Fig. 6: Full-length C1ql1 assembles into higher-order oligomers for cluster formation and recruitment with BAI3.

a Sequence alignment showing the two conserved cysteine residues indicated by orange triangles in the NT region among C1ql family members. b aSEC analyses of bacteria-expressed full-length C1ql1 proteins in the absence or presence of DTT in the buffer. Three distinct peaks were observed in the elution profile, indicating its high-oligomer, hexamer, and trimer forms, respectively. c, d Negative-staining EM analyses of the different assemblies of the full-length, wild-type C1ql1 protein samples in fractions P3 (c) and P2 (d). In the P2 fraction, two types of hexamers were observed, indicated by green (gC1q-mediated hexamers) and red arrowheads (NT-mediated hexamers). To enhance the contrast, the representative images were low-pass filtered to 10 Å. e EM analyses of protein particles from the P2 fraction using 2D-classification. f Negative-staining EM analysis of the particles in the P1 fraction. The cluster-like patterns were circled by black lines. A magnified view of one of the particle clusters observed in the P1 fraction. A serpentine structure is observed, in which two adjacent hexamers resembling gC1q- and NT-mediated hexamers are labeled by green and red arrowheads, respectively. The representative images were low-pass filtered to 10 Å. g Cell surface recruitments of HEK293T cell-secreted full-length C1ql1 and its C2S mutant by BAI3 on the cell surfaces. The corresponding line analyses of GFP and Flag intensities along the cell surfaces were indicated by white dashed lines as shown on the right. h Statistical analysis of the relative intensity ratio of Flag to GFP on the cell surface derived by quantifying the fluorescent signals as shown in (g). Data were collected from 20 cells in each condition. Bars represent the means ± SEM. The unpaired Student’s t-test two-sided analysis was used to define a statistically significant difference. i NS-EM analyses of the full-length C1ql1-C2S mutant secreted from HEK293F cells. gC1q-mediated hexamer particles were indicated by green arrowheads.