De novo centriole formation in human cells is error-prone and does not require SAS-6 self-assembly
- National Yang-Ming University, Taiwan
- The Rockefeller University, United States
- Academia Sinica, Taiwan
- Memorial Sloan-Kettering Cancer Center, United States
Figures
Figure 1 with 2 supplements
De novo centrosome formation in the absence of SAS-6 self-oligomerization.
(A) Wild-type (WT) or acentrosomal (p53-/-; SAS-6 -/-; clone #1) RPE1 cells were stained with anti-centrin (green) and γ-tubulin (red). DNA (DAPI, blue). (B) Western blot analysis of WT or SAS-6-/- …
Figure 1—figure supplement 1
Characterization of SAS6-/-; p53-/- cell lines (clone #1 and #2).
(A, B) Sequence analyses of SAS-6 and p53 alleles in clone #1 and clone #2 as indicated. The sequence of wild-type and edited alleles are in black and blue, respectively. The lesion (deletion or …
Figure 1—figure supplement 2
Induction of de novo centrosome formation in clone #2 SAS6-/-; p53-/- cells in the absence of SAS-6 self-oligomerization.
(A, B) Wild-type (WT) or clone #2 acentrosomal (p53-/-; SAS-6 -/-) RPE1 cells were stained with indicated antibodies during interphase (A) and mitosis (B). DNA (DAPI, blue). (C, D) Clone #2 …
Figure 2 with 1 supplement
De novo centrioles formed in the absence of SAS-6 self-oligomerization can ciliate and duplicate.
(A) Isogenic SAS-6-/-; p53-/- cells carrying indicated SAS-6 constructs (see ‘Methods’) were arrested in S phase and induced to express indicated SAS-6 mutants for 16 hr and then immunostained with …
Figure 2—figure supplement 1
Characterization of de novo centrioles formed in the absence of SAS-6 self-oligomerization.
p53-/-; SAS-6 -/- cells (clone #1) exogenously expressing full-length SAS-6 (FL) or various SAS-6 mutants as indicated were analyzed by immunofluorescence with indicated antibodies.
Figure 3
SAS-6 self-assembly is not essential for the ninefold symmetry of centrioles.
Isogenic SAS-6-/-; p53-/- cells carrying indicated SAS-6 constructs (see ‘Methods’) were induced to express indicated SAS-6 mutants for 3 days, and then processed for serial sectioning electron …
Figure 4
Centrioles formed through de novo assembly are error-prone.
(A–C) Isogenic SAS-6-/-; p53-/- cells carrying indicated SAS-6 constructs (see ‘Methods’) were induced to express indicated SAS-6 mutants for 3 days, and then processed for serial sectioning …
Videos
Video 1
EM tomography of a normal centriole from SAS-6DM4 expressing cells (related to Figure 3).
Note that a centriole on the right in cross-sectional views contains nine MT triplets.
Video 2
EM tomography of an abnormal centriole from SAS-6DM4 expressing cells (related to Figure 4).
Note that a centriole on the right in cross-sectional views contains eight MT triplets.
