Fig 1: Vasculitis pathology and description of ARPC1B variants in patients.Patient 1 (a,c) and Patient 2 (b,d) developed skin lesions associated with small vessel vasculitis. Patient 1 (a) had a complicated ulcerating lesion after a skin biopsy preceded by the more typical skin lesions seen in Patient 2 (b). Images a,b are not included in the Creative Commons license for the article. Small vessel vasculitis was confirmed by skin biopsies (c,d); arrowheads indicate: (1) epidermis, (2) dermal–epidermal junctions, (3) dermis. At low magnification (c,d, left panels) areas of leukocytoclastic vasculitis (4) were evident, which at higher magnification (c,d, right panels) showed vessel wall destruction (5) and neutrophil infiltration (6). (e) ARPC1B is located on Chromosome 7 (position numbering relative to GRCh37), immediately preceded by ARPC1A. (f) Nucleotide positions of identified mutations (black arrows) relative to ARPC1B coding exons (accession #: NM_005720.3). Patient 1 is homozygous for c.269_270dupCT, Patient 2 is homozygous for two missense variants (c.314C>T and c.712G>A). (g) ARPC1B has 6 WD40 repeat domains forming a ß-propeller required for Arp2/3 complex function. The amino acid change caused by the mutation in Patient 1 causes a frame shift predicted to yield a protein lacking the last five WD40 domains; both mutations carried by Patient 2 affect WD40 domains. Adapted from http://smart.embl-heidelberg.de/smart/show_motifs.pl; ARC1B_HUMAN, O15143.
Fig 2: Effects of SKAP2 RNAi on ARP2 and WAVE2 expression. (A) Subcellular localization of ARP2 after SKAP2 siRNA injection. ARP2 was mainly distributed at the membrane in the control oocytes, whereas ARP2 expression was barely detectable in the siRNA-injected group. Green: ARP2; blue: chromatin. Bar = 20 µm. (B) Localization of WAVE2 after SKAP2 siRNA injection. WAVE2 was expressed around the spindle, whereas no specific localization of WAVE2 was observed around spindle in the siRNA-injected group. Red: WAVE2; blue: chromatin. Bar = 20 µm. (C) The fluorescence intensity of ARP2 in the SKAP2 siRNA-injected oocytes was decreased. (D) The fluorescence intensity of WAVE2 in SKAP2 siRNA-injected oocytes was significantly reduced. (E) ARP2 expression was reduced after SKAP2 siRNA injection by western blotting examination, as the relative intensity of ARP2 protein was significantly decreased. (F) WAVE2 expression was decreased after SKAP2 siRNA injection by western blotting analysis, as the relative intensity of WAVE2 protein was significantly reduced. *: significant difference (P < 0.05).
Fig 3: ARPC1B and Arp2/3 complex are deficient in ARPC1B mutant platelets.(a) Crystal structure of mammalian Arp2/3 showing relative location of component proteins from the protein data bank (PDB1K8K, http://www.rcsb.org/pdb/explore/explore.do?structureId=1K8K; Robinson et al.16). (b) Immunoblot analysis of platelet lysates showed ARPC1B to be absent in Patient 1 (P1) and greatly reduced in Patients 2 and 3 (P2, P3) compared to normal (N), while levels of ARPC2, ARPC3, ARPC5, ARP2 and ARP3 were normal (gamma tubulin used as loading control, see Supplementary Fig. 3a). (c) Platelet ARPC1A was increased in all three patients relative to normal, while WASP expression was equivalent (GAPDH used as loading control). (d) Immunoblot analysis of a normal platelet lysate after native gel electrophoresis showed a band corresponding to Arp2/3 complex detected by probing for ARPC1B (shown) or other Arp2/3 components (Supplementary Fig. 3d). This band was resolved on a second dimension SDS–PAGE gel, and immunoblotting confirmed the presence of Arp2/3 components (ARPC1B, ARPC2, ARP2 and ARP3 shown). (e) Immunoblotting of platelet lysates after native gel electrophoresis for ARPC5 (left) showed a greatly reduced level of Arp2/3 in Patient 1 (P1) platelets relative to normal (N). ARPC1A (right) was detected in the Arp2/3 complex in Patient 1 (P1) but not in normal (N) platelet lysate (native GAPDH tetramer used as loading control).
Fig 4: Arp2/3 is needed for CCV biogenesis.(A and B) CK-666 treatment prevents CCV expansion. Vero cells at 24 hr post-infection were treated with CK-666 or chloramphenicol for 2 days. Cells were fixed and fluorescently stained for CD63 and F-actin. (C and D) CK-666 treatment causes CCV collapse. Vero cells at 2 dpi were treated with CK-666 for 24 hr, then fixed along with the corresponding 3 dpi DMSO-treated control or washed to remove CK-666 and allowed an additional 24 hr recovery before fixation. Cells were fluorescently stained for F-actin and CD63. Histograms depict the means of the percentage of cells with normal CCV expansion or CCV area ± SD of = 60 cells for at least 3 independent experiments. Statistical significance was determined by the Student’s t-test (****P <0.0001). NMII, C. burnetii Nine Mile phase II strain. Scale bar, 5 µm.
Fig 5: Arp2/3 is required for endocytic trafficking of transferrin in C. burnetii infected cells.(A-C) Two dpi Vero cells treated with CK-666 or DMSO for 24 hr were incubated with 488-transferrin (488Tfr) for 1 hr, washed with PBS, then fixed and immunostained for CD63 and C. burnetii. CK-666 inhibits trafficking of 488Tfr to CCVs, decreasing the amount of 488Tfr on the CCV and colocalization with CCV CD63 clusters. Graphs represent the means ± SD of = 60 cells from 3 independent experiments. Statistical significance was determined by the Student’s t-test (****P <0.0001). NMII, C. burnetii Nine Mile phase II strain. Scale bar, 5 µm.
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