Fig. 3: NtDD dimerization and its role in PGL-1 self-assembly.
From: C. elegans germ granules require both assembly and localized regulators for mRNA repression
a Structural model of the NtDD dimer. Subunits in yellow and brown. b, c Enlargement of dimer interface (red box in a). PGL-1 amino acids (b) K126 and K129, and (c) R123 interact with apposing subunit side chains. Residue labels in yellow or brown to indicate their representative subunits. d–f Size exclusion chromatography and multi-angle light scattering (SEC-MALS) of recombinant PGL-1 NtDD (d) wild-type (44,170 (±6.509%) Da), (e) K126E K129E (23,740 (±2.455%) Da), and (f) R123E (23,210 (±2.099%) Da) proteins. Differential refractive index (left y-axis) in arbitrary units (blue). Molecular weight (MW, right y-axis) in dalton (Da, red). Wild-type protein measured the approximate size of a dimer, while both mutant proteins measured approximately as monomers. BSA control protein analyzed by SEC-MALS in Supplementary Fig. 5g. g Diagram of C. elegans PGL-1 C-terminally tagged with GFP. N-terminal dimerization domain (NtDD, yellow), central dimerization domain (CDD, orange), C-terminal region (C-region), RGG repeats (blue), and GFP (green). h–k Representative images of (h) GFP-tagged PGL-1, (i) GFP alone, and GFP-tagged PGL-1, (j) K126E K129E, and (k) R123E mutants expressed in Chinese Hamster Ovary (CHO) cells. Cell cultures were imaged live, and GFP-positive cells counted for the presence or absence of granules. Images show the majority result (percentages noted above image). Fisher’s exact test versus GFP: PGL-1 GFP (p-value < 0.0001), PGL-1 K126E K129E (p-value = 0.3958), PGL-1 R123E (>0.9999). Hoechst (DNA) in blue. GFP in green. Scale bar, 10 µm.
