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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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eGFP-WASpI294T expression retards proliferation and induces apoptosis. (A) U937 cells transduced with eGFP (MOI of 0.2 and 1), eGFP-WASp (MOI of 1 and 5), or eGFP-WASpI294T (MOI of 1 and 5) were analyzed for eGFP expression by flow cytometry 3, 6, 8, 10, 13, 15, and 17 d after transduction (each bar represents the mean ± SD value of the percentage of eGFP+ cells in the culture for independent triplicates from each day). (B) Apoptosis assessed by flow cytometry (annexin V and 7AAD staining) 8 d after transduction at the lower MOIs shown in A. (C) Apoptosis (annexin V+) quantified in triplicate samples 6, 8, 10, and 13 d after transduction at the lower MOIs shown in A. *, P < 0.005 compared with untransduced U937 cells in the same culture. eGFP was transduced at a lower MOI than other constructs, as eGFP expression was 5–10-fold higher than all other eGFP fusion proteins.
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fig2: eGFP-WASpI294T expression retards proliferation and induces apoptosis. (A) U937 cells transduced with eGFP (MOI of 0.2 and 1), eGFP-WASp (MOI of 1 and 5), or eGFP-WASpI294T (MOI of 1 and 5) were analyzed for eGFP expression by flow cytometry 3, 6, 8, 10, 13, 15, and 17 d after transduction (each bar represents the mean ± SD value of the percentage of eGFP+ cells in the culture for independent triplicates from each day). (B) Apoptosis assessed by flow cytometry (annexin V and 7AAD staining) 8 d after transduction at the lower MOIs shown in A. (C) Apoptosis (annexin V+) quantified in triplicate samples 6, 8, 10, and 13 d after transduction at the lower MOIs shown in A. *, P < 0.005 compared with untransduced U937 cells in the same culture. eGFP was transduced at a lower MOI than other constructs, as eGFP expression was 5–10-fold higher than all other eGFP fusion proteins.

Mentions: Proliferation of myeloid cells in culture was severely compromised in primary WASpI294T patient progenitors, suggesting that there is an intrinsic restriction (19). To characterize the mechanisms underlying the lack of proliferation, U937 cell cultures transduced with eGFP, eGFP-WASp, and eGFP-WASpI294T were analyzed over a 17-d period. eGFP-WASpI294T expression decreased the proliferation of these cells, as untransduced cells in the same culture outgrew the eGFP-WASpI294T–expressing cells, resulting in a reduction in the percentage of eGFP-WASpI294T cells over time (Fig. 2 A). This diminished proliferation was not a spurious result of lentiviral transduction, as neither eGFP nor eGFP-WASp exhibited this effect (Fig. 2 A). In conjunction with the slower growth of the eGFP-WASpI294T–expressing cells, there was a significant increase in apoptosis (Fig. 2, B and C). The inhibition of proliferation and induction of apoptosis caused by eGFP-WASpI294T was confirmed in HT1080 fibroblasts, a nonhematopoietic cell line that does not normally express WASp (Fig. S1, A and B, available at http://www.jem.org/cgi/content/full/jem.20062324/DC1). These results establish a direct link between expression of auto-active WASp and the reduced proliferation and increased apoptosis seen in XLN.


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)

eGFP-WASpI294T expression retards proliferation and induces apoptosis. (A) U937 cells transduced with eGFP (MOI of 0.2 and 1), eGFP-WASp (MOI of 1 and 5), or eGFP-WASpI294T (MOI of 1 and 5) were analyzed for eGFP expression by flow cytometry 3, 6, 8, 10, 13, 15, and 17 d after transduction (each bar represents the mean ± SD value of the percentage of eGFP+ cells in the culture for independent triplicates from each day). (B) Apoptosis assessed by flow cytometry (annexin V and 7AAD staining) 8 d after transduction at the lower MOIs shown in A. (C) Apoptosis (annexin V+) quantified in triplicate samples 6, 8, 10, and 13 d after transduction at the lower MOIs shown in A. *, P < 0.005 compared with untransduced U937 cells in the same culture. eGFP was transduced at a lower MOI than other constructs, as eGFP expression was 5–10-fold higher than all other eGFP fusion proteins.
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Related In: Results  -  Collection

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fig2: eGFP-WASpI294T expression retards proliferation and induces apoptosis. (A) U937 cells transduced with eGFP (MOI of 0.2 and 1), eGFP-WASp (MOI of 1 and 5), or eGFP-WASpI294T (MOI of 1 and 5) were analyzed for eGFP expression by flow cytometry 3, 6, 8, 10, 13, 15, and 17 d after transduction (each bar represents the mean ± SD value of the percentage of eGFP+ cells in the culture for independent triplicates from each day). (B) Apoptosis assessed by flow cytometry (annexin V and 7AAD staining) 8 d after transduction at the lower MOIs shown in A. (C) Apoptosis (annexin V+) quantified in triplicate samples 6, 8, 10, and 13 d after transduction at the lower MOIs shown in A. *, P < 0.005 compared with untransduced U937 cells in the same culture. eGFP was transduced at a lower MOI than other constructs, as eGFP expression was 5–10-fold higher than all other eGFP fusion proteins.
Mentions: Proliferation of myeloid cells in culture was severely compromised in primary WASpI294T patient progenitors, suggesting that there is an intrinsic restriction (19). To characterize the mechanisms underlying the lack of proliferation, U937 cell cultures transduced with eGFP, eGFP-WASp, and eGFP-WASpI294T were analyzed over a 17-d period. eGFP-WASpI294T expression decreased the proliferation of these cells, as untransduced cells in the same culture outgrew the eGFP-WASpI294T–expressing cells, resulting in a reduction in the percentage of eGFP-WASpI294T cells over time (Fig. 2 A). This diminished proliferation was not a spurious result of lentiviral transduction, as neither eGFP nor eGFP-WASp exhibited this effect (Fig. 2 A). In conjunction with the slower growth of the eGFP-WASpI294T–expressing cells, there was a significant increase in apoptosis (Fig. 2, B and C). The inhibition of proliferation and induction of apoptosis caused by eGFP-WASpI294T was confirmed in HT1080 fibroblasts, a nonhematopoietic cell line that does not normally express WASp (Fig. S1, A and B, available at http://www.jem.org/cgi/content/full/jem.20062324/DC1). These results establish a direct link between expression of auto-active WASp and the reduced proliferation and increased apoptosis seen in XLN.

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