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. Author manuscript; available in PMC: 2015 Oct 6.
Published in final edited form as: Curr Biol. 2014 Sep 11;24(19):2327–2334. doi: 10.1016/j.cub.2014.08.029

Figure 3. Pcnt interacts with Centriolin, Cep215 and ninein, is required for their localization to the mitotic spindle poles, and contributes to spindle orientation.

Figure 3

(A) Ninein and Cep215 (green) are significantly displaced from mitotic spindle poles in Pcnt−/− MEFs compared to Pcnt+/+ MEFs. DNA, blue. Bar, 5 μm. Quantification in Figure S3D.

(B-C) Immunoprecipitation from Pcnt+/+ MEFs compared to Pcnt−/− MEFs reveals protein complexes: Pcnt and Cep215 (also see in Figure S3G-H); Cep215 and ninein; Dynein, Cep215, and Pcnt.

(D) Pcnt−/− MEFs show significant displacement of centriolin (green) from mitotic spindle poles compared to Pcnt+/+ MEFs. DNA, blue. Bar, 5 μm.

(E-F) Immunoprecipitation from human epithelial cells demonstrates an interaction between Pcnt and centriolin (E, reciprocal immunoprecipitation in Figure S4B). An interaction was observed in cells stabily expressing Centriolin-mVenus [21] and Cep215.

(G-H) Cells expressing Cep215 c-term (c-term; aa1201-1822; reported in [22]) have an increase in Centriolin recruitment to spindle poles that was comparable to the increased recruitment of the c-terminus of Cep215 (p<0.001 as marked, n.s. is non-significant, representative of three experiments). A loss of endogenous Cep215 at the spindle pole was observed using an antibody targeting the Cep215 n-terminal domain. No effect was observed on Pcnt localization when Cep215 c-term was expressed.

(I) Centriolin or Pcnt-depleted cells show increased spindle misorientation. Shown are maximum projections of z-axis; centrosome (5051, red), NuMA (green). Note that NuMA localization is unaffected at both spindle poles and cell cortex (yellow arrows, x/y-axis maximum projection).

(J) Quantification showing a significant increase (>5°) of spindle angle in cells depleted of either centriolin or Pcnt (~3-fold, n=3 experiments, p<0.001 comparing spindle angles >5° across treatments. n>20 cells counted/experiment).

(K) Model represents a Pcnt-mediated interaction between Cep215 and dynein (as demonstrated by immunoprecipitation in 3C), and Ninein interacting peripherally with Cep215 (shown both by immunoprecipitation in 3B, S3G, and genetically in S3I). Pcnt-anchored Cep215 further interacts with Centriolin (3E-F) through the c-terminal domain of Cep215 (4G-H).