Videos

Human U2OS G3BP1/2 knockout cells co-expressing stress granule-nucleating factor, G3BP (green), and P-body-nucleating protein, LSM14A (red), post-stress (arsenite treatment). The strong adhesion between the two condensates is readily observable (1-hour video), which is likely due to overlap between the RNP-interaction networks critical to formation of both condensates.

G3BP1/2 knockout U2OS cells with reconstituted optogenetic stress granules. 1-hour time lapse post-stress (arsenite); optogenetic activatation-deactivation cycling using Corelet technology. Green = ferritin Core oligomerization platform, red = G3BP DelNTF2 bait.

Human U2OS G3BP1/2 knockout cells expressing G3BP1 DelNTF2 Corelets (green, ferritin Core oligomerization platform; red, DelNTF2 optogenetic bait) with stress granule (SG) RNA-binding protein marker, HNRNPA1 (purple). Here, cells were pre-treated with arsenite for one-hour, which blocks translation and releases RNA into the cytoplasm, and subjected to iterative activation-deactivation cycles. The optogenetic SGs get larger with each cycle, as RNA floods into the cell and increases the network's connectivity.

G3BP1/2 knockout U2OS cells with reconstituted optogenetic stress granules (post-stress, arsenite); optogenetic activation-deactivation cycling, using Corelet technology. Green = ferritin Core oligomerization platform, red = G3BP DelNTF2 bait. Opto-SGs fuse and relax to sphere in liquid-like manner.

This is a fun experiment that we haven't followed up on extensively. Shown are G3BP1/2 knockout U2OS cells expressing G3BP1 DelNTF2 Corelets (Core oligomerization recruitment platform, green; optogenetic DelNTF2 bait, red) exposed to stress/arsenite + continuous blue-light activation for 1-hour. At end, we reverted the oligomerized RNA-binding domain to its monomeric form. The result? Explosion of individual stress granules into a swirling cacophony of mRNPs.

Human U2OS G3BP1/2 knockout cells expressing G3BP1 DelNTF2 Corelets (green, ferritin Core oligomerization platform; red, DelNTF2 optogenetic bait) with stress granule (SG)-nucleating factor, UBAP2L (purple). Here, cells were pre-treated with arsenite for one-hour, which blocks translation and releases RNA into the cytoplasm. UBAP2L nucleates small SG-like assemblies in the absence of G3BP1 DelNTF2 oligomerization. However, these assemblies are a bit different than canonical SGs with regard to protein/RNA components (in fact, a hybrid between stress granules and the related P-bodies). No worries, we arrive with blue-light triggered G3BP DelNTF2 optogenetic stress granules, in hand, and pull the pieces together. But upon disintegration of the glue? Chaos.

Similar experiment to previous video.

Human U2OS G3BP1/2 knockout cells expressing CAPRIN1 RNA-binding domain (RBD) Corelets (green, ferritin Core oligomerization platform; red, RBD optogenetic RBD bait) in the absence of stress/arsenite. The optogenetically-induced phase separated RNPs form on the surface of microtubules. Not shown: abrogation of droplets by transcription inhibition (result: MT-binding only), promotion by RNA influx (result: larger droplets, no MT-binding). Appears to be a tug of war between microtubules- vs. RNA-binding. Classic disordered RGG protein. Want to study this? All yours.