Analysis of Protein Turnover by Quantitative SNAP-Based Pulse-Chase Imaging

Assessment of protein dynamics in living cells is crucial for understanding their biological properties and functions. The SNAP-tag, a self labeling suicide enzyme, presents a tool with unique features that can be adopted for determining protein dynamics in living cells. Here we present detailed protocols for the use of SNAP in fluorescent pulse-chase and quench-chase-pulse experiments. These time-slicing methods provide powerful tools to assay and quantify the fate and turnover rate of proteins of different ages. We cover advantages and pitfalls of SNAP-tagging in fixed- and live-cell studies and evaluate the recently developed fast-acting SNAPf variant. In addition, to facilitate the analysis of protein turnover datasets, we present an automated algorithm for spot recognition and quantification.

Temporal Control of Leaf Complexity by miRNA-Regulated Licensing of Protein Complexes

The tremendous diversity of leaf shapes has caught the attention of naturalists for centuries. In addition to interspecific and intraspecific differences, leaf morphologies may differ in single plants according to age, a phenomenon known as heteroblasty. In Arabidopsis thaliana, the progression from the juvenile to the adult phase is characterized by increased leaf serration. A similar trend is seen in species with more complex leaves, such as the A. thaliana relative Cardamine hirsuta, in which the number of leaflets per leaf increases with age. Although the genetic changes that led to the overall simpler leaf architecture in A. thaliana are increasingly well understood, less is known about the events underlying age-dependent changes within single plants, in either A. thaliana or C. hirsuta. Here, we describe a conserved miRNA transcription factor regulon responsible for an age-dependent increase in leaf complexity. In early leaves, miR319-targeted TCP transcription factors interfere with the function of miR164-dependent and miR164-independent CUC proteins, preventing the formation of serrations in A. thaliana and of leaflets in C. hirsuta. As plants age, accumulation of miR156-regulated SPLs acts as a timing cue that destabilizes TCP-CUC interactions. The destabilization licenses activation of CUC protein complexes and thereby the gradual increase of leaf complexity in the newly formed organs. These findings point to posttranslational interaction between unrelated miRNA-targeted transcription factors as a core feature of these regulatory circuits.