Date of Award
Doctor of Philosophy (PhD)
Jim O. Vigoreaux
Matthew D. Liptak
The evolutionary success of Insecta has been attributed largely to the development of efficient means of motility: flight powered by muscle architecture harboring a largely conserved yet tunable system of power relay. The indirect flight muscle (IFM) of Drosophila melanogaster is a well-studied model for dissection of the structural and mechanical means by which muscle operates and evolves. Striated muscle, conserved throughout Animalia, is demarcated by an ordered array of thick- and thin-filaments prominently composed of the proteins myosin and actin. Flightin (fln) is a myosin binding thick filament protein essential for IFM stability, structure and function. The manner by which fln contacts myosin and relevance of its highly conserved domain (WYR) has not been fully elucidated. This dissertation presents the culmination of an effort to elucidate fln’s role in the thick filament and the nature and involvement of the novel WYR domain. Cardiac myosin binding protein-C (cMyBP-C), exclusive to vertebrates, and fln, exclusive to Pancrustacea bind a common site in the light meromyosin (LMM) region of myosin and have been hypothesized to have partially overlapping functions within the thick filament. To evaluate this, IFM sarcomeres and thick filaments from D. melanogaster mutant and transgenic strains with and without additional cMyBP-C expression were examined by transmission electron microscopy (TEM) and atomic force microscopy (AFM), respectively. cMyBP-C, like fln, is found to influence sarcomere length and contribute to thick filament flexural rigidity. This suggests a shared influence on thick filament properties though cMyBP-C did not fully rescue the fln0 phenotype. Adding depth to the fln-LMM relationship, we examined the structure and function of WYR. The structure of WYR, determined by circular dichroism (CD), is mostly aperiodic, with 30% antiparallel β content. A putative model of WYR secondary structure is presented, derived from CD findings and interpreted on the basis of WYR’s primary sequence and the potential contributions of its aromatic and polar residue electronic state transitions. Employing both cosedimentation and CD, we find that WYR binds the LMM and induces structural change. The WYR-LMM structure depict the LMM as decreasing in ɑ-helical nature and increasing in coiled-coil character and sedimentation assays demonstrate increased prevalence of macroscopic assemblies upon the association. Data from a structural study of the waterbug IFM thick filament was processed to reveal fln association to regions depicting coiled-coil unwinding. The portions of the LMM interfacing with fln were associated to the myosin sequence, revealing specific amino acids over which fln is in close proximity. We identify five interfaces, one of which is heptad mapped and reveals an LMM binding region shared between fln and cMyBP-C. Given the importance of fln to IFM function and the conservation of the WYR domain through Pancrustacea, the convergent effects of fln and cMyBP-C along with LMM structural change induced by WYR presents a positional and structural basis over which the thick filament experiences context-dependent tuning. Our findings depict fln as a cinch connecting multiple myosin dimers via the LMM, and support its intimate involvement in thick filament assembly. This work describes WYR on a multiscale, considering the nanoscopic mechanisms that underpin macroscopic biological phenomena. WYR is an important agent by which structural and mechanical adaptations are incorporated into the IFM hierarchy, relevant to the rise of flight within Insecta. Further dissection of WYR’s function and relationship to the LMM should provide insight pertinent to the scaling of mechanical processes by structural design and have bearing in studies beyond the IFM and insect adaptation.
Number of Pages
Menard, Lynda, "Structural Characterization Of The Novel Flightin Domain Wyr And Its Defining Role In The Thick Filament Structure And Mechanics" (2021). Graduate College Dissertations and Theses. 1341.
Available for download on Wednesday, June 16, 2021