Centrosome function is critical during terminal erythroid differentiation
2022; Springer Nature; Volume: 41; Issue: 14 Linguagem: Inglês
10.15252/embj.2021108739
ISSN1460-2075
Autores Tópico(s)Epigenetics and DNA Methylation
ResumoArticle9 June 2022Open Access Transparent process Centrosome function is critical during terminal erythroid differentiation Péter Tátrai Péter Tátrai Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, UK Contribution: Conceptualization, Resources, Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Fanni Gergely Corresponding Author Fanni Gergely [email protected] orcid.org/0000-0002-2441-8095 Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, UK Department of Biochemistry, University of Oxford, Oxford, UK Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Péter Tátrai Péter Tátrai Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, UK Contribution: Conceptualization, Resources, Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Fanni Gergely Corresponding Author Fanni Gergely [email protected] orcid.org/0000-0002-2441-8095 Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, UK Department of Biochemistry, University of Oxford, Oxford, UK Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Péter Tátrai1,3 and Fanni Gergely *,1,2 1Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, UK 2Department of Biochemistry, University of Oxford, Oxford, UK 3Present address: Solvo Biotechnology, Budapest, Hungary *Corresponding author. Tel: +44 01865613279; E-mail: [email protected] The EMBO Journal (2022)41:e108739https://doi.org/10.15252/embj.2021108739 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Red blood cells are produced by terminal erythroid differentiation, which involves the dramatic morphological transformation of erythroblasts into enucleated reticulocytes. Microtubules are important for enucleation, but it is not known if the centrosome, a key microtubule-organizing center, is required as well. Mice lacking the conserved centrosome component, CDK5RAP2, are likely to have defective erythroid differentiation because they develop macrocytic anemia. Here, we show that fetal liver-derived, CDK5RAP2-deficient erythroid progenitors generate fewer and larger reticulocytes, hence recapitulating features of macrocytic anemia. In erythroblasts, but not in embryonic fibroblasts, loss of CDK5RAP2 or pharmacological depletion of centrosomes leads to highly aberrant spindle morphologies. Consistent with such cells exiting mitosis without chromosome segregation, tetraploidy is frequent in late-stage erythroblasts, thereby giving rise to fewer but larger reticulocytes than normal. Our results define a critical role for CDK5RAP2 and centrosomes in spindle formation specifically during blood production. We propose that disruption of centrosome and spindle function could contribute to the emergence of macrocytic anemias, for instance, due to nutritional deficiency or exposure to chemotherapy. Synopsis Immature erythroid cells differentiate into red blood cells by undergoing a defined number of cell divisions followed by ejection of their nuclei. This study investigates the role of centrosomes in this process using ex vivo differentiation of fetal liver-derived erythroid progenitors in mice. The conserved centrosomal protein CDK5RAP2 facilitates erythroid differentiation both ex vivo and in vivo Centrosomes and CDK5RAP2 are crucial for normal spindle assembly in erythroblasts but not in embryonic fibroblasts Erythroblasts lacking CDK5RAP2 or centrosomes develop tetraploidy TP53 is activated in erythroblasts lacking CDK5RAP2 or centrosomes but is not responsible for the observed defects in erythropoiesis Unlike embryonic fibroblasts, erythroblasts do not expand their centrosomes in mitosis Introduction The centrosome is a small non-membranous subcellular organelle, which comprises two cylindrical centrioles that are embedded in a protein-rich matrix called the pericentriolar material (PCM). Through nucleating and tethering microtubules (MT), the centrosome acts as an important microtubule organizing center (MTOC) in both proliferating and non-proliferating cells. Centrosomes undergo a duplication cycle that coincides with DNA replication in the S-phase and involves the templated assembly of a single procentriole per each old centriole. The master regulator of centriole biogenesis is Polo-like kinase 4 (PLK4) that acts together with the essential structural proteins SAS-6 and STIL (Yamamoto & Kitagawa, 2020). During G2, centrosomes mature by increasing the size and nucleating capacity of the PCM, and upon entry into mitosis, the two centrosomes separate to facilitate bipolar spindle assembly. γ-tubulin-mediated MT nucleation and anchorage at the centrosome is mainly driven by CEP192 and further enhanced by AURORA-A and PLK1 kinase cascade in mitosis (Gomez-Ferreria et al, 2007; Lee & Rhee, 2011; Joukov et al, 2014; O'Rourke et al, 2014). The tight bond between parental centrioles and their procentriole is dissolved in mitosis, thereby allowing daughter centrioles to accumulate PCM in the following cell cycle. CEP215/CDK5RAP2 (cyclin-dependent kinase 5 regulatory subunit-associated protein 2) and pericentrin (PCNT) are important for PCM assembly and form the mitotic PCM scaffold (Megraw et al, 1999; Sawin et al, 2004; Fong et al, 2008; Lee & Rhee, 2011; Conduit et al, 2014; Woodruff et al, 2015; Feng et al, 2017). CDK5RAP2 and PCNT are interdependent for their centrosomal localization in mitosis but CDK5RAP2 seems non-essential for γ-tubulin recruitment during the mammalian cell cycle (Haren et al, 2009; Kim & Rhee, 2014; Gavilan et al, 2018). In addition, CDK5RAP2 connects centrioles with the PCM at mitotic spindle poles (Lucas & Raff, 2007; Barr et al, 2010; Chavali et al, 2016) and promotes centrosome cohesion from G1- through S- and G2-phases (Graser et al, 2007). Although the majority of proliferating animal cells contain centrosomes, these organelles have been suggested to play cell-type-specific roles such as their contribution to T-cell-mediated killing (Stinchcombe et al, 2006). Whether centrosomes have additional cell-type-specific functions in the hematopoietic lineage, and in particular, during red blood cell development, is not known. The latter is especially of interest because adult mice with mutations in the PCM component Cdk5rap2 have fewer but bigger red blood cells (RBC), which is defined as macrocytic anemia (Russell, 1979; Lizarraga et al, 2010). Erythropoiesis describes the process of red blood cell development. In mice, definitive erythropoiesis, the process whereby erythroid precursors differentiate into mature enucleated red blood cells, begins in the fetal liver. In the adult, the main site of steady-state erythropoiesis is the bone marrow, however, under anemic stress, red blood cells can be produced in the spleen (stress erythropoiesis). Hematopoietic stem cells differentiate into committed erythroid progenitors called BFU-E (burst-forming unit) and CFU-E (colony-forming unit) after their ability to form morphologically distinct colonies in semi-solid media (Koury, 2016). During terminal erythroid differentiation, progenitors undergo four to five cell divisions until they eject their nucleus to become reticulocytes (Zhang et al, 2003; Sankaran et al, 2012). These terminal cell divisions are unusual as they yield daughter cells that are morphologically and functionally different from their mothers. In addition, as they differentiate, erythroblasts (EBs) progressively decrease in cell size, condense their chromatin, and accumulate hemoglobin. Before enucleation, EBs exit the cell cycle, which has been attributed to accumulation of the cyclin-dependent kinase (CDK) inhibitors P27 (Hsieh et al, 2000; Li et al, 2006) and P18 (Han et al, 2017). P27 and P18 expression is controlled by the erythroid-specific transcription factor EKLF/KLF1 (Tallack et al, 2007; Gnanapragasam et al, 2016). The different stages of terminal erythroid differentiation can be distinguished based on expression of surface markers such as the transferrin receptor CD71/CD44 and the erythroid-specific marker glycophorin A (TER119). TER119 is present throughout terminal differentiation from early erythroblasts to mature red blood cells but absent in erythroid progenitors (Fig 1H) (Chen et al, 2009). Figure 1. Ex vivo differentiation of Cdk5rap2null erythroblasts recapitulates key features of macrocytic anemia Schematic of Cdk5rap2null (Cdk5rap2tm1b) allele generated from the EUCOMM-knockout first allele by Cre-mediated deletion of exon 5. Immunoblot showing CDK5RAP2 levels in Cdk5rap2 wild-type (WT), heterozygous (HET), and null erythroid progenitors (EPs) isolated from fetal livers. Actin was used as loading control. ** indicates non-specific band. Quantification of mean protein levels from (B). Numbers in brackets correspond to number of embryos analyzed. Immunofluorescence images of Cdk5rap2 WT, HET, and null erythroid progenitors isolated from fetal livers. Progenitors were stained for CDK5RAP2 (grey), γ-tubulin (magenta), and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 3 μm. Insets show higher magnification of centrosomes. Scale bar, 1 μm. Quantification of mean centrosomal signal intensities of CDK5RAP2 and γ-tubulin from (D). Numbers in brackets correspond to number of embryos analyzed with a total number of 470 (WT), 406 (HET), and 379 (null) progenitors. Complete blood count analysis from adult mice with genotypes as indicated. The number of mice analyzed is shown in brackets. RBC = red blood cell. MCV = mean corpuscular volume. RDW = red blood cell distribution width. HCT = hematocrit. MCH = mean corpuscular hemoglobin. Quantification of serum erythropoietin (EPO) levels from adult mice with genotypes as indicated. The number of mice analyzed is shown in brackets. Schematic of the ex vivo differentiation culture system. ImageStream images of ex vivo cultured enucleating EBs and reticulocytes. Cells were stained for TER119 (erythroid marker, green) and DNA (Hoechst, blue). BF: bright field. Scale bar, 5 μm. Quantification of enucleating EBs and reticulocytes after 48 h (T48) in ex vivo culture. Genotypes are as indicated. The numbers in brackets correspond to the number of embryos analyzed. Quantification of enucleating EB size from (I). The numbers in brackets refer to the number of embryos analyzed. Data information: Box plots show 5th and 95th (whiskers) and 25th, 50th, and 75th percentiles (boxes). Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos (C, E, I, and J) or number of mice (F and G). Statistical significances were determined by one-way ANOVA test with Tukey's (C, E, F, G, J) or Kruskal–Wallis test with Dunn's (K) multiple comparisons. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Download figure Download PowerPoint Erythroid enucleation can be divided into three stages: nuclear polarization, extrusion, and physical cell separation. In preparation for erythroid enucleation, histone deacetylation promotes chromatin condensation leading to reduced nuclear size and transcriptional inactivation (Popova et al, 2009; Ji et al, 2010). During polarization, the nucleus migrates toward one side of the cell in an MT-dependent manner and gets ejected by F-actin polymerization and actomyosin contraction forces (Konstantinidis et al, 2012; Ubukawa et al, 2012; Wang et al, 2012; Kobayashi et al, 2016; Nowak et al, 2017). Organelles, including centrosomes, are cleared during and following enucleation by autophagy-dependent and -independent degradation (Watanabe et al, 2016; Moras et al, 2017). The final step of enucleation, the separation of the nucleus from the nascent reticulocyte, is mediated by vesicle and vacuole trafficking (Keerthivasan et al, 2010; Konstantinidis et al, 2012). Macrophages, which associate with differentiating EBs in the erythroblastic island, engulf the extruded nuclei (pyrenocytes) and enable release of reticulocytes into the bloodstream where they further mature into erythrocytes. Several signaling and cytoskeletal components have already been assigned roles during enucleation. Indeed, it has been previously reported that late-stage EBs (i.e., EBs that undergo one last division with their daughters subsequently enucleating) contain one or two γ-tubulin-positive foci, indicative of the presence of MTOCs (Konstantinidis et al, 2012; Wang et al, 2012; Kobayashi et al, 2016). Furthermore, classical electron microscopy studies identified centrioles in enucleating EBs from rabbit bone marrow (Skutelsky & Danon, 1970). However, the functional relevance of these MTOCs/centrosomes in terminal erythroid differentiation and enucleation is not known. Previous studies using small molecule inhibitors of centrosome-associated mitotic kinases (e.g., PLK1, AURORA-A) and MT motors (e.g., EG5) in a human erythroid culture system concluded that MTOCs/centrosomes were dispensable for EB enucleation (Ubukawa et al, 2012; Kobayashi et al, 2016). Inhibiting these pleiotropic regulators may not fully block centrosome function, and thus, contribution by the MTOC/centrosome remains unclear. Here, we employ an ex vivo differentiation system of erythroid progenitors isolated from mouse fetal liver to probe the function of centrosomes, and the PCM in particular, during erythroid differentiation and enucleation. Using a small molecule inhibitor to induce centrosome depletion or by genetic removal of the PCM component Cdk5rap2, we show that faithful regulation of spindle assembly in late-stage EBs is a prerequisite for efficient enucleation. Together, our findings elucidate the underlying cellular mechanism for the macrocytic anemia observed in mice in the absence of CDK5RAP2. Results Cdk5rap2null mice exhibit macrocytic anemia We set out to interrogate the role of centrosomes during erythropoiesis. The starting point for this project was the Cdk5rap2/Cep215tm1a mouse strain, generated by EUCOMM, which carries a LacZ gene-trapping cassette (Skarnes et al, 2011). By crossing these mice to PGK-Cre mice, we generated a strain where exon 5 of Cdk5rap2 is deleted resulting in a LacZ-tagged null allele (also called tm1b) (Fig 1A). Using our previously published polyclonal N-terminal antibody against human CDK5RAP2 (Barr et al, 2010), we could not detect a protein product of the expected size in cell lysates of Cdk5rap2tm1b erythroid progenitors, the cell population representing the majority of hematopoietic progenitors cells in the fetal liver (Zhang et al, 2003). Likewise, no signal was visible in the centrosomes of these progenitors (Fig 1B–E). In vitro transcription/translation (IVT) from cDNA spanning different mouse exons revealed that the protein sequence encoded by exon 7 of mouse Cdk5rap2 is the main recognition site of this antibody (Fig EV1A and B). On immunoblots of cell lysates, this antibody recognized an additional band below 190 kDa, which appeared identical across all Cdk5rap2 genotypes both in erythroid progenitors and mouse embryonic fibroblasts (MEFs) (Figs 1B and EV1C). To assess the specificity of this band, we tested three commercial antibodies against C-terminal sequences of human CDK5RAP2 but none recognized murine CDK5RAP2. In native gel electrophoresis, our antibody recognized a single band in cell lysates of wild-type MEFs, which was missing from Cdk5rap2tm1b MEFs (Fig EV1D), suggesting that the band below 190 kDa is unique to denaturing conditions and may be non-specific. Consistently, in immunofluorescence, our antibody stained interphase and mitotic centrosomes of wild-type but not CDK5RAP2-deficient MEFs (Fig EV1E and F). We cannot exclude that N-terminally truncated protein products lacking the first 220 amino acids (corresponding to exons 1–7) are expressed in the mutants, but from genomic databases we found no evidence for splice variants of Cdk5rap2 that lack exon 7 or where translation starts downstream of exon 7, and so we refer to this strain as Cdk5rap2null hereafter. Click here to expand this figure. Figure EV1. Hematopoietic progenitor pools are largely normal in Cdk5rap2null mice A. Immunoblot showing the detection of mCherry-tagged IVT. Ponceau-S staining was used to compare equal loading. 7* marks the alternative start site in exon 7. B. Schematic representation showing the antigen and the epitope of CDK5RAP2 N-terminal antibody as suggested from IVT experiments in (A). C. Immunoblot showing CDK5RAP2 levels in Cdk5rap2 wild-type (WT) and null mouse embryonic fibroblasts (MEFs). Actin was used as loading control. ** indicates unspecific band. D. Immunoblot of native gel showing CDK5RAP2 levels in Cdk5rap2 wild-type (WT) and null mouse embryonic fibroblasts (MEFs). Tubulin was used as loading control. E. Immunofluorescence images of interphase (I) or mitotic (M) Cdk5rap2 WT and null mouse embryonic fibroblasts (MEFs). MEFs were stained for CDK5RAP2 (grey), γ-tubulin (magenta), and DNA (Hoechst, blue). Images are maximum-intensity projections of deconvolved z-stacks. Scale bar, 4 μm. Insets show higher magnification of centrosomes. Scale bar, 500 nm. F. Quantification of mean centrosomal signal intensities of CDK5RAP2 from (E). Numbers in brackets correspond to number of MEF lines analyzed with 168 (WT) and 154 (null) interphase cells and 59 (WT) and 56 (null) mitotic cells. G, H. Quantification of hematopoietic stem and progenitor cells (G) and erythroblast stages (H) in bone marrow (BM) of 10- to 13-week-old mice. Genotypes are as indicated. The number in brackets refers to the number of mice analyzed. HSC = hematopoietic stem cells. MPP = multipotent hematopoietic progenitors. HPC = hematopoietic progenitor cells. I. Quantification of erythroblast stages in E13.5 fetal livers. Genotypes are as indicated. The number in brackets refers to the number of embryos analyzed. J. Quantification of cell size of TER119pos cells in bone marrow (BM) of 10-week-old mice. Genotypes are as indicated. The number in brackets refers to the number of mice analyzed. Data information: Box plots show 5th and 95th (whiskers) and 25th, 50th, and 75th percentiles (boxes). Bar graph in J displays mean ± s.d. Statistical analysis was based on the number of MEF lines (F), the number of mice (G, H, and J), or the number of embryos (I). Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test (F and I), Mann–Whitney test (G and H), or two-tailed unpaired Student's t-test (J). **P ≤ 0.01. Download figure Download PowerPoint Cdk5rap2null mice exhibit mild macrocytic normochromic anemia similarly to their parental Cdk5rap2tm1a(EUCOMM)Wtsi strain (International Mouse Phenotypic Service (IMPC), mousephenotype.org). Anemia is defined as a decrease in total amount of red blood cells (RBC) or hemoglobin levels. Cdk5rap2null mice have fewer but bigger red blood cells (RBC), characteristic of macrocytic anemia (Fig 1F). Their red blood cell size is also more variable, as indicated by a higher red cell distribution width (RDW) value. Because mice lacking CDK5RAP2 have fewer but larger RBC, their hematocrit (HCT) levels are normal. Likewise, total hemoglobin levels in blood are also unaffected (described as normochromic) because CDK5RAP2-deficient RBC accumulate greater hemoglobin mass per cell (mean corpuscular hemoglobin, MCH) but are reduced in numbers (Fig 1F). The same phenotype was observed in the Hertwig's anemia (an/an) mouse model (Russell, 1979), which was generated by mutagenesis and subsequently shown to carry an in-frame deletion of exon 4 in Cdk5rap2 (Lizarraga et al, 2010). Anemia can trigger a compensatory mechanism through increased release of the cytokine erythropoietin (EPO) into the blood to stimulate stress erythropoiesis in the spleen. Consistent with anemic stress, we found elevated EPO levels in Cdk5rap2null mice (Fig 1G). Additionally, mild-to-moderate hyperplasia and extramedullary (i.e., outside of the bone marrow) hematopoiesis were observed in the spleen of homozygous Cdk5rap2tm1a(EUCOMM)Wtsi mice (IMPC, mousephenotype.org). In Cdk5rap2null adult bone marrow, hematopoietic stem cell and progenitor populations appeared normal (Fig EV1G) with no evidence for an erythroid differentiation block in either the bone marrow or the fetal liver (Fig EV1H and I). Nonetheless, consistent with macrocytic anemia, TER119-positive cells in Cdk5rap2null bone marrow were larger than control (Fig EV1J). We therefore reasoned that the defect responsible for macrocytic anemia in Cdk5rap2null was likely to arise in late terminal erythroid differentiation, possibly during enucleation. To investigate the underlying mechanism, we employed an ex vivo differentiation system that recapitulates key stages of terminal erythroid differentiation (Zhang et al, 2003). Erythroid progenitors were isolated from the fetal liver, the site of fetal definitive erythropoiesis, and differentiated ex vivo over 48 h (T48) (Fig 1H). We found that EBs lacking CDK5RAP2 are impaired in enucleation; both enucleating EB and reticulocyte populations were reduced at the end point of the culture (Fig 1I and J). In addition, we observed an increase in the size of the enucleating EBs (Fig 1K). These results are in complete agreement with the anemia observed in adult Cdk5rap2null mice (Fig 1F). In summary, mice lacking CDK5RAP2 show a mild macrocytic anemia and this phenotype can be recapitulated using an ex vivo erythroid differentiation system. Centrosomes persist during enucleation CDK5RAP2 is a highly conserved centrosomal protein, and therefore, its function in terminal erythroid differentiation is likely to be linked to centrosomes. However, little is known of what happens to centrosomes during this process. We therefore characterized levels and distribution of key PCM proteins including CDK5RAP2 during terminal erythroid differentiation in wild-type EBs using stimulated emission depletion (STED) super-resolution microscopy. Several PCM proteins are known to adopt a ring-shaped pattern corresponding to toroidal protein assembly around the cylindrical wall of an intact centriole (Lawo et al, 2012; Sonnen et al, 2012). Consistent with continued presence of intact centrioles during erythroid differentiation, such ring-shaped patterns of PCM proteins were detectable in both non-enucleating and enucleating EBs/reticulocytes (Fig 2A). However, as EBs progressed through differentiation, the PCM became smaller, whereas centriole diameter remained constant (Fig EV2A). CDK5RAP2 together with other PCM components PCNT and CEP192 co-localized with γ-tubulin in interphase centrosomes throughout erythroid differentiation (Fig EV2B). In line with a decrease in PCM size, levels of CDK5RAP2, PCNT, CEP192, and γ-tubulin were reduced in enucleating EBs and reticulocytes compared to non-enucleating EBs (Fig EV2A and C). We next determined the number of centrosomes in these populations. Consistent with the presence of two loosely linked parental centrioles, most non-enucleating and enucleating EBs, and reticulocytes, contained two γ-tubulin foci. We also noted a small increase in single centriole-containing cells in the enucleating and reticulocyte population (Fig EV2D). In these cases, the two centrioles may be too close to be resolved but it is also feasible that some cells contain a single centriole or two centrioles with only one incorporating PCM. Figure 2. Cdk5rap2null erythroblasts undergo fewer divisions and enucleate prematurely Deconvolved STED images of centrosomes in ex vivo cultured wild-type non-enucleating EBs (non-enucl.) or enucleating EBs/reticulocytes (enucl./RetiC). Cells were stained for γ-tubulin (magenta) and centriolar (CP110, CEP135) or PCM (CDK5RAP2, PCNT) proteins (cyan). Scale bar, 400 nm. Quantification of number of cell divisions after 24 (T24) and 48 (T48) hours. Ex vivo cultured erythroid progenitors with indicated genotypes were labeled with PKH26 to measure cell divisions. The numbers in brackets refer to the number of embryos analyzed. Schematic showing how ex vivo cultured erythroid progenitors were tracked in bright-field time-lapse microscopy experiments. Briefly, the two daughter cells produced by the first cytokinesis and their progeny were followed through terminal erythroid differentiation. Whenever a cell performed cytokinesis, enucleation, or death, this was recorded as an event (ev). Examples depict paths taken by four daughter cells. Quantification of the frequency of cytokinesis, enucleation, and death at each event (ev) in Cdk5rap2 wild-type or null erythroid progenitors as shown in (C). Four embryos for each genotype were analyzed. Quantification of cell cycle duration of Cdk5rap2 wild-type or null erythroid progenitors. The enucleation event was used as reference point to align previous divisions (see schematic in C). The number in brackets refers to the number of embryos analyzed. Data information: Box plots show 5th and 95th (whiskers) and 25th, 50th, and 75th percentiles (boxes). Bar graphs display mean ± s.d. All statistical analysis was based on the number of embryos. Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test (B) or Mann–Whitney U-test (D and E). *P ≤ 0.05, **P ≤ 0.01. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Intact centrosomes are present in enucleating erythroblasts but their PCM is reduced in size Quantification of signal diameters for centriolar (dot) and PCM proteins (ring) from Fig 2A. The numbers in brackets correspond to the number of centrosomes analyzed in one experiment. Immunofluorescence images of ex vivo cultured wild-type non-enucleating EBs (non-enucl.) or enucleating EBs/reticulocytes (enucl./RetiC). Cells were stained for γ-tubulin (magenta), protein of interest (POI, CDK5RAP2, PCNT, or CEP192 in grey), TER119 (erythroid marker, green), and DNA (Hoechst, blue). Scale bar, 2 μm. Quantification of mean centrosomal signal intensities of PCM proteins from (B). The numbers in brackets refer to the number of cells analyzed in one experiment. Quantification of centrosome number in ex vivo cultured non-enucleating and enucleating erythroblasts as well as reticulocytes. Four litters with a total number of 2,618 (non-enucl.), 715 (enucl.), and 877 (RetiC) cells were analyzed. Data information: Box plots show 5th and 95th (whiskers) and 25th, 50th, and 75th percentiles (boxes). Statistical analysis was based on the number of centrosomes (A), the number of cells (C), or the number of litters (D). Statistical significances were determined by Mann–Whitney test (A and C) or One-way ANOVA with Tukey's multiple comparisons test (D). *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. Download figure Download PowerPoint In summary, we find that intact centrosomes are maintained during erythroid differentiation. Because cell size of EBs reduces during differentiation, interphase PCM size also decreases, consistent with a previously reported link between centrosome and cell size in C. elegans embryos (Decker et al, 2011). Cdk5rap2null erythroblasts undergo fewer divisions and enucleate prematurely Having established that CDK5RAP2 localization is sustained throughout erythroid differentiation, we next sought to identify its functional contribution to the process. In cells lacking CDK5RAP2, we found no difference in the number of committed erythroid progenitor cells (BFU-E and CFU-E) (Fig EV3A and B), suggesting that the abnormalities in red blood cell number and size are more likely to arise from defects in terminal erythroid differentiation or enucleation. We speculated that the appearance of bigger cells in the absence of CDK5RAP2 might result from a reduction in the number of cell divisions during differentiation. To address this, we used the PKH26 membrane dye, which gets diluted with each division, and therefore its intensity inversely correlates with the number of cell divisions. Progenitors are known to divide four to five times before enucleation (Zhang et al, 2003; Sankaran et al, 2012). Indeed, PKH26 labeling of wild-type erythroid progenitors revealed that cells complete on average five divisions by the 48-h time point (T48). By contrast, during the same period, erythroid progenitors lacking CDK5RAP2 undergo only four divisions on average. This difference becomes apparent only at T48 because by the 24-h time point (T24) cells complete on average three divisions independent of their genotype (Fig 2B). Click here to expand this figure. Figure EV3. Cell cycle analysis of Cdk5rap2null erythroid progenitors during ex vivo differentiation A, B. Quantification of ex vivo cultured BFU-E and CFU-E (A) as well as mature CFU-E (B) progenitor populations after 24 h (T24). Genotypes are as indicated. Number of embryos analyzed is shown in brackets. C, D. Quantification of apoptotic (AnnexinVpos) TER119pos cells at T24 (C) and T48 (D) of ex vivo culture. Genotypes are as indicated. Number of embryos analyzed is shown in brackets. T24 = 24 h. T48 = 48 h. E. Quantification of cell cycle profil
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