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Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia.

Moulding DA, Blundell MP, Spiller DG, White MR, Cory GO, Calle Y, Kempski H, Sinclair J, Ancliff PJ, Kinnon C, Jones GE, Thrasher AJ - J. Exp. Med. (2007)

Bottom Line: This caused enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis.Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis.These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization.

View Article: PubMed Central - PubMed

Affiliation: Wolfson Centre for Gene Therapy of Childhood Disease, UCL Institute of Child Health, University College London, London, UK.

ABSTRACT
Specific mutations in the human gene encoding the Wiskott-Aldrich syndrome protein (WASp) that compromise normal auto-inhibition of WASp result in unregulated activation of the actin-related protein 2/3 complex and increased actin polymerizing activity. These activating mutations are associated with an X-linked form of neutropenia with an intrinsic failure of myelopoiesis and an increase in the incidence of cytogenetic abnormalities. To study the underlying mechanisms, active mutant WASp(I294T) was expressed by gene transfer. This caused enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis. Cells became binucleated, suggesting a failure of cytokinesis, and micronuclei were formed, indicative of genomic instability. Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis. During mitosis, filamentous actin was abnormally localized around the spindle and chromosomes throughout their alignment and separation, and it accumulated within the cleavage furrow around the spindle midzone. These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization.

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WASpI294T induces cytogenetic abnormalities in patient-derived B-LCLs, transduced cell lines, and primary HPCs. (A) Normal interphase cell showing two copies of RB1 (red signals) and two copies of the control probe BCR (green signals). (B) Metaphase tetraploid cell showing four copies of RB1 (two of which are amplified; arrows indicate the two chromosomes with increased signal intensity) and four copies of BCR. (C) Partial metaphase cell and adjoining interphase cell. Amplification of RB1 is seen on one of the two copies of chromosome 13 (arrowed, increased signal intensity) and also confirmed in the interphase cell by four copies of the RB1 (arrowed) compared with two copies of the control probe, BCR. (D) Abnormal interphase cell showing loss of one copy of RB1. (E) eGFP-WASpI294T–expressing U937 cells (from Fig. 2 A; MOI of 5) show increased DNA content assessed by flow cytometry of independent triplicates propidium iodide stained 13 d after transduction. (F) Percentage of U937 cells on cytospins with multiple nuclei (gray bars) and micronuclei (black bars). Data is the average from days 3, 7, 10, and 14. (G) Images of cytospins from U937 (eGFP-WASpI294T; MOI of 5, day 6). Cells showed multiple nuclei (black arrows), micronuclei (dashed arrows), and asymmetric three-way division (white arrow). (H) Percentage multinucleate human primary CD34+ cells on cytospins (days 7 and 10; n = 1). (I) CD34+ HPCs show binucleation when expressing eGFP-WASpI294T. *, P < 0.001; +, P < 0.01 compared with eGFP, eGFP-WASp, or untransduced U937 cells. Bars: B, G, and I, 20 μm; A, C, and D, 10 μm.
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fig3: WASpI294T induces cytogenetic abnormalities in patient-derived B-LCLs, transduced cell lines, and primary HPCs. (A) Normal interphase cell showing two copies of RB1 (red signals) and two copies of the control probe BCR (green signals). (B) Metaphase tetraploid cell showing four copies of RB1 (two of which are amplified; arrows indicate the two chromosomes with increased signal intensity) and four copies of BCR. (C) Partial metaphase cell and adjoining interphase cell. Amplification of RB1 is seen on one of the two copies of chromosome 13 (arrowed, increased signal intensity) and also confirmed in the interphase cell by four copies of the RB1 (arrowed) compared with two copies of the control probe, BCR. (D) Abnormal interphase cell showing loss of one copy of RB1. (E) eGFP-WASpI294T–expressing U937 cells (from Fig. 2 A; MOI of 5) show increased DNA content assessed by flow cytometry of independent triplicates propidium iodide stained 13 d after transduction. (F) Percentage of U937 cells on cytospins with multiple nuclei (gray bars) and micronuclei (black bars). Data is the average from days 3, 7, 10, and 14. (G) Images of cytospins from U937 (eGFP-WASpI294T; MOI of 5, day 6). Cells showed multiple nuclei (black arrows), micronuclei (dashed arrows), and asymmetric three-way division (white arrow). (H) Percentage multinucleate human primary CD34+ cells on cytospins (days 7 and 10; n = 1). (I) CD34+ HPCs show binucleation when expressing eGFP-WASpI294T. *, P < 0.001; +, P < 0.01 compared with eGFP, eGFP-WASp, or untransduced U937 cells. Bars: B, G, and I, 20 μm; A, C, and D, 10 μm.

Mentions: Cytogenetic analysis of bone marrow samples from the index patient revealed loss of chromosome 13 in 3/16 metaphase cells, one of which also had monosomy 14 (19). Retrospective FISH investigations for locus-specific changes revealed the discreet presence of tetraploid cells that were confirmed at the metaphase level. The generation of cytogenetic abnormalities was further investigated in a WASpI294T B lymphoblastoid cell line (LCL; derived from patient peripheral blood). Cytogenetic analysis was performed by locus-specific identifier–FISH using probes for RB1 (chromosome 13), BCR (chromosome 22), and the chromosome 7 α-satellite region and locus D7S486 (Fig. 3, A–D, and Tables I and II). The WASpI294T B-LCL population displayed a variety of cytogenetic abnormalities, including tetraploidy (Fig. 3 B), amplification of RB1 (Fig. 3 C), and loss of one copy of RB1 (Fig. 3 D). Analysis of metaphase cells showed there was a significant increase in both tetraploidy and amplification of RB1 (Table I). A significant proportion of interphase WASpI294T B-LCLs also appeared to be tetraploid, as they exhibited tetrasomy for both RB1 and BCR (Table I). Interphase WASpI294T cells also showed a greater frequency of cells that had lost or gained copies of RB1 without changes in BCR copy number (Table I). Similar rates of cytogenetic abnormalities were seen using the D7S486 probe (Table I). The frequency of asynchronous replication (where one allele has replicated and the other has not, generating a characteristic singlet/doublet hybridization signal) was similar in both WASpI294T B-LCLs and control B-LCLs (Tables I and II). In summary WASpI294T is associated with a substantial increase in cytogenetic abnormalities, with tetraploidy being the most common defect observed.


Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia.

Moulding DA, Blundell MP, Spiller DG, White MR, Cory GO, Calle Y, Kempski H, Sinclair J, Ancliff PJ, Kinnon C, Jones GE, Thrasher AJ - J. Exp. Med. (2007)

WASpI294T induces cytogenetic abnormalities in patient-derived B-LCLs, transduced cell lines, and primary HPCs. (A) Normal interphase cell showing two copies of RB1 (red signals) and two copies of the control probe BCR (green signals). (B) Metaphase tetraploid cell showing four copies of RB1 (two of which are amplified; arrows indicate the two chromosomes with increased signal intensity) and four copies of BCR. (C) Partial metaphase cell and adjoining interphase cell. Amplification of RB1 is seen on one of the two copies of chromosome 13 (arrowed, increased signal intensity) and also confirmed in the interphase cell by four copies of the RB1 (arrowed) compared with two copies of the control probe, BCR. (D) Abnormal interphase cell showing loss of one copy of RB1. (E) eGFP-WASpI294T–expressing U937 cells (from Fig. 2 A; MOI of 5) show increased DNA content assessed by flow cytometry of independent triplicates propidium iodide stained 13 d after transduction. (F) Percentage of U937 cells on cytospins with multiple nuclei (gray bars) and micronuclei (black bars). Data is the average from days 3, 7, 10, and 14. (G) Images of cytospins from U937 (eGFP-WASpI294T; MOI of 5, day 6). Cells showed multiple nuclei (black arrows), micronuclei (dashed arrows), and asymmetric three-way division (white arrow). (H) Percentage multinucleate human primary CD34+ cells on cytospins (days 7 and 10; n = 1). (I) CD34+ HPCs show binucleation when expressing eGFP-WASpI294T. *, P < 0.001; +, P < 0.01 compared with eGFP, eGFP-WASp, or untransduced U937 cells. Bars: B, G, and I, 20 μm; A, C, and D, 10 μm.
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fig3: WASpI294T induces cytogenetic abnormalities in patient-derived B-LCLs, transduced cell lines, and primary HPCs. (A) Normal interphase cell showing two copies of RB1 (red signals) and two copies of the control probe BCR (green signals). (B) Metaphase tetraploid cell showing four copies of RB1 (two of which are amplified; arrows indicate the two chromosomes with increased signal intensity) and four copies of BCR. (C) Partial metaphase cell and adjoining interphase cell. Amplification of RB1 is seen on one of the two copies of chromosome 13 (arrowed, increased signal intensity) and also confirmed in the interphase cell by four copies of the RB1 (arrowed) compared with two copies of the control probe, BCR. (D) Abnormal interphase cell showing loss of one copy of RB1. (E) eGFP-WASpI294T–expressing U937 cells (from Fig. 2 A; MOI of 5) show increased DNA content assessed by flow cytometry of independent triplicates propidium iodide stained 13 d after transduction. (F) Percentage of U937 cells on cytospins with multiple nuclei (gray bars) and micronuclei (black bars). Data is the average from days 3, 7, 10, and 14. (G) Images of cytospins from U937 (eGFP-WASpI294T; MOI of 5, day 6). Cells showed multiple nuclei (black arrows), micronuclei (dashed arrows), and asymmetric three-way division (white arrow). (H) Percentage multinucleate human primary CD34+ cells on cytospins (days 7 and 10; n = 1). (I) CD34+ HPCs show binucleation when expressing eGFP-WASpI294T. *, P < 0.001; +, P < 0.01 compared with eGFP, eGFP-WASp, or untransduced U937 cells. Bars: B, G, and I, 20 μm; A, C, and D, 10 μm.
Mentions: Cytogenetic analysis of bone marrow samples from the index patient revealed loss of chromosome 13 in 3/16 metaphase cells, one of which also had monosomy 14 (19). Retrospective FISH investigations for locus-specific changes revealed the discreet presence of tetraploid cells that were confirmed at the metaphase level. The generation of cytogenetic abnormalities was further investigated in a WASpI294T B lymphoblastoid cell line (LCL; derived from patient peripheral blood). Cytogenetic analysis was performed by locus-specific identifier–FISH using probes for RB1 (chromosome 13), BCR (chromosome 22), and the chromosome 7 α-satellite region and locus D7S486 (Fig. 3, A–D, and Tables I and II). The WASpI294T B-LCL population displayed a variety of cytogenetic abnormalities, including tetraploidy (Fig. 3 B), amplification of RB1 (Fig. 3 C), and loss of one copy of RB1 (Fig. 3 D). Analysis of metaphase cells showed there was a significant increase in both tetraploidy and amplification of RB1 (Table I). A significant proportion of interphase WASpI294T B-LCLs also appeared to be tetraploid, as they exhibited tetrasomy for both RB1 and BCR (Table I). Interphase WASpI294T cells also showed a greater frequency of cells that had lost or gained copies of RB1 without changes in BCR copy number (Table I). Similar rates of cytogenetic abnormalities were seen using the D7S486 probe (Table I). The frequency of asynchronous replication (where one allele has replicated and the other has not, generating a characteristic singlet/doublet hybridization signal) was similar in both WASpI294T B-LCLs and control B-LCLs (Tables I and II). In summary WASpI294T is associated with a substantial increase in cytogenetic abnormalities, with tetraploidy being the most common defect observed.

Bottom Line: This caused enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis.Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis.These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization.

View Article: PubMed Central - PubMed

Affiliation: Wolfson Centre for Gene Therapy of Childhood Disease, UCL Institute of Child Health, University College London, London, UK.

ABSTRACT
Specific mutations in the human gene encoding the Wiskott-Aldrich syndrome protein (WASp) that compromise normal auto-inhibition of WASp result in unregulated activation of the actin-related protein 2/3 complex and increased actin polymerizing activity. These activating mutations are associated with an X-linked form of neutropenia with an intrinsic failure of myelopoiesis and an increase in the incidence of cytogenetic abnormalities. To study the underlying mechanisms, active mutant WASp(I294T) was expressed by gene transfer. This caused enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis. Cells became binucleated, suggesting a failure of cytokinesis, and micronuclei were formed, indicative of genomic instability. Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis. During mitosis, filamentous actin was abnormally localized around the spindle and chromosomes throughout their alignment and separation, and it accumulated within the cleavage furrow around the spindle midzone. These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization.

Show MeSH
Related in: MedlinePlus