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Loss of the Arp2/3 complex component ARPC1B causes platelet abnormalities and predisposes to inflammatory disease

View Article: PubMed Central - PubMed

ABSTRACT

Human actin-related protein 2/3 complex (Arp2/3), required for actin filament branching, has two ARPC1 component isoforms, with ARPC1B prominently expressed in blood cells. Here we show in a child with microthrombocytopenia, eosinophilia and inflammatory disease, a homozygous frameshift mutation in ARPC1B (p.Val91Trpfs*30). Platelet lysates reveal no ARPC1B protein and greatly reduced Arp2/3 complex. Missense ARPC1B mutations are identified in an unrelated patient with similar symptoms and ARPC1B deficiency. ARPC1B-deficient platelets are microthrombocytes similar to those seen in Wiskott–Aldrich syndrome that show aberrant spreading consistent with loss of Arp2/3 function. Knockout of ARPC1B in megakaryocytic cells results in decreased proplatelet formation, and as observed in platelets from patients, increased ARPC1A expression. Thus loss of ARPC1B produces a unique set of platelet abnormalities, and is associated with haematopoietic/immune symptoms affecting cell lineages where this isoform predominates. In agreement with recent experimental studies, our findings suggest that ARPC1 isoforms are not functionally interchangeable.

No MeSH data available.


Spreading behaviour and distribution of ARPC5 and actin in normal and ARPC1B-deficient platelets.Platelets in platelet-rich plasma were allowed to spread on fibrinogen for 30 min before fixation, staining and imaging by laser fluorescence structured illumination microscopy. (a) Each set of panels shows a wide field image (bars=5 μm) and (b) higher magnification images of representative spread platelets stained for CD61/fibrinogen receptor (red), ARPC5 (green) and F-actin (magenta; bars=2 μm). Most normal platelets show circular lamellipodia, peripheral ARPC5 and F-actin present at the lamellipodial edge and in stress fibres and nodules (see also Fig. 6). In contrast, at his time point many ARPC1B- platelets (from Patient 1) have not spread, or show abnormal shapes and subcellular distributions of ARPC5 and F-actin (note: close up panels are collages necessitated by the sparse distribution of spread platelets; all others are cropped fields). ARPC1B-deficient platelets (from Patient 2) also show limited spreading and unusual spread morphologies, including extremely long filopodia containing long unbranched actin fibres.
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f7: Spreading behaviour and distribution of ARPC5 and actin in normal and ARPC1B-deficient platelets.Platelets in platelet-rich plasma were allowed to spread on fibrinogen for 30 min before fixation, staining and imaging by laser fluorescence structured illumination microscopy. (a) Each set of panels shows a wide field image (bars=5 μm) and (b) higher magnification images of representative spread platelets stained for CD61/fibrinogen receptor (red), ARPC5 (green) and F-actin (magenta; bars=2 μm). Most normal platelets show circular lamellipodia, peripheral ARPC5 and F-actin present at the lamellipodial edge and in stress fibres and nodules (see also Fig. 6). In contrast, at his time point many ARPC1B- platelets (from Patient 1) have not spread, or show abnormal shapes and subcellular distributions of ARPC5 and F-actin (note: close up panels are collages necessitated by the sparse distribution of spread platelets; all others are cropped fields). ARPC1B-deficient platelets (from Patient 2) also show limited spreading and unusual spread morphologies, including extremely long filopodia containing long unbranched actin fibres.

Mentions: We examined the consequences of ARPC1B deficiency for platelet spreading on fibrinogen-treated surfaces using high-resolution fluorescence microscopy and scanning electron microscopy to monitor cell morphology and intracellular localization/distribution of tubulin, F-actin and Arp2/3 components. Comparisons of both washed platelets (Fig. 6) and platelets in plasma (Fig. 7) showed that maximally spread cells from normal donors formed typical near-circular lamellipodia. As expected from experimental observations36, normal platelet lamellipodia had peripheral localization of F-actin and ARPC5 (Fig. 7), and also displayed prominent F-actin stress fibres and podosome-like nodules. In contrast, maximally spread ARPC1B- and ARPC1B-deficient platelets typically formed spiky structures with tubulin-rich tips (Figs 6 and 7) that contained fewer and often elongated F-actin fibres and showed little evidence of podosome-like nodule formation. The spread platelet surface area was significantly reduced in ARPC1B- and -deficient platelets compared to normal (Supplementary Fig. 4). Allowing ARPC1B- and -deficient platelets more time to spread did not alter their observed behaviour. These observations indicate a profound loss of actin branching required for lamellipodia formation3032 in ARPC1B-deficient platelets, despite their increased ARPC1A content. This is consistent with experimental observations that isoforms of Arp2/3 containing ARPC1B are significantly better than complexes containing ARPC1A at promoting the rapid assembly of stable branched actin networks24.


Loss of the Arp2/3 complex component ARPC1B causes platelet abnormalities and predisposes to inflammatory disease
Spreading behaviour and distribution of ARPC5 and actin in normal and ARPC1B-deficient platelets.Platelets in platelet-rich plasma were allowed to spread on fibrinogen for 30 min before fixation, staining and imaging by laser fluorescence structured illumination microscopy. (a) Each set of panels shows a wide field image (bars=5 μm) and (b) higher magnification images of representative spread platelets stained for CD61/fibrinogen receptor (red), ARPC5 (green) and F-actin (magenta; bars=2 μm). Most normal platelets show circular lamellipodia, peripheral ARPC5 and F-actin present at the lamellipodial edge and in stress fibres and nodules (see also Fig. 6). In contrast, at his time point many ARPC1B- platelets (from Patient 1) have not spread, or show abnormal shapes and subcellular distributions of ARPC5 and F-actin (note: close up panels are collages necessitated by the sparse distribution of spread platelets; all others are cropped fields). ARPC1B-deficient platelets (from Patient 2) also show limited spreading and unusual spread morphologies, including extremely long filopodia containing long unbranched actin fibres.
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Related In: Results  -  Collection

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f7: Spreading behaviour and distribution of ARPC5 and actin in normal and ARPC1B-deficient platelets.Platelets in platelet-rich plasma were allowed to spread on fibrinogen for 30 min before fixation, staining and imaging by laser fluorescence structured illumination microscopy. (a) Each set of panels shows a wide field image (bars=5 μm) and (b) higher magnification images of representative spread platelets stained for CD61/fibrinogen receptor (red), ARPC5 (green) and F-actin (magenta; bars=2 μm). Most normal platelets show circular lamellipodia, peripheral ARPC5 and F-actin present at the lamellipodial edge and in stress fibres and nodules (see also Fig. 6). In contrast, at his time point many ARPC1B- platelets (from Patient 1) have not spread, or show abnormal shapes and subcellular distributions of ARPC5 and F-actin (note: close up panels are collages necessitated by the sparse distribution of spread platelets; all others are cropped fields). ARPC1B-deficient platelets (from Patient 2) also show limited spreading and unusual spread morphologies, including extremely long filopodia containing long unbranched actin fibres.
Mentions: We examined the consequences of ARPC1B deficiency for platelet spreading on fibrinogen-treated surfaces using high-resolution fluorescence microscopy and scanning electron microscopy to monitor cell morphology and intracellular localization/distribution of tubulin, F-actin and Arp2/3 components. Comparisons of both washed platelets (Fig. 6) and platelets in plasma (Fig. 7) showed that maximally spread cells from normal donors formed typical near-circular lamellipodia. As expected from experimental observations36, normal platelet lamellipodia had peripheral localization of F-actin and ARPC5 (Fig. 7), and also displayed prominent F-actin stress fibres and podosome-like nodules. In contrast, maximally spread ARPC1B- and ARPC1B-deficient platelets typically formed spiky structures with tubulin-rich tips (Figs 6 and 7) that contained fewer and often elongated F-actin fibres and showed little evidence of podosome-like nodule formation. The spread platelet surface area was significantly reduced in ARPC1B- and -deficient platelets compared to normal (Supplementary Fig. 4). Allowing ARPC1B- and -deficient platelets more time to spread did not alter their observed behaviour. These observations indicate a profound loss of actin branching required for lamellipodia formation3032 in ARPC1B-deficient platelets, despite their increased ARPC1A content. This is consistent with experimental observations that isoforms of Arp2/3 containing ARPC1B are significantly better than complexes containing ARPC1A at promoting the rapid assembly of stable branched actin networks24.

View Article: PubMed Central - PubMed

ABSTRACT

Human actin-related protein 2/3 complex (Arp2/3), required for actin filament branching, has two ARPC1 component isoforms, with ARPC1B prominently expressed in blood cells. Here we show in a child with microthrombocytopenia, eosinophilia and inflammatory disease, a homozygous frameshift mutation in ARPC1B (p.Val91Trpfs*30). Platelet lysates reveal no ARPC1B protein and greatly reduced Arp2/3 complex. Missense ARPC1B mutations are identified in an unrelated patient with similar symptoms and ARPC1B deficiency. ARPC1B-deficient platelets are microthrombocytes similar to those seen in Wiskott–Aldrich syndrome that show aberrant spreading consistent with loss of Arp2/3 function. Knockout of ARPC1B in megakaryocytic cells results in decreased proplatelet formation, and as observed in platelets from patients, increased ARPC1A expression. Thus loss of ARPC1B produces a unique set of platelet abnormalities, and is associated with haematopoietic/immune symptoms affecting cell lineages where this isoform predominates. In agreement with recent experimental studies, our findings suggest that ARPC1 isoforms are not functionally interchangeable.

No MeSH data available.