaments. However, the length regulation exerted by cMyBP-C is surpassed by FLN when FLN is present, either by direct binding competition to a common myosin binding site or another regulatory mechanism. The PL for fln + ;cMyBPC + obtained by AFM was significantly higher than PL for fln 0 ;fln + , suggesting that cMyBP-C contributes to filament stiffness when expressed ectopically in IFM. However, the cMyBP-C effect is seen only in the presence of FLN as PL of fln 0 ;cMyBPC + was not different that PL of fln 0 . Our observations suggest that the presence of FLN influences the effects that cMyBP-C has on the mechanical properties of the thick filaments. This may possibly be due to FLN stabilizing the thick filaments to permit a more ideal environment for cMyBP-C binding. From these studies we conclude that 80 doi:10.1017/S1431927613002390 Microsc. Microanal. 19 (Suppl 2), 2013 © Microscopy Society of America 2013 https://doi.org/10.1017/S1431927613002390 Downloaded from https://www.cambridge.org/core. IP address: 54.163.42.124, on 29 May 2020 at 22:59:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms.