Journal: Molecular Biology of the Cell

Article Title: PPP1R35 ensures centriole homeostasis by promoting centriole-to-centrosome conversion

doi: 10.1091/mbc.e18-08-0525

Figure Lengend Snippet: Figure 1. PPP1R35 is enriched on daughter centrioles (A) Schematic outlining the SILAC screen used to classify centrosomal proteins (Tanos et al., 2013; Wang et al., 2013). Two populations of differentially labeled centrosomes were purified in equal amount. One population of centrosomes (labeled with light isotopes) was isolated from cells arrested in S-phase, while the other (labeled with heavy isotopes) was from cells arrested in S-phase and overexpressing Plk4. The former consisted mainly of one mother and one daughter centriole pair, while the latter was made up of one mother and 4-6 daughter centrioles complex, or what commonly known as the centriole rosette. The mixture was subject to a quantitative proteomics analysis SILAC, and proteins that exhibited a high heavy:light isotope ratio was considered as daughter centriole protein candidates. A list of candidates identified by the high H/L ratios is shown. Highlighted in red is PPP1R35, which has a H/L ratio similar to that of the daughter centriole protein SAS-6. (B) PPP1R35 localizes to all centrioles throughout the cell cycle, but is more enriched at newborn daughter centrioles in S/G2 phase. Immunofluorescence (IF) images of RPE1 cells at different stages of the cell cycle showing centrin2-GFP (green), PPP1R35 (red), SAS-6 (blue, pseudo-colored) and DAPI (blue). Mother (M) and daughter (D) centrioles were marked. Scale bar, 5 μm, 1 μm in inset. (C) Co-localization of PPP1R35 (red) with SAS-6 (blue) at the centriole rosette in PLK4 overexpressing cells. Centrin2-GFP marks centrioles. Scale bar, 5 μm, 1 μm in inset.

Article Snippet: Antibodies used in this study were listed with the information on working dilution and source in parentheses – anti-centrin2 (mouse, 1:1000, 04-1624, Millipore), anti--tub (mouse, 1:500, sc51715, Santa Cruz Biotechology), anti-BrdU (rat, 1:500, MCA2060T, AbD Serotec), anti-SAS-6 (mouse, 1:200, sc-81431, Santa Cruz Biotechnolocy), anti-HA (mouse, 1:1000, MMSH101P, Covance; rat, 1:1000, 11867423001, Roche Diagnostic), anti-c-myc (mouse, 1;1000, sc-40, Santa Cruz Biotechnolocy), anti-STIL (rabbit, 1:200, A302-441A, Bethyl Laboratories), anti-CP110 (rabbit, 1:200, 12780-1-AP, Proteintech), anti-CEP135 (rabbit, 1:200, kind gift from Erich Nigg at U. of Basel), anti-CEP120 (rabbit, 1:5, kind gift from Moe R. Mahjoub at Washington U.), anti-tubulin (mouse, 1:10,000, T6199, Sigma), anti-centrobin (rabbit, 1:5, kind gift from K. Rhee at Seoul National U.), anti-C-Nap1 (rabbit, 1:200, produced against the human C-Nap1 as previously described(Tsou and Stearns, 2006)), anti-CEP295 (rabbit, 1:1000, ab122490, Abcam).

Techniques: Multiplex sample analysis, Labeling, Purification, Isolation, Quantitative Proteomics, Immunofluorescence

Journal: Journal of Cell Science

Article Title: Enhancement of CEP215 dynamics for spindle pole assembly during mitosis

doi: 10.1242/jcs.263542

Figure Lengend Snippet: Generation of CEP215-truncated mutants with defects in molecular dynamicity. (A) Schematic diagram illustrating the CEP215 protein with functional sites (CM1 and CM2), protein-binding sites, coiled-coil domains (CCDs), intrinsically disordered regions (IDRs) and the truncated mutant sites used in this study. (B) The number of cells with (w/) cytoplasmic clusters of FLAG–CEP215–CRY2-truncated mutant proteins was counted. * P <0.05 (one-way ANOVA with Tukey's post hoc test). (C) FRAP signals of the mCh–CEP215–CRY2 mutant proteins at the cytoplasm were measured for up to 600 s. w/o, without. (D) Stably expressed FLAG–mCh–CEP215 mutants in p53 KO ; CEP215 KO HeLa cells were subjected to immunoblot analyses with anti-CEP215, FLAG and GAPDH antibodies. Blot representative of four experimental repeats. (E) FRAP analyses of the FLAG–mCh–CEP215 proteins were carried out at mitotic phase centrosomes of the rescued cells. GFP–centrin2 marks the centrosomes. The analyzed cells were photographed with differential interference contrast microscopy. Scale bars: 1 μm (FRAP); 10 μm (DIC). (F) The FRAP signals of FLAG–mCh–CEP215 (WT, ΔCCD5 and L1867S) at mitotic phase centrosomes were measured for up to 160 s. For B, C and F, at least 90 (B), 30 (C) and 10 (F) cells per experimental group were counted in three independent experiments. Values are mean±s.e.m. C and F show arbitrary units.

Article Snippet: For immunostaining analyses, we used rabbit antibodies against CEP215/CDK5RAP2 (06-1398; Merck Millipore; 1:100), PCNT , CEP192 (A302-324A; Bethyl Laboratory; 1:100), α-tubulin (ab18251; Abcam), γ-tubulin (ab11317; Abcam; 1:100); mouse antibodies against centrin2 (04-1624; Merck Millipore), α-tubulin (T6199; Sigma; 1:100), FLAG (F3165; Sigma; 1:100); and goat anti-FLAG (ab1257; Abcam; 1:100) antibodies.

Techniques: Functional Assay, Protein Binding, Mutagenesis, Stable Transfection, Western Blot, Microscopy

Journal: The FASEB Journal

Article Title: Roles of Cep215/Cdk5rap2 in establishing testicular architecture for mouse male germ cell development

doi: 10.1096/fj.202401541R

Figure Lengend Snippet: The numbers of germ, Sertoli, and Leydig cells in Cep215 KO testes. (A) Immunohistochemistry of the testes from Cep215 KO mice at P7 and P17 with Sox9 (green) and γH2AX (red) antibodies. (B) The number of Sertoli and germ cells per tubule was counted. At least 1200 cells per experimental group were counted ( n = 3). (C) Immunohistochemistry of the testes from Cep215 KO mice at P7, P17, and adult with HSD3β antibody. (D) The number of Leydig cells per section was counted (bars). The cross‐sectional area was also measured (dots). At least 90 cells per section were counted ( n = 3). (A, C) Scale bars: 50 μm. (B, D) Values are means and SEM. The statistical significance was determined by two‐way ANOVA (B) and unpaired t ‐test (D). * p < .05. ns, not significant.

Article Snippet: The Cep215 and Pcnt antibodies were previously described., The antibodies were commercially purchased as listed below: Cep215 (06‐1398; Millipore), γH2AX (05‐636; Upstate), Sycp3 (ab15093; Abcam), centrin‐2 (04‐1624; Millipore), γ‐tubulin (ab11316‐100; Abcam), γ‐tubulin (ab11317; Abcam), claudin‐11 (36‐4500; Invitrogen), connexin43 (3512; Cell signaling), Sox9 (AB5535; Millipore), vimentin (ab92547; Abcam), Gapdh (AM4300; Invitrogen), and HSD3β (GTX102744; Genetex).

Techniques: Immunohistochemistry

Journal: Journal of Cell Science

Article Title: Enhancement of CEP215 dynamics for spindle pole assembly during mitosis

doi: 10.1242/jcs.263542

Figure Lengend Snippet: Spindle pole recruitment of the FLAG–mCh–CEP215 mutant proteins. (A) The FLAG–mCh–CEP215 mutant replacement HeLa cells were treated with proTAME for the metaphase arrest. The cells were co-immunostained with anti-CEP215 and PCNT antibodies. DNA was stained with DAPI. Scale bar: 10 μm. (B,C) Spindle pole intensities of FLAG–mCh–CEP215 (B) and endogenous PCNT (C) were measured and presented as box plots. (D) Cell cycle of the FLAG–mCh–CEP215 mutant replacement cells were synchronized with a double-thymidine block-and-release method and co-immunostained with anti-CEP215, PCNT, γ-tubulin and CEP192 antibodies. The area of the specific PCM proteins were measured. For B–D, at least 50 cells per experimental group were counted in three independent experiments. In B and C, box represents the 25–75th percentiles, and the median is indicated. The whiskers show the values for the 5th to 95th percentiles. D shows mean±s.e.m. * P <0.05 (one-way ANOVA with Tukey's post hoc test). a.u., arbitrary units. (E) Model. (a) PCM proteins are recruited to the centrosomes at late G2 phase. Molecular dynamics of CEP215 is upregulated for formation of compact, functional spindle poles at M phase. (b) In the absence of CEP215, PCM proteins are insufficiently recruited to the centrosomes, resulting in spindle defects. (c) PCM proteins are not properly recruited to the centrosomes when molecular dynamicity of CEP215 is reduced in mitotic centrosomes, resulting in spindle defects.

Article Snippet: For immunostaining analyses, we used rabbit antibodies against CEP215/CDK5RAP2 (06-1398; Merck Millipore; 1:100), PCNT , CEP192 (A302-324A; Bethyl Laboratory; 1:100), α-tubulin (ab18251; Abcam), γ-tubulin (ab11317; Abcam; 1:100); mouse antibodies against centrin2 (04-1624; Merck Millipore), α-tubulin (T6199; Sigma; 1:100), FLAG (F3165; Sigma; 1:100); and goat anti-FLAG (ab1257; Abcam; 1:100) antibodies.

Techniques: Mutagenesis, Staining, Blocking Assay, Functional Assay