Figure 2. Multivalent interactions drive... | Rockefeller University Press

$Multivalent interactions drive the condensed phase formation of the IRSp53/Shank3 mixture. (A) Schematic diagram showing the domain organization of Shank3. Multiple sequence alignment of the proline-rich motif (PRM), which is responsible for IRSp53 to interact with different isoforms of Shank from human (h), mouse (m), and rat (r). (B) Fluorescence images showing IRSp53 and Shank3 are colocalized and enriched in the condensate droplets. IRSp53 and Shank3 were mixed at an equimolar ratio at 15 μM. The dashed box is selected for zoom-in view of the droplet fusion process in C. (C) Phase droplets that are in close contact fuse into larger ones over time. (D) FRAP experiments showing fluorescence recovery of IRSp53 and Shank3 in a condensed droplet after photobleaching. Fluorescence recovery curves represent the averaged signals from three droplets, and data are presented as mean ± SD. (E) Sedimentation-based experiments showing that IRSp53 and Shank3 undergo phase separation in a concentration-dependent manner. The amounts of proteins in the condensed phase/pellet fraction (P) and in the dilute phase/supernatant fraction (S) are quantified. Data are obtained from three batches of independent experiments and are presented as mean ± SD. (F) ITC-based measurement of the binding between IRSp53 and Shank3 WT (black titration curve) or ΔPRM (red titration curve). 200 μM of Shank3 was titrated into 20 μM of IRSp53 with 200 mM NaCl in the binding buffer. (G) Fluorescence imaging analysis showing that a series of Shank3 mutants with progressively weakened phase separation with IRSp53. 15 μΜ Alexa Fluor 488–labeled IRSp53 was mixed with 15 μΜ unlabeled various truncations of Shank3. Identical imaging settings were used for all groups. (H) Fluorescence images showing that the preformed IRSp53/Shank3 condensate droplets could be gradually dispersed by the addition of the PRM1 peptide. Both IRSp53 and Shank3 were at 15 μΜ. (I) Fluorescence imaging analysis showing that the BAR domain–mediated dimerization is critical for IRSp53/Shank3 phase separation. Myo-CC restores the oligomerization status of IRSp53 (also see Fig. S2), and the replacement of BAR with Myo-CC could rescue the loss of phase separation of IRSp53 with Shank3 resulting from the BAR domain deletion. Two protein concentrations, 15 and 30 μM, were assayed, and both were mixed at an equimolar ratio. Source data are available for this figure: SourceData F2.$

Figure 2.

Multivalent interactions drive the condensed phase formation of the IRSp53/Shank3 mixture. (A) Schematic diagram showing the domain organization of Shank3. Multiple sequence alignment of the proline-rich motif (PRM), which is responsible for IRSp53 to interact with different isoforms of Shank from human (h), mouse (m), and rat (r). (B) Fluorescence images showing IRSp53 and Shank3 are colocalized and enriched in the condensate droplets. IRSp53 and Shank3 were mixed at an equimolar ratio at 15 μM. The dashed box is selected for zoom-in view of the droplet fusion process in C. (C) Phase droplets that are in close contact fuse into larger ones over time. (D) FRAP experiments showing fluorescence recovery of IRSp53 and Shank3 in a condensed droplet after photobleaching. Fluorescence recovery curves represent the averaged signals from three droplets, and data are presented as mean ± SD. (E) Sedimentation-based experiments showing that IRSp53 and Shank3 undergo phase separation in a concentration-dependent manner. The amounts of proteins in the condensed phase/pellet fraction (P) and in the dilute phase/supernatant fraction (S) are quantified. Data are obtained from three batches of independent experiments and are presented as mean ± SD. (F) ITC-based measurement of the binding between IRSp53 and Shank3 WT (black titration curve) or ΔPRM (red titration curve). 200 μM of Shank3 was titrated into 20 μM of IRSp53 with 200 mM NaCl in the binding buffer. (G) Fluorescence imaging analysis showing that a series of Shank3 mutants with progressively weakened phase separation with IRSp53. 15 μΜ Alexa Fluor 488–labeled IRSp53 was mixed with 15 μΜ unlabeled various truncations of Shank3. Identical imaging settings were used for all groups. (H) Fluorescence images showing that the preformed IRSp53/Shank3 condensate droplets could be gradually dispersed by the addition of the PRM1 peptide. Both IRSp53 and Shank3 were at 15 μΜ. (I) Fluorescence imaging analysis showing that the BAR domain–mediated dimerization is critical for IRSp53/Shank3 phase separation. Myo-CC restores the oligomerization status of IRSp53 (also see Fig. S2), and the replacement of BAR with Myo-CC could rescue the loss of phase separation of IRSp53 with Shank3 resulting from the BAR domain deletion. Two protein concentrations, 15 and 30 μM, were assayed, and both were mixed at an equimolar ratio. Source data are available for this figure: SourceData F2.

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