Journal: The Journal of Cell Biology

Article Title: Liquid–liquid phase separation facilitates the biogenesis of secretory storage granules

doi: 10.1083/jcb.202206132

Figure Lengend Snippet: In vitro characterization of CG LLPS. (A) Scheme used for purifying GFP and 6x-His-tagged CGA or CGB, respectively. Stable lines expressing CGB-GFP and CGA-GFP under a doxycycline-inducible promoter are treated with doxycycline and the calcium ionophore A23187 to induce secretion of the respective proteins in serum-free medium, which is then used for purification using Ni-NTA affinity columns. (B) Plot of CGA generated using PONDR depicting disordered regions in the proteins. Almost 90% of CGA is disordered when analyzed using the VL-XT algorithm. (C) Coomassie-stained gel depicting purified CGA-GFP. Images showing different droplets of CGA-GFP at different protein concentrations at pH 6.1 in presence of 5% PEG 8000. Note that the size of the condensates decreases with decreasing protein concentration. (D) Representative images of solutions containing CGA-GFP 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. Droplets of CGA-GFP were induced at 4 µM protein concentration in presence of 5% dextran. (E) Images obtained from plating a solution of CGB-GFP (2.5 µM final concentration) to monitor the presence or absence on liquid-like condensates either without or with 250 µM or 5 mM calcium. Note that droplet formation is induced only in the presence of 5 mM calcium. (F) Phase diagram obtained by varying calcium and CGB concentrations after plating solutions on imaging dishes and observing after 15–20 min. Red circles indicate conditions where no droplets were seen, and green circles indicate conditions which favored presence of CGB droplets. (G) Images obtained from plating a solution of CGA-GFP (2.5 µM final concentration) in presence of either 250 µM, 20 or 40 mM calcium to test for the presence or absence of droplet formation. No droplets are seen even at 40 mM which is the highest calcium concentration. (H) Representative images of CGB-GFP (2.5 µM) in presence of different concentrations of zinc. At high concentrations zinc induces formation of insoluble aggregates but at low concentration, it induces CGB-GFP droplets. Source data are available for this figure: .

Article Snippet: For cloning of sfGFP and 6X-His tagged constructs of human CGA (pBT-PAF-CGA_sfGFP_6XHis) and CGB (pBT-PAF-CGB_sfGFP_6XHis), coding regions of CGA and CGB were cloned by PCR amplifications from expression plasmids from Origene (cat no. RC RC200492; CGA and cat no. RC201744; CGB).

Techniques: In Vitro, Expressing, Purification, Generated, Staining, Protein Concentration, Concentration Assay, Imaging

Journal: The Journal of Cell Biology

Article Title: Liquid–liquid phase separation facilitates the biogenesis of secretory storage granules

doi: 10.1083/jcb.202206132

Figure Lengend Snippet: Ectopic expression of soluble secreted proteins in INS1 832/13 cells results in their routing to insulin granules. (A and B) Representative images from INS1 832/13 cells expressing LyzC-GFP (A; green) or EqSol-GFP (B; green) and stained with insulin antibody (red) to observe the localization of the ectopically expressed proteins with respect to insulin granules. Images are average projections from two slices from a confocal stack. Arrowheads point to cytoplasmic insulin granules which also shows the presence of LyzC-GFP and EqSOL-GFP respectively, in E and F. (C) Representative images from INS1 832/13 cells stably expressing CatD-GFP (green) and labeled with CGB antibody to observe the localization of ectopically expressed CatD-GFP with respect to SGs. Images are average projections from two slices from a confocal stack. Arrowheads point to some of the cytoplasmic SG, which shows colocalization of CGB and CatD-GFP. (D) Representative images from INS1 832/13 expressing HA-tagged version of the calcium ATPase, SPCA1 (green) and stained using CGB antibody (red). Images are a single slice from a confocal stack. Note that overexpressed SPCA1 remains localized at the Golgi apparatus with no signal seen from the CGB containing SGs. (E) Representative images from HeLa cells stably expressing CGA-GFP and transfected with LyzC-RFP. Images are a single slice from a confocal stack imaged in the airy-scan mode. The arrowheads point to some of the ectopic granule-like structures seen in HeLa cells upon expression of CGA-GFP. Note that LyzC-RFP gets routed to these ectopic granule-like structures. (F) Images extracted from live imaging of HeLa cells co-expressing Halo-RUSH-CGB (red) and LyzC-GFP (green) before and after addition of biotin for 52 min when CGB appears at the Golgi. (G) Images extracted from live imaging of HeLa cells co-expressing RUSH-CGB (red) and LyzC-GFP (green) after biotin addition and images after arrival of CGB at the Golgi. Arrow heads point to colocalizing structures at the Golgi and vesicles in the cytoplasm. (H) Western blot at the top shows bands for LyzC-GFP, probed using α-GFP antibody, in supernatant and lysates from INS1 832/13 cells stable expressing SNAP-tagged proinsulin. The basal condition represents cells grown in 3 mM glucose in serum-free medium and the stimulated condition represents cells grown in 15 mM glucose in serum-free medium, also containing 35 mM potassium chloride. Note the stronger band intensity in the supernatant in stimulated condition compared to the basal condition, although the levels in cell lysates are the same. The blot in the bottom left detects the presence of SNAP-tagged C-peptide, probed using α-SNAP-tag antibody, which is used as a proxy to measure insulin secretion. Again, the signal intensity of the band is stronger in stimulated condition as compared to the basal condition. The blot on the bottom right depicts actin bands in cell lysates obtained from basal and stimulated conditions. The graph quantifies secretion of LyzC-GFP normalized with levels in cell lysates in basal and stimulated conditions. Value of the band intensity in secreted compared to the band intensity in cell lysates was set to 1 for stimulated condition in each experiment. Data is represented as mean ± SD from three independent experiments. Statistical analysis was performed by two-tailed one-sample t test *P = 0.019. Source data are available for this figure: .

Article Snippet: For cloning of sfGFP and 6X-His tagged constructs of human CGA (pBT-PAF-CGA_sfGFP_6XHis) and CGB (pBT-PAF-CGB_sfGFP_6XHis), coding regions of CGA and CGB were cloned by PCR amplifications from expression plasmids from Origene (cat no. RC RC200492; CGA and cat no. RC201744; CGB).

Techniques: Expressing, Staining, Stable Transfection, Labeling, Transfection, Imaging, Western Blot, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Liquid–liquid phase separation facilitates the biogenesis of secretory storage granules

doi: 10.1083/jcb.202206132

Figure Lengend Snippet: Proinsulin co-traffics with CGB in vivo and is recruited to droplets in vitro. (A) Schematic depiction of dual pulse chase experiment in INS1 832/13 cells expressing SNAP tagged insulin (proCpepSNAP) and CLIP tagged CGB (CGB-CLIP). Cells are initially incubated with a non-fluorescent blocking probe to mask the existing proteins in the cells. After 2 h, cells are incubated with medium containing SNAP 505 and CLIP-TMR to label the newly synthesized proteins (20 min). After three washes in growth medium, cells are fixed immediately (0 h chase) when majority of the cargo is at the Golgi apparatus or after a chase of 2 h where most of the cargo has moved to the SG in the cytoplasm. (B) Top panel shows confocal images form INS1 832/13 cells expressing SNAP tagged insulin (proCpepSNAP; green) and CLIP tagged CGB in red and fixed immediately after labeling with fluorescent probes, SNAP-505 and CLIP-TMR to monitor the Golgi resident (peri-nuclear) pool of the proteins. Bottom panels show images after a 2 h chase and the arrows point to some of the colocalizing structures which are cytoplasmic SGs. (C) INS1 832/13 wild-type (top) and CGA/CGB dKO (bottom) cells fixed and labeled with antibodies to TGN38 (red) and PC2 (green). Left and the middle images are extracted from a 3D projection. The image on the right represents surfaces which were created using the TGN38 staining (red outline) on deconvolved images in Imaris. The TGN38 volume mask was then used to generate distinct surfaces in the PC2 channel. (D) A scatter plot (median) depicting differences in the numbers of PC2 surfaces between wild-type and CGA/CGB dKO cells from 22 wild-type and 24 dKO cells. Statistical analysis was performed using Mann–Whitney test. ***P < 0.001. (E) Graph showing normalized glucose stimulated insulin secretion (GSIS; stimulated/basal) in wild-type, CGA/CGB dKO cells. Data is represented as mean ± SD from six independent experiments. Statistical analysis was performed using unpaired two-tailed t test. ***P < 0.001. (F) CGB-GFP (16 µM; green) was mixed with Cy3 tagged proinsulin (1 µM; red) in (i). Tagged proinsulin gets recruited to the CGB-GFP droplets as evident from the colocalization image. When GFP (16 µM; green) is mixed with Cy3 tagged proinsulin (1 µM; red) in (ii), no droplets are seen either with GFP or proinsulin indicating that GFP or Cy3-proinsulin are incapable of forming droplets on their own at these concentrations.

Article Snippet: For cloning of sfGFP and 6X-His tagged constructs of human CGA (pBT-PAF-CGA_sfGFP_6XHis) and CGB (pBT-PAF-CGB_sfGFP_6XHis), coding regions of CGA and CGB were cloned by PCR amplifications from expression plasmids from Origene (cat no. RC RC200492; CGA and cat no. RC201744; CGB).

Techniques: In Vivo, In Vitro, Pulse Chase, Expressing, Incubation, Blocking Assay, Synthesized, Labeling, Staining, MANN-WHITNEY, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Liquid–liquid phase separation facilitates the biogenesis of secretory storage granules

doi: 10.1083/jcb.202206132

Figure Lengend Snippet: Localization of endogenous granin proteins with insulin in INS1 832/13 cells and validation of CGA/CGB double KO cells. (A–C) INS1 832/13 cells fixed and labeled with antibodies to insulin in or CGB (A), CGB (B) and SCG III (C) in green. Insulin puncta at the Golgi apparatus in the perinuclear region, outlined using the dashed lines in the merge colocalize with each of the granin proteins. For CGB and CGA, images are a single slice from a confocal stack and in case of SCG III, an average projection from three consecutive slices from a confocal image. (D) Representative images of INS1 832/13 cells (wild type; top) and CGA/CGB double knockout (dKO; bottom) stained for CGA and GM130 antibodies to validate absence of CGA staining from the dKO cells. (E) Western blot (top) shows cell lysates from INS1 832/13 wild-type and dKO cells probed using CGB antibody. Note the absence of band in dKO cells which have been highlighted using the red rectangle. The bottom blot is probing of the same membrane for actin, which is used as a loading control. (F) qPCR to monitor the reduction in transcripts for Chga and Chgb in wild-type and dKO cells. Values are represented as mean ± SD from three independent experiments and expressed relative to Gapdh. Relative values for wild-type cells are normalized to 1. Statistical analysis was performed using unpaired two-tailed t test ***P < 0.001. Source data are available for this figure: .

Article Snippet: For cloning of sfGFP and 6X-His tagged constructs of human CGA (pBT-PAF-CGA_sfGFP_6XHis) and CGB (pBT-PAF-CGB_sfGFP_6XHis), coding regions of CGA and CGB were cloned by PCR amplifications from expression plasmids from Origene (cat no. RC RC200492; CGA and cat no. RC201744; CGB).

Techniques: Biomarker Discovery, Labeling, Double Knockout, Staining, Western Blot, Membrane, Control, Two Tailed Test