Figure 1.
CGs undergo LLPS in vitro at a mildly acidic pH, while calcium is dispensable. (A) Plots of CGB generated using PONDR depicting disordered regions in the proteins. N-terminal half of CGB is completely disordered, when analyzed using the VL-XT algorithm. (B) Coomassie -stained gel depicting purified CGB-GFP. The images show droplets of CGB-GFP (left) at 10 µM protein concentration at pH 6.1 without a crowding agent. The same concentrations of super folder-GFP do not form droplets at pH 6.1 (right). (C) A panel of images extracted from a movie showing the biophysical behavior of CGB-GFP (2.5 µM) droplets induced by 1% PEG 8000. Two droplets, which come in proximity undergo fusion and the larger droplet subsequently relaxes into a spherical shape. (D) A panel of images monitoring recovery of fluorescence of CGB-GFP droplets after bleaching a small region within the droplets. Note the rapid recovery of fluorescence (more than 60%) in the bleached region within a minute. (E) Graph quantifying the fluorescence recovery in time. Data represented as mean ± SD (error blanket) from seven droplets. (F) Representative images of solutions containing CGB-GFP (10 µM) buffered at either pH 6.1 (left) or pH 7.3 (right). Droplet formation occurs at pH 6.1 and not at pH 7.3. (G) Representative images of CGB-GFP (2.5 µM) protein without any divalent cation (minus) or in presence of 20 mM calcium, and zinc, respectively. CGB-GFP solution (∼10 µM concentration) was centrifuged to preclear of existing droplets and diluted to a final concentration of 2.5 µM. While calcium induces CGB-GFP droplets, zinc induces formation of insoluble aggregates. (H) Schematic representation of CGB, depicting five exons. Patches in red are stretches enriched in acidic amino acids. S* and Y* denote phosphorylation and sulfation respectively on these residues. KR or RK are the dibasic sites in the protein which are predominantly concentrated in the C-terminal half of the protein. (I) Representative Coomassie-stained image of mutant form of CGB, CGB_5(ED)/A-GFP to depict its purity. In this mutant the 5 (ED) stretches have been replaced with alanine. The fluorescence image shows condensates of CGB_5(ED)/A when equilibrated at pH 6.1 at 10 µM protein concentration. (J) Comparison of CGB-GFP and CGB_5(ED)/A-GFP in presence of 20 mM calcium concentrations. Note that at 2.5 µM protein concentration, calcium can induce droplet formation with only CGB-GFP (left), but now with CGB_5(ED)/A-GFP (right). (K and L) Snapshots captured at different time points after photobleaching a region within a calcium-induced CGB-GFP droplet (top) or a zinc induced CGB-GFP aggregate (bottom) are shown in (K). While there is more than 60% fluorescence recovery within the bleached region in the calcium induced droplet, recovery within the bleached region in the aggregate is only 10% as seen in the graph in (L). Red curve denotes recovery of calcium-induced droplets and blue curve denotes zinc-induced aggregates. Data is represented as mean ± SD (error blanket) from seven calcium-induced droplets and six zinc-induced aggregates. Source data are available for this figure: SourceData F1.

CGs undergo LLPS in vitro at a mildly acidic pH, while calcium is dispensable. (A) Plots of CGB generated using PONDR depicting disordered regions in the proteins. N-terminal half of CGB is completely disordered, when analyzed using the VL-XT algorithm. (B) Coomassie -stained gel depicting purified CGB-GFP. The images show droplets of CGB-GFP (left) at 10 µM protein concentration at pH 6.1 without a crowding agent. The same concentrations of super folder-GFP do not form droplets at pH 6.1 (right). (C) A panel of images extracted from a movie showing the biophysical behavior of CGB-GFP (2.5 µM) droplets induced by 1% PEG 8000. Two droplets, which come in proximity undergo fusion and the larger droplet subsequently relaxes into a spherical shape. (D) A panel of images monitoring recovery of fluorescence of CGB-GFP droplets after bleaching a small region within the droplets. Note the rapid recovery of fluorescence (more than 60%) in the bleached region within a minute. (E) Graph quantifying the fluorescence recovery in time. Data represented as mean ± SD (error blanket) from seven droplets. (F) Representative images of solutions containing CGB-GFP (10 µM) buffered at either pH 6.1 (left) or pH 7.3 (right). Droplet formation occurs at pH 6.1 and not at pH 7.3. (G) Representative images of CGB-GFP (2.5 µM) protein without any divalent cation (minus) or in presence of 20 mM calcium, and zinc, respectively. CGB-GFP solution (∼10 µM concentration) was centrifuged to preclear of existing droplets and diluted to a final concentration of 2.5 µM. While calcium induces CGB-GFP droplets, zinc induces formation of insoluble aggregates. (H) Schematic representation of CGB, depicting five exons. Patches in red are stretches enriched in acidic amino acids. S* and Y* denote phosphorylation and sulfation respectively on these residues. KR or RK are the dibasic sites in the protein which are predominantly concentrated in the C-terminal half of the protein. (I) Representative Coomassie-stained image of mutant form of CGB, CGB_5(ED)/A-GFP to depict its purity. In this mutant the 5 (ED) stretches have been replaced with alanine. The fluorescence image shows condensates of CGB_5(ED)/A when equilibrated at pH 6.1 at 10 µM protein concentration. (J) Comparison of CGB-GFP and CGB_5(ED)/A-GFP in presence of 20 mM calcium concentrations. Note that at 2.5 µM protein concentration, calcium can induce droplet formation with only CGB-GFP (left), but now with CGB_5(ED)/A-GFP (right). (K and L) Snapshots captured at different time points after photobleaching a region within a calcium-induced CGB-GFP droplet (top) or a zinc induced CGB-GFP aggregate (bottom) are shown in (K). While there is more than 60% fluorescence recovery within the bleached region in the calcium induced droplet, recovery within the bleached region in the aggregate is only 10% as seen in the graph in (L). Red curve denotes recovery of calcium-induced droplets and blue curve denotes zinc-induced aggregates. Data is represented as mean ± SD (error blanket) from seven calcium-induced droplets and six zinc-induced aggregates. Source data are available for this figure: SourceData F1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal