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

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 WASpI294T 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.

Phosphorylation of WASp is a key regulator of activity and stability in vivo

“…Y291E-hWASp has previous been shown to exhibit enhanced actin polymerization activity in vitro (14). Expression of this mutant in U937 cells resulted in increased levels of dysregulated F-actin, and higher levels of apoptosis and multinucleation compared to wtWASp as has been described previously for other activating mutants (26) (Fig. S4 A-C).…”

Wiskott-Aldrich syndrome gene mutations modulate cancer susceptibility in the p53±murine model

“…This is associated with aberrant segregation of chromosomes to the daughter cells and impaired cytokinesis during mitosis, leading to increased cell death. 14-16 Moreover, somatic XLN mutations in WASp correlate with poor prognosis in patients with juvenile myelomonocytic leukemia and the XLN-WASp expressing leukemic clone becomes dominant in these patients. 17 …”

Activating WASP mutations associated with X-linked neutropenia result in enhanced actin polymerization, altered cytoskeletal responses, and genomic instability in lymphocytes

“…Moreover, pharmacologically induced actin polymerization leads to increased apoptosis of monocyte cells (Moulding et al, 2007), suggesting that an increased load of polymerized actin renders cells prone to undergoing apoptosis. We examined whether increased polymerized actin in WASP-I296T lymphocytes was associated with increased apoptosis.…”

“…Although deficiency in neutrophil elastase, HAX1, and glucose-6-phosphatase catalytic subunit 3 can be explained by increased apoptosis of neutrophils and their precursors, it is difficult to predict how constitutively active WASP may induce neutropenia and how the function of other hematopoietic cells are affected. We have recently provided a mechanism for induction of neutropenia in which forced expression of WASP-I294T in a monocyte cell line induced increased polymerized actin, delayed cell-cycle progression, increased apoptosis, and genomic instability with multinucleated and tetraploid cells (Moulding et al, 2007).…”

“…These processes are associated with increased genomic instability (Ganem and Pellman, 2007;Moulding et al, 2007). To directly determine a potential role of WASP-I296T in the maintenance of genomic stability, we used a telomere-FISH assay that combines DAPI staining of chromosomes with a telomerespecific peptide nucleic acid probe to assay metaphase chromosomes for chromosomal abnormalities (Zha et al, 2007).…”