In a study aimed at defining conditions conducive to σW expression in B. subtilis, by Cao et al [59], an elegant disc-diffusion reporter-gene fusion method was employed.
Specifically, in one such study by Cao et al [59], an elegant disc-diffusion reporter-gene fusion method was employed to define conditions conducive to the expression of σW in B. subtilis.
Spatial resolution is typically compromised by diffusion of reporter molecules prior to deposition, and the lack of orthogonal deposition chemistries dictates that in situ amplification be performed serially for multiplexed studies, leading to progressive sample degradation and lengthy protocols.
By expanding the scale to wide fields of cysts, we generated morphogen diffusion gradients, controlling reporter gene expression.
Thus, it would correspond to one extreme for a protein tracer reporter for intracellular diffusion.
Purified HGF or NK4 were added at opposite ends, allowing diffusion over a field of reporter cysts, while taking timelapse GFP fluorescent images.
The small size of NLS-mCherry allows diffusion across the NPC, whereas nuclear accumulation of the reporter protein relies on active nuclear import.
Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporter and showed major restriction to lateral diffusion when photobleached in large fields.
Likewise, the path followed by these cells can be observed in living tissue, as well as in the diffusion of signals through interstitial space, for example, using hydrogen peroxide reporters [ 2].
Combining a rigorously characterized setup and an adequate reporter strategy, we could for the first time quantify the accessibility of the nuclear landscape and the diffusion coefficient of small molecules, up to eGFP tetramers, depending on the chromatin compaction level.
Since our goal was to quantify the diffusion of small molecules of various sizes, we decided to use eGFP mono-, di-, tri- and tetramers as mobility reporters.
