Exploring The Hippo Signalling Pathway Through High Resolution Electron Microscopy
Over recent years, the Hippo signalling pathway has emerged as a regulator of key developmental processes, including organ size and tissue growth. Initially identified in the fruit fly, Drosophila melanogaster, this evolutionarily conserved signalling pathway comprises of over 35 components, each fitting into a complex network feeding a central kinase cascade. Deregulation in Hippo signalling has been observed in several types of human cancers; therefore, it is crucial to understand the mechanisms by which the Hippo pathway components regulate tissue growth. Confocal microscopy approaches have been used to visualise protein dynamics, localisation, and abundance, and have provided important insights into Hippo signalling, however, these are limited by their resolution of 200nm. This often makes it difficult to definitively assess protein localisation at specific subcellular domains. Electron microscopy (EM), alternatively, provides high resolution (up to 0.2nm), ultra-structural details and has the potential to generate new insights into the subcellular localisation of Hippo pathway proteins and hence Hippo signal transduction. A recently developed EM technique which fuses an ascorbate peroxidase (APEX2) with a GFP binding peptide (GBP) can work with a growing library of GFP-bound proteins and allows for a versatile reporter tool to visualise GFP-bound protein localisation in EM.