Disruption of Drosophila larval muscle structure and function by UNC45 knockdown.

BACKGROUND: Proper muscle function is heavily dependent on highly ordered protein complexes. UNC45 is a USC (named since this region is shared by three proteins UNC45/CRO1/She4P) chaperone that is necessary for myosin incorporation into the thick filaments. UNC45 is expressed throughout the entire Drosophila life cycle and it has been shown to be important during late embryogenesis when initial muscle development occurs. However, the effects of UNC45 manipulation at later developmental times, after muscle development, have not yet been studied. MAIN RESULTS: UNC45 was knocked down with RNAi in a manner that permitted survival to the pupal stage, allowing for characterization of sarcomere organization in the well-studied third instar larvae. Second harmonic generation (SHG) microscopy revealed changes in the striated pattern of body wall muscles as well as a reduction of signal intensity. This observation was confirmed with immunofluorescence and electron microscopy imaging, showing diminished UNC45 signal and disorganization of myosin and z-disk proteins. Concomitant alterations in both synaptic physiology and locomotor function were also found. Both nerve-stimulated response and spontaneous vesicle release were negatively affected, while larval movement was impaired. CONCLUSIONS: This study highlights the dependency of normal sarcomere structure on UNC45 expression. We confirm the known role of UNC45 for myosin localization and further show the I-Z-I complex is also disrupted. This suggests a broad need for UNC45 to maintain sarcomere integrity. Newly discovered changes in synaptic physiology reveal the likely presence of a homeostatic response to partially maintain synaptic strength and muscle function.

Dual- and single-shot susceptibility ratio measurements with circular polarizations in second-harmonic generation microscopy.

Polarization-resolved second-harmonic generation (P-SHG) microscopy is a technique capable of characterizing nonlinear optical properties of noncentrosymmetric biomaterials by extracting the nonlinear susceptibility tensor components ratio χzzz2'/χzxx2' , with z-axis parallel and x-axis perpendicular to the C6 symmetry axis of molecular fiber, such as a myofibril or a collagen fiber. In this paper, we present two P-SHG techniques based on incoming and outgoing circular polarization states for a fast extraction of χzzz2'/χzxx2' : A dual-shot configuration where the SHG circular anisotropy generated using incident right- and left-handed circularly-polarized light is measured; and a single-shot configuration for which the SHG circular anisotropy is measured using only one incident circular polarization state. These techniques are used to extract the χzzz2'/χzxx2' of myosin fibrils in the body wall muscles of Drosophila melanogaster larva. The results are in good agreement with values obtained from the double Stokes-Mueller polarimetry. The dual- and single-shot circular anisotropy measurements can be used for fast imaging that is independent of the in-plane orientation of the sample. They can be used for imaging of contracting muscles, or for high throughput imaging of large sample areas.

Live imaging of contracting muscles with wide-field second harmonic generation microscopy using a high power laser.

Wide-field second harmonic generation (SHG) microscopy was developed using a high-power (> 4 W) and high-repetition-rate (MHz range) laser oscillator to achieve fast SHG imaging over a large area (400 µm × 400 µm). The microscope was used for high spatial resolution imaging of contracting muscles in live Drosophila melanogaster larvae. Anisotropic and isotropic bands of striated muscle were distinguished, allowing accurate determination of sarcomere length and SHG intensity from individual sarcomeres. Therefore, wide-field SHG microscopy has applications in basic contractility research and studying arrhythmias, muscular dystrophies and pharmaceutical effects on the muscle contraction dynamics of sarcomeres.

Examination of Drosophila eye development with third harmonic generation microscopy.

Third harmonic generation (THG) microscopy can exploit endogenous harmonophores such as pigment macromolecules for enhanced image contrast, and therefore can be used without exogenous contrast agents. Previous studies have established that carotenoid compounds are ideal harmonophores for THG microscopy; we therefore sought to determine whether THG from endogenous carotenoid-derived compounds, such as retinal in photoreceptor cells, could serve as a new label-free method for developmental studies. Here we study the development of the pupal eye in Drosophila melanogaster and determine the localization of rhodopsin using THG microscopy technique. Additionally, by altering the chromophore or the opsin protein we were able to detect changes in both the retinal distribution morphology and in THG intensity age-dependent profiles. These results demonstrate that THG microscopy can be used to detect altered photoreceptor development and may be useful in clinically relevant conditions associated with photoreceptor degeneration.

Second harmonic generation double stokes Mueller polarimetric microscopy of myofilaments.

The experimental implementation of double Stokes Mueller polarimetric microscopy is presented. This technique enables a model-independent and complete polarimetric characterization of second harmonic generating samples using 36 Stokes parameter measurements at different combinations of incoming and outgoing polarizations. The degree of second harmonic polarization and the molecular nonlinear susceptibility ratio are extracted for individual focal volumes of a fruit fly larva wall muscle.