RESUMO
Nuclear transport is an essential part of eukaryotic cell function. Here, we present scFRAP, a model-assisted fluorescent recovery after photobleaching (FRAP)- based method to determine nuclear import and export rates independently in individual live cells. To overcome the inherent noise of single-cell measurements, we performed sequential FRAPs on the same cell. We found large cell-to-cell variation in transport rates within isogenic yeast populations. For passive transport, the variability in NPC number might explain most of the variability. Using this approach, we studied mother-daughter cell asymmetry in the active nuclear shuttling of the transcription factor Ace2, which is specifically concentrated in daughter cell nuclei in early G1. Rather than reduced export in the daughter cell, as previously hypothesized, we found that this asymmetry is mainly due to an increased import in daughters. These results shed light on cell-to-cell variation in cellular dynamics and its sources.
RESUMO
The diversity and flexibility of life offers a wide variety of molecules and systems useful for biosensing. A biosensor device should be robust, specific and reliable. Inorganic arsenic is a highly toxic water contaminant with worldwide distribution that poses a threat to public health. With the goal of developing an arsenic biosensor, we designed an incoherent feed-forward loop (I-FFL) genetic circuit to correlate its output pulse with the input signal in a relatively time-independent manner. The system was conceived exclusively based on the available BioBricks in the iGEM Registry of Standard Biological Parts. The expected behavior in silico was achieved; upon arsenic addition, the system generates a short-delayed reporter protein pulse that is dose dependent to the contaminant levels. This work is an example of the power and variety of the iGEM Registry of Standard Biological Parts, which can be reused in different sophisticated system designs like I-FFLs. Besides the scientific results, one of the main impacts of this synthetic biology project is the influence it had on team's members training and career choices which are summarized at the end of this article.
RESUMO
The unfolded protein response (UPR) and the Akt signaling pathway share several regulatory functions and have the capacity to determine cell outcome under specific conditions. However, both pathways have largely been studied independently. Here, we asked whether the Akt pathway regulates the UPR. To this end, we used a series of chemical compounds that modulate PI3K/Akt pathway and monitored the activity of the three UPR branches: PERK, IRE1 and ATF6. The antiproliferative and antiviral drug Akt-IV strongly and persistently activated all three branches of the UPR. We present evidence that activation of PERK/eIF2α requires Akt and that PERK is a direct Akt target. Chemical activation of this novel Akt/PERK pathway by Akt-IV leads to cell death, which was largely dependent on the presence of PERK and IRE1. Finally, we show that hypoxia-induced activation of eIF2α requires Akt, providing a physiologically relevant condition for the interaction between Akt and the PERK branch of the UPR. These data suggest the UPR and the Akt pathway signal to one another as a means of controlling cell fate.