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. Author manuscript; available in PMC: 2016 May 23.
Published in final edited form as: Nat Cell Biol. 2015 Nov 23;18(1):87–99. doi: 10.1038/ncb3274

Figure 5. Ana1 loads Asl to daughter centriole for centriole-to-centrosome conversion in cultured Drosophila cells.

Figure 5

(a) D.Mel-2 cells were depleted of endogenous Asl and immunostained to reveal Asl, Dplp, Sas-6 and DNA. Note: Asl loses its ring structure whereas Dplp remains intact. 86% (n=28) of interphase centrosomes fail to recruit Sas-6 to a site for pro-centriole formation.

(b) D.Mel-2 cells were depleted of GST (control) or Ana1 and immunostained to reveal Ana1, Dplp and DNA. Cells with single centrosome were selected indicating compromised duplication. Depletion of Ana1 does not affect existing centrioles but prevents Ana1 recruitment onto newly formed daughters.

(c) Ana1-depleted cells were immunostained to reveal Dplp, DNA and Sas-6, Ana2 or Sas-4. Recruitment of Sas-6, Ana2 and Sas-4 to interphase daughter centrioles is not affected.

(d) Control or Ana1-depleted cells were immunostained to reveal Asl, Dplp and DNA or Sas-4, Asl and DNA. Depletion of Ana1 leads to failure of Asl recruitment to daughters in metaphase and anaphase. Recruitment of Dplp is similarly impaired in metaphase. Sas-4 is seen on mitotic mother and daughter whereas Asl only on mother in the absence of Ana1. Arrow indicates Dplp on daughter centriole.

(e) D.Mel-2 cells were depleted of GST or Ana1 and immunostained to reveal Sas-6, γ-tubulin and DNA. Note: in control telophase cells, mother and daughter (new mother-to-be) centrioles are both surrounded by γ-tubulin (upper panel, 30 cases out of 30); daughter centrioles in Ana1-depleted cells fail to recruit γ-tubulin by this stage (lower panel, 8 cases out of 8).

(f) D.Mel-2 cells were depleted of GST or Cep135 and immunostained to reveal Dplp, DNA and Ana1 or Asl. Note: 73% (n=67) and 78% (n=18) of metaphase centrosomes fail to load Ana1 and Asl to daughter centrioles, respectively.

(g) D.Mel-2 cells were depleted of GST or Ana1 and immunostained to reveal Cep135, Sas-6 and DNA. Loading of Cep135 to the daughter is impaired by Ana1 depletion. Error bars, SEM (Standard Error of the Mean); **, p<0.005 (two-tailed student’s t-test); n=20 and 21 centrosomes pooled across 3 independent experiments for GST and Ana1 RNAi, respectively.

Scale bars, 5 μm for complete images of cells and 500 nm for magnified centrioles.