Grimm, J. | Search bioRxiv

bioRxiv2017

Cohesin Can Remain Associated With Chromosomes During DNA Replication

To ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication. We used unlabelled HaloTag ligand following fluorescent labelling to block newly synthesised Halo-tagged Scc1 protein from incorporating fluorescent dye (pulse-chase or pcFRAP). In cells whose cohesin turnover is inactivated by deletion of WAPL, Scc1 remains associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to unreplicated DNA.

cell biology↗

bioRxiv2017

Stochastic Protein Labeling Enables Long-term Single Molecule Observation In Vivo

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic titration range of >10,000 fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a waterfall mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.

biophysics↗