Fig 2: Actin isoform regulation in ACTB-AST patient fibroblasts. a RNA-Seq analysis of actin transcripts in P4 and P5 compared to the healthy control (log2 fold change ± s.e.m.). Bar graphs show the combined results from both patients relative to the control. Overlayed dot plots show the individual patient data calculated from three technical replicates (p < 0.0001 unless stated as ns); b Representative western blots show reduced ß-CYA, increased ?-CYA and a-SMA in P4 and P5 fibroblasts compared to the control (C). Double the sample amount was required to detect a-SMA (#); c Densitometry analysis of P4 and P5 signals expressed as mean (±s.d.) relative to the control (9–10 replicates from 6 lysates; Kruskal–Wallis test); d Reduced ß-CYA:?-CYA ratios are detected in P4 and P5 fibroblasts by immunofluorescence microscopy (mean ± s.d.; image numbers analyzed from 3 experiments are given in brackets; one-way ANOVA); e Representative maximum intensity projections show ß-CYA (top) and ?-CYA (bottom) distribution within z-stack slices 1–2 of C, P4, P5, and BWCFF control fibroblasts. Yellow arrows indicate cells where thick basal ß-CYA bundles are abundant. Scale bars are 15 µm; f Immunofluorescence microscopy shows increased a-SMA expression in P4 and P5 fibroblasts. Inserts show a-SMA incorporation in ACTB-AST sub-nuclear basal filaments. Scale bars are 15 µm; g Normalized a-SMA fluorescence intensity in individual cells (mean ± s.d.; the number of cells analyzed from 3 experiments is given in brackets; Kruskal–Wallis test); h The percentage of cells per image in which a-SMA is incorporated into basal sub-nuclear filaments (median and IQR; 14–16 images from 3 experiments; Kruskal-Wallis test); i P5 cells co-stained for ß-CYA or ?-CYA (left, green in merge) and a-SMA (mid, magenta in merge). Scale bars are 20 µm; j Greater overlap of a-SMA with ß-CYA is observed in P5 cells (median and IQR; 33–36 cells from 3 experiments; Mann–Whitney test). In all cases, *p < 0.05, ***p < 0.001, ****p < 0.0001 and ns, not significant

Fig 3: ACTB-AST patient platelets have disordered actin and microtubule cytoskeletons. a–c Assessment of the actin and microtubule cytoskeletons in platelets purified from healthy control (C), patient 3 (P3), patient 4 (P4), and patient 5 (P5) EDTA-peripheral blood (1 experimental replicate); a Representative maximum intensity projections (top row) are given for ß-CYA (green) and ?-CYA (magenta) labeled samples, showing reduced ß-CYA:?-CYA ratios in patient samples. Mid-stack slices of the marked regions of interest (white squares) are shown below, demonstrating cortical redistribution of actin isoforms in patient thrombocytes; b Mid-stack slices show strong cortical recruitment of NM-2A and a-actinin 1, and a moderate cortical enrichment of Filamin A in patient platelets; c Representative images demonstrate the highly disordered nature of ß-tubulin in patient-derived platelets compared to those from a healthy control. 3–6 images (total of 20–60 platelets) were assessed for each protein of interest. All scale bars represent 2 µm

Fig 4: ACTB-AST patients display minor facial anomalies and thrombocytopenia with enlarged platelets. a Craniofacial appearance of patient 3 (left, P3, p.Ala331Val_fs*27) at 5 years of age, patient 4 (mid, P4, p.Ala331Val_fs*27) at 31 years of age and patient 5 (right, P5, p.Ser338_Ile341del) at 4 year 10 months. Flared eyebrows (P3 and P4), straight eyebrows (P4 and P5), telecanthus (all), epicanthal folds (P3 and P5), upslanting palpebral fissures (P3 and P4), a broad nasal tip (P3 and P5), a bulbous nose (P4), a thin upper vermillion border (all) and prominent chin (P4) are observed in these patients; b, c CD61-labeled platelets purified from a healthy control (C), P3, P4 and P5 were analyzed by immunofluorescence microscopy; b Representative images show that platelets in patient samples vary from normal to large in size. Scale bars represent 5 µm; c Particle analysis shows a significant shift in the size distribution and average diameter of patient platelets compared with a healthy control. Individual data points are plotted with the median and IQR. The number of platelets analyzed from 1 experiment is given in brackets below each condition. Significance was determined with the Kruskal–Wallis and Dunn’s multiple comparisons tests, where ****p < 0.0001

Fig 5: ICAM-2 WT and variants co-precipitated with a-actinin. A) IP/IB experiments with control cell lines demonstrate the expected association of ICAM-2, a-actinin and actin in lysates from SK-N-ASpIRES.ICAM-2 cells (labeled as WT), but not SK-N-ASpIRESneo cells (neo). Results for these control cell lines were published previously [13]. B) ICAM-2 glycosylation site variants associated simultaneously with a-actinin and actin. Immunoprecipitations (IP) were performed using whole cell lysates and a mouse monoclonal antibody to a-actinin (MAB1682, Millipore). Following protein separation by electrophoresis, immunoblots (IB) were performed with antibodies to a-actinin (sc-7454, Santa Cruz Biotech), ICAM-2 (AF244, R&D Systems), or actin (4968, Cell Signaling). The presence of ICAM-2 WT and variants in each preparation was confirmed by immunoblot analysis of input preparations (a representative blot is shown in Figure 2C) and also by immunoblot analysis of the proteins remaining in the supernatant following precipitation (not shown), to confirm the presence of sufficient/excess ICAM-2 protein or variant in each preparation used for immunoprecipitation experiments.