Abstract
BackgroundProper 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 resultsUNC45 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.ConclusionsThis 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.
Highlights
Proper muscle function is heavily dependent on highly ordered protein complexes
In both vertebrates and invertebrates UNC45 is composed of three domains [7, 12]: an Nterminal tetratricopeptide repeat (TPR) domain that interacts with Heat Shock Protein 90 [12], a central domain with an unknown function and a UCS domain (aptly named since this region is shared by three proteins (UNC45, CRO1 and She4P)) which interact with myosin and facilitate myosin function [13]
In order to assess the contribution of UNC45 to muscle development and function, we first tested knockdown of UNC45 with three widely used muscle driver lines in the UAS-Gal4 expression system
Summary
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. 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 found. UNC45 has been of keen interest in studying muscle development
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