Fig. 4 | Nature Communications

Fig. 4

From: Hexameric NuMA:LGN structures promote multivalent interactions required for planar epithelial divisions

Fig. 4

Binding of dimeric NuMA to LGNTPR generates protein networks. a NuMA:LGN form hetero-hexamers in mitotic cells. HEK293T cells stably depleted of endogenous NuMA and stably expressing NuMA1821–2115 were transiently co-transfected with plasmids expressing human GFP-LGN wild-type and LGN-3xFLAG wild-type or GFP-LGN-ΔOLIGO and LGN-ΔOLIGO-3xFLAG. After 48 h, mitotic lysates were immunoprecipitated (IP) with anti-GFP antibodies conjugated to agarose beads, and immunoblotted with the indicated antibodies. Cells transfected with LGN-3xFLAG constructs were used as specificity control of the IP, and wild-type HEK293T cells were loaded to monitor NuMA depletion (IB anti-NuMA at a molecular weight of 250 kDa) and NuMA1821–2115 expression (IB anti-NuMA at a molecular weight of 37 kDa). b Scheme of NuMA full-length, the C-terminal part of NuMA coiled-coil, and the NuMA chimera used to assemble NuMA:LGNTPR multimers in vitro. c SLS profile of NuMA1592–1694 showing an average molecular mass of 24 kDa along the peak, as expected for a dimeric construct. d SEC elution profiles of the NuMA chimeric construct encompassing the coiled-coil and the LGNBD in complex with LGNTPR (LGN1–409, blue trace) or LGNTPR-ΔOLIGO (LGN13–350, yellow trace). Complexes of NuMA-chimera with the oligomerization-deficient LGNTPR construct elute later indicating that they have an average molecular weight lower than the ones assembled with LGNTPR, as confirmed by the SLS analyses of Fig. 4e–g. The elution profile of globular molecular weight markers is shown in gray dotted lines. e SLS profile of the NuMA-chimera:LGNTPR sample indicating a polydispersed population of complexes with molecular masses in the range of 4.6 MDa to 130 kDa. f Scheme of different oligomers resulting from the assembly of NuMA with LGNTPR. The minimal stoichiometry of dimeric NuMA engaged in hetero-hexamers with LGNTPR is 6:6 (left). When each of the dimeric NuMA chains belongs to a different LGNTPR donut, higher order protein networks with diverse stoichiometry are possible (right). g SLS analysis of NuMA-chimera:LGN-TPR-ΔOLIGO showing that in the absence of NuMA:LGN oligomerization complexes with an average molecular mass of 136 kDa are formed, corresponding to 2:2 hetero-tetramers

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