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.

Clustering of Rab11 vesicles in influenza A virus infected cells creates hotspots containing the 8 viral ribonucleoproteins

Influenza A virus is an important human pathogen causative of yearly epidemics and occasional pandemics. The ability to replicate within the host cell is a determinant of virulence, amplifying viral numbers for host-to-host transmission. This process requires multiple rounds of entering permissive cells, replication, and virion assembly at the plasma membrane, the site of viral budding and release. The assembly of influenza A virus involves packaging of several viral (and host) proteins and of a segmented genome, composed of 8 distinct RNAs in the form of viral ribonucleoproteins (vRNPs). The selective assembly of the 8-segment core remains one of the most interesting unresolved problems in virology. The recycling endosome regulatory GTPase Rab11 was shown to contribute to the process, by transporting vRNPs to the periphery, giving rise to enlarged cytosolic puncta rich in Rab11 and the 8 vRNPs. We recently reported that vRNP hotspots were formed of clustered vesicles harbouring protruding electron-dense structures that resembled vRNPs. Mechanistically, vRNP hotspots were formed as vRNPs outcompeted the cognate effectors of Rab11, the Rab11-Family-Interacting-Proteins (FIPs) for binding, and as a consequence impair recycling sorting at an unknown step. Here, we speculate on the impact that such impairment might have in host immunity, membrane architecture and viral assembly.