A novel role for survivin in erythroblast enucleation

Keerthivasan, Ganesan; Liu, Hui; Gump, Jacob M.; Dowdy, Steven F.; Wickrema, Amittha; Crispino, John D.

doi:10.3324/haematol.2011.061093

Cited by 37 publications

(29 citation statements)

References 34 publications

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“…Several lines of evidence support the hypothesis that formation of cytoplasmic vesicles and autophagic vacuoles contributes to enucleation [4,5]. Recently, Keerthivasan et al [4,5] showed that vesicle trafficking, in particular the formation and movement of vacuoles at the junction of the nucleus and the cytoplasm, is a critical component of mammalian erythroblast enucleation. There is likewise evidence supporting a link between autophagy and vesicle trafficking [43].…”

Section: Discussionmentioning

confidence: 90%

Molecular signature of erythroblast enucleation in human embryonic stem cells

et al. 2015

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While enucleation is a critical step in the terminal differentiation of human red blood cells, the molecular mechanisms underlying this unique process remain unclear. To investigate erythroblast enucleation, we studied the erythroid differentiation of human embryonic stem cells (hESCs), which provide a unique model for deeper understanding of the development and differentiation of multiple cell types. First, using a two-step protocol, we demonstrated that terminal erythroid differentiation from hESCs is directly dependent on the age of the embryoid bodies. Second, by choosing hESCs in two extreme conditions of erythroid culture, we obtained an original differentiation model which allows one to study the mechanisms underlying the enucleation of erythroid cells by analyzing the gene and miRNA (miR) expression profiles of cells from these two culture conditions. Third, using an integrated analysis of mRNA and miR expression profiles, we identified five miRs potentially involved in erythroblast enucleation. Finally, by selective knockdown of these five miRs we found miR-30a to be a regulator of erythroblast enucleation in hESCs.

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Section: Discussionmentioning

confidence: 90%

Molecular signature of erythroblast enucleation in human embryonic stem cells

et al. 2015

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“…Throughout terminal erythropoiesis, erythroblasts undergo gradual nuclear condensation that is associated with decreased cell size. This is followed by the extrusion of the pycnotic nucleus in the final stage of terminal differentiation that involves a highly regulated asymmetric cell division processes (Ji et al, 2008; Keerthivasan et al, 2010; Ji et al, 2011; Zhang et al, 2011; Keerthivasan et al, 2012; Konstantinidis et al, 2012; Ubukawa et al., 2012). …”

Section: Introductionmentioning

confidence: 99%

Nuclear Condensation during Mouse Erythropoiesis Requires Caspase-3-Mediated Nuclear Opening

et al. 2016

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SUMMARY Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step towards chromatin condensation during erythropoiesis in mice.

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“…Recent studies revealed that multiple signaling pathways are involved in the generation of enucleated erythroid cells. 6,7 These include histone deacetylation, 8,9 actin cytoskeleton, [10][11][12][13] cytokinesis, 14,15 cell-matrix interactions, 16 specific microRNAs 17 and vesicle trafficking. 18 Many of these studies used a mouse fetal liver erythroblast culture system, which makes it possible to analyze erythroid cell differentiation and enucleation both quantitatively and step by step.…”

Section: Introductionmentioning

confidence: 99%

Targeted shRNA screening identified critical roles of pleckstrin-2 in erythropoiesis

et al. 2014

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ABSTRACTliver erythroblast in vitro culture technology together with a targeted array-based high throughput screening system and discovered more than 30 genes that have novel functions in the early and late stages of terminal erythropoiesis. Among these genes, we identified pleckstrin-2 (plek2), which was previously reported to be involved in T-cell cytoskeleton reorganization, 23 play critical roles in terminal erythropoiesis. We demonstrated that plek2 is important in the regulation of actin cytoskeleton, cell differentiation and apoptosis. We also found that plek2 blocks apoptosis in the early stage of terminal erythropoiesis through preventing cofilin's mitochondrial entry. Furthermore, we dissected these functions of plek2 in the early and late stages of terminal erythropoiesis reflecting different intracellular levels of reactive oxygen species (ROS). Methods MaterialsRabbit polyclonal antibody against plek2 was purchased from Proteintech. Rabbit polyclonal antibodies against cofilin and phospho-cofilin were purchased from Cell Signaling Technology. Monoclonal antibody against Hsc70 was purchased from Santa Cruz Biotechnology. All the antibodies for flow cytometric analysis were purchased from eBioscience. Alexa Fluor 488 phalloidin and Alexa Fluor 544 for immunofluorescence were purchased from Invitrogen Molecular Probes. The Hairpin-pLKO.1 lentiviruses were from the Broad Institute (Cambridge, MA, USA). Purification and culture of fetal liver cells for targeted screeningPurification of mouse fetal liver erythroblast progenitors (CFU-E; TER119-negative cells) was based on previously described procedures. 10 Details of procedures including viral transduction and the screening protocol are available in the Online Supplementary Appendix. Retroviral shRNA constructionRetroviral shRNA oligonucleotides against plek2 were designed using an automated shRNA selection web server (http://jura.wi.mit.edu/bioc/siRNAext/home.php). 24 Cloning of shRNA into MSCV-U3-H1 was performed as previously described. 25 The shRNA nucleotide sequences used in the experiments are as follows. Non-targeting shRNA: 5'-GGACTAGTCTCCACGGTGA -3'. Plek2 shRNA: 5' -CAACTCAGTATCTCTGGGA -3'. Flow cytometric analysis and immunoprecipitationFlow cytometric analysis of differentiation and enucleation of cultured mouse fetal erythroblasts was as previously described. Immunofluorescence stain and microscopic analysisDetails of the protocol for immunofluorescence stain and microscopic analysis are available in the Online Supplementary Appendix. Quantitative analysis of intracellular reactive oxygen speciesThe intracellular ROS levels were determined using CM-H2DCFDA (Molecular Probes). Briefly, erythroid cells were incubated with 10 mM CM-H2DCFDA or DMSO control. After incubation for 30 min at 37°C, the cells were harvested, washed, and resuspended in PBS. The levels of fluorescence were immediately detected using flow cytometric analysis. Results Targeted shRNA screening identified genes that play novel functions in the early and late stages of ter...

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A novel role for survivin in erythroblast enucleation

Cited by 37 publications

References 34 publications

Molecular signature of erythroblast enucleation in human embryonic stem cells

Molecular signature of erythroblast enucleation in human embryonic stem cells

Nuclear Condensation during Mouse Erythropoiesis Requires Caspase-3-Mediated Nuclear Opening

Targeted shRNA screening identified critical roles of pleckstrin-2 in erythropoiesis

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