| Popis: |
We are using fluorescence microscopy to characterize the interaction of single active MLCK molecules with smooth muscle myosin (SMM) filaments and acto-SMM bundles in an in vitro¬ model system to determine the mechanism by which MLCK phosphorylates SMM. Since ATP is required for the SMM ATPase and the MLCK kinase activity, this requires unphosphorylated SMM filaments that are stable in the presence of ATP, a condition that normally causes disassembly. We have prepared and characterized modified SMM filaments for this study. They are 1) covalently labeled with NHS-Rhodamine for ease of imaging, 2) lightly cross-linked with EDC on the heavy chain so they are stable to ATP-induced disassembly, 3) similar to native SMM filaments in length, 4) fully regulated by phosphorylation measured by acto-SMM ATPase activity, and the ability to move actin in an in vitro motility assay, and 5) able to dissociate from actin in an [ATP]-dependent manner. Interestingly, these modified SMM filaments are capable of bundling actin filaments in the presence of ATP, calmodulin (CaM), and Ca2+; conditions that are sufficient for MLCK to phosphorylate SMM. SMM filaments preferentially bind to actin bundles rather than single actin filaments, and as ATP is titrated into a preparation of acto-SMM bundles, they dissociate leaving short actin filaments which are predominantly bound to SMM filaments. We have imaged the interaction of single quantum dot-labeled MLCK (QD-MLCK) molecules with these SMM filaments and acto-SMM bundles using total internal reflection fluorescence (TIRF) microscopy to visualize single QD-MLCK. We observed QD-MLCK colocalizing with SMM filaments and acto-SMM bundles and will characterize the QD-MLCK interaction with both proteins in the presence of ATP before and after activation of MLCK by Ca2+-CaM. |
