PhD Exit Seminar – Abiramy Karunendiran (Stewart Lab)

Sarcomere Structure in Drosophila Musculature Studied with Polarimetric Second Harmonic Generation Microscopy 

Thursday, September 10, 2020 at 1:10pm 

Join Zoom Meeting:

https://utoronto.zoom.us/j/99127382979

Meeting ID: 991 2738 2979

Abstract

Nonlinear optical microscopy is a powerful imaging modality that enables label free imaging with endogenous signal generated from the protein arrays that constitute biological structures and can be used to reveal structural and functional information in a system. Second Harmonic Generation (SHG) Microscopy is a parametric process that does not require photon absorption for signal generation, providing the added benefit of having a significantly reduced photo-bleaching effect. SHG signal is observed in non-centrosymmetric molecules like myosin and can be used to directly visualize muscle structure. Additionally, polarimetric SHG imaging allows for the quantification of the microstructure of the sample. Muscle function depends on highly ordered protein complexes. UNC45 is a USC chaperone necessary for myosin incorporation into thick filaments. UNC45 is expressed throughout the entire Drosophila life cycle and has been shown to be important during late embryogenesis when initial muscle development occurs. However, the effects of UNC45 manipulation at later developmental

times have not yet been studied. The focus of my thesis is to utilize both traditional and nonlinear optical microscopy to investigate changes in myosin filament organization and interactions between protein complexes and how it relates to muscle function. UNC45 was knocked down with RNAi in a way that permitted survival to the pupal stage. Immunofluorescence and electron microscopy imaging showed diminished expression of UNC45 and disorganized myosin filaments and Z-disk proteins. Associated changes in synaptic physiology and impaired locomotor behavior were also observed. These observations were confirmed and further analyzed on a whole larval scale using the newly developed polarimetric wide-field SHG microscopy, revealing changes in the striated pattern of somatic muscles and a reduced signal intensity. Polarimetric parameters were used to study chiral and achiral properties of larval muscles. Respectively R- and C-ratios were determined using scanning SHG microscopy. R-ratio values were higher in the mutant compared to control, suggesting that not only myosin, but also a second protein structure was producing a second harmonic response. CD image and C-ratio revealed the bimodal distribution of myosin filaments and changes in chiral structure in UNC45 knockdown larva. Overall, this technique provides insight on how second harmonic properties changes with myosin filament structure. This opens new perspective to study the dynamic properties of muscle structure during contraction.