Light-triggered myosin activation for probing dynamic cellular processes.

Myosin II is an ATPase motor protein essential for many cellular functions including cell migration and division. In nonmuscle cells, myosin modulates protrusions at the leading edge and promotes retraction at the trailing edge during migration, while during cytokinesis, myosin is required for contraction of the cleavage furrow. For nonmuscle myosin, these varied functions are regulated by phosphorylation of the associated myosin regulatory light chain (mRLC) protein at Ser19, which activates the myosin complex to promote myosin assembly, cell contractility, and stress fiber formation. Upon phosphorylation of the mRLC at both Thr18 and Ser19, these activities are further enhanced. 6 ] The dramatic effects of phosphorylation can also be recapitulated in vitro. Specifically, myosin and the proteolytic derivative heavy meromyosin (HMM), 7 ] which contains only one-third of the Cterminal myosin tail, exhibit low in vitro activities when associated with the nonphosphorylated mRLC. Phosphorylation of Ser19 amplifies actin-activated ATPase activities 10 – 1000-fold 8 ] and leads to myosin-mediated actin translocation. While myosin has been studied extensively for almost five decades, questions surrounding the dynamic interactions of the protein within live cells remain. Methods currently used to study myosin and modulate activity include gene deletions or siRNAmediated knockdown of gene expression, overexpression of kinases that phosphorylate the mRLC, 10 ] and small molecule inhibitors of myosin, 11 ] mRLC kinases, 12 ] and myosin phosphatase. While these methods have provided a wealth of valuable information about myosin, they do not enable studies of the spatial dynamics of myosin regulation because localized activation cannot be achieved. Additionally, genetic approaches provide imprecise temporal control over protein function, preventing realtime studies of the protein. Thus, we sought to develop chemical tools to overcome these drawbacks and to complement the existing approaches by enabling direct and controlled myosin activation through the semisynthesis of a photoactivated mRLC. The lightmediated activation is achieved by the incorporation of a photolabile protecting group, or “caging group,” onto the essential phosphate of pSer19 within the full-length mRLC. The caging group masks the phosphate functionality and renders the protein biologically inactive until irradiation removes the masking group and releases the active native phosphoprotein. By using light as the trigger for phosphorylation, this strategy offers a kinase-independent method to activate myosin with precise spatial and temporal resolution and enables researchers to obtain real-time information about the downstream effects of myosin phosphorylation within a complex network of interactions. The 1-2-(nitrophenyl)ethyl (NPE) caging group has been employed for cellular applications because it is efficiently released, under biologically-compatible conditions, at 365 nm. Peptides and proteins containing NPE-caged phosphorylated amino acids have been successfully exploited for the study of many diverse systems. Additionally, a general method for incorporating NPEcaged thiophosphoamino acids, which, upon irradiation, function similarly to the corresponding phosphorylated species but with greater resistance to phosphatases, has been reported and can be used for advancing studies of myosin. Herein we report the development of a chemical approach to investigate myosin function through the preparation of unnatural amino acid mutants of the mRLC. We present an efficient semisynthesis of full-length mRLC through expressed protein ligation for the site-specific incorporation of phosphorylation at Ser19 (pSer19) and Thr18 (pThr18) and the genesis of caged phosphoserine (cpSer) and caged thiophosphoserine (c(S)pSer) at position 19. Caging of pSer19 eliminates myosin and HMM activities, and irradiation releases the native phospho-mRLC to restore activity to nearly native phosphorylated levels (Figure 1). Microinjection of myosin exchanged with the caged protein into live cells and subsequent irradiation releases the phosphoprotein within cells. This tool is poised to facilitate future investigations of the downstream effects of myosin activation.