Supplementary Figure 1 Pfn1, but not other Pfn isoforms are expressed in

Transcription

1 Supplementary Figure 1 Pfn1, but not other Pfn isoforms are expressed in platelets. (a) RT-PCR of Pfn isoforms in control mouse platelets, Pfn1 -/- platelets and control heart. Expected band size for Pfn1 in control platelets 305 bp and 113 bp in Pfn1 -/- platelets. No other isoforms were detected in platelets. (b) Whole platelet or (c) bone marrow MK proteins were separated by SDS-PAGE and immunoblotted with an anti-pfn1 antibody.

2 Supplementary Figure 2 Microthrombocytopenia in Pfn1 -/- mice shown in blood smears. A 5 µl drop of blood was pulled longitudinally across a glass slide using a second glass slide at a 45 angle and stained according to Pappenheim. Scale bars represent 10 µm. Images are representative of 5 vs 5 individuals.

3 Supplementary Figure 3 Reduced platelet size in 6-weeks old Pfn1 -/- and Pfn +/- mice. Fluorescence-activated cell sorter analyses. (a) Peripheral platelet counts and platelet size of control, Pfn1 +/- and Pfn1 -/- platelets. Values are mean ± SD (n = 5 vs 5). FSC indicates forward scatter. (b) Peripheral platelet counts and platelet size of 6-weeks old mice (n = 7 vs 7). Values are mean ± SD. (c) Surface expression levels of prominent platelet surface receptors. Values are mean fluorescence intensity ± SD (n = 6 vs 6). Unpaired Student s-t-test, (*), P < 0.05; (**), P < 0.01; (***), P < 0.001; n.s. non-significant.

4 Supplementary Figure 4 Increased number, but normal localisation of Pfn1 -/- bone marrow MKs. (a) Number of MKs were determined per visual field of 328 x 246 µm. Values are mean ± SD of at least 6 vs 6 individuals. (b) Bone marrow MKs were stained with a megakaryocyte-specific antibody (anti-gpiib/iiia) and DNA with propidium iodide. DNA distribution was determined by flow cytometric analysis. Values are mean ± SD (n = 10 vs 14). (c and d) Total numbers of bone marrow MKs were determined per visual field of x µm by using confocal images with immunostained MKs and vessels. MKs closer than 1 µm to the vessel were counted as MKs at the sinusoids. Sin = sinusoid, BC = bone marrow compartment, Norm = round MKs, Pre = MKs that pre-released proplatelets or platelets into bone marrow. Values are mean ± SD (n = 252 vs 383 MKs) (e) Confocal images of bone marrow-derived MKs spread on

5 fibrinogen. Scale bar represents 15 µm. Images are representative of at least 5 vs 5 individuals. Unpaired Student s-t-test, (*), P < 0.05; (**), P < 0.01; (***), P < 0.001; n.s. non-significant.

6 Supplementary Figure 5 Defective podosome formation of Pfn1 -/- MKs. Confocal images of bone marrow-derived MKs spread (3 h) on collagen I and stained for F-actin, DAPI and phospho-asap, WASp or vinculin. Scale bar represents 25 µm. Images are representative of at least 5 vs 5 individuals.

7 Supplementary Figure 6 Decreased actin assembly in Pfn1 -/- platelets. (a) Whole platelet lysates were separated by SDS-PAGE and immunoblotted with anti-actin or tubulin antibody and normalized to controls. Band intensities were determined by ImageJ software. Values are mean ± SD (n = 4 vs 4). (b and c) Washed platelets, resting or activated with the indicated agonists were fixed, permeabilized, stained with phalloidin-fitc, and analysed by flow cytometry. (b) The mean phalloidin-fitc fluorescence intensity of resting platelets was determined. Values are mean ± SD (n = 3 vs 3) (c) The ratio of polymerized actin in activated vs. resting platelets was determined. Thr = thrombin, Rhd = rhodocytin, CRP = collagen-related peptide. Values are mean ± SD (n = 3 vs 3). (d) Microtubule coils were determined from at least 120 platelets per condition.

8 Values are mean ± SD. Unpaired Student s-t-test, (*), P < 0.05; (**), P < 0.01; (***), P <

9 Supplementary Figure 7 Pfn1 expression in human platelets and mouse MK. Determination of Pfn1 expression in (a) cultured fetal liver cell-derived MKs with formed proplatelets and (b) human platelets by using confocal images. F-actin (red), α-tubulin (green), Pfn1 (cyan) and DAPI (blue). Scale bars represent (a) 10 µm and (b) 3 µm. Images are representative of at least 3 individuals.

10 Supplementary Figure 8 Pre-treatment with colchicine cannot rescue filopodia formation of Pfn1 -/- platelets on von-willebrand factor. Control and Pfn1 -/- platelets were pretreated with 10 µm colchicine, spread (15 min) on a von-willebrand factor-coated surface, (a) stained for F-actin and α-tubulin and (b) filopodia formation was quantified. Scale bar represents 3 µm. Values are mean ± SD (n = 3 vs 3 individuals). Unpaired Student s-t-test, (***), P <

11 Supplementary Figure 9 Pfn1 -/- platelets fail to reorganize their microtubule coils on CRP. Control and Pfn1 -/- platelets spread (15 min) on a CRP-coated surface were stained for F-actin (red) and α-tubulin (green) and analysed by confocal microscopy (upper panels) or processed for rapid-freezing electron microscopic analysis of the cytoskeleton (lower panels). Arrow indicates disintegrated appearing microtubule coils that could not be reorganized upon spreading. Scale bars represent 3 µm in confocal images, and 1 µm in TEM images. Images are representative of at least 5 vs 5 individuals.

12 Supplementary Figure 10 Pfn1 -/- platelets contain highly acetylated microtubules that cannot be reorganized. (a) Resting control and Pfn1 -/- platelets contain highly acetylated microtubules. Whilst control platelets completely deacetylate and reorganize their microtubules to allow efficient spreading, Pfn1 -/- platelets maintain a number of stable, highly acetylated microtubules when spread (15 min) on (b) fibrinogen-, (c) von-willebrand factor- or (d) collagenrelated peptide-coated surfaces. Spread control and Pfn1 -/- platelets were stained for F-actin (red) and acetylated-tubulin (green). Scale bars represent 3 µm. Images are representative of at least 5 vs 5 individuals.

13 Supplementary Figure 11 The actin cytoskeleton does not contribute to increased microtubule stability in Pfn1 -/- platelets. Control and Pfn1 -/- platelets were preincubated for 30 min at 37 C either with (a) 10 µm colchicine, (b) 10 µm taxol or (c) 5 µm Lat A prior to spreading (15 min) on (a and b) a fibrinogencoated surface or (c) incubation at 4 C. Platelets were stained for F-actin (red) and α- (green, b and c) or acetylated-tubulin (green; a). Scale bars represent 3 µm. Images are representative of at least 5 vs 5 individuals.

14 Supplementary Figure 12 Toxin-mediated stabilization decreases the mean microtubule coil area. (a) Control platelets were treated with 10 µm taxol and/or 200 ng ml -1 TSA for 30 min, fixed on PLL-coated slides and stained for α-tubulin (green) and F-actin (red). Scale bars represent 3 µm. (b) F-actin content of toxintreated platelets was determined by flow cytometry. (c) Normal and aberrant shape of platelets was quantified. Values are mean ± SD (n = 5 vs 5). Unpaired Student s-t-test, (**), P < 0.01.

15 Supplementary Figure 13 Microtubules in Wasp -/- platelets can be reorganized upon different challenges. (a) Cryo sections of whole femora revealed a premature platelet release within the bone marrow of Wasp -/- mice. Endothelium is stained in red (CD105), MKs and platelets in green (GPIbα) and nuclei are highlighted in blue (DAPI). Scale bars represent 50 µm. (b) Densitometry of actin and tubulin via Western blot using count-adjusted Wasp +/+ (black bar) and Wasp - /- platelets (dark grey bar). Values are mean ± SD (n = 3 vs 3 samples per group). (c) Platelets were allowed to spread (15 min) on a fibrinogen-coated surface or (d) chilled at 4 C and fixed on a PLL-coated surface. F-actin is highlighted in red and α-tubulin in green. Scale bars represent 3 µm. Images are representative of at least 3 vs 3 individuals. Unpaired Student s-t-test, (***), P <

16 Supplementary Figure 14 Platelets of Wiskott-Aldrich syndrome patients and carriers resemble the microtubule phenotype of Pfn1 -/- platelets. (a) On poly-llysine immobilized resting platelets of a WAS family consisting of a healthy father (WASP y/+ ), a heterozygous mother (carrier, WASP +/- ) and a son, patient 3 suffering from WAS (WASp y/- ) were stained for F-actin (red) and different tubulin isoforms (green) and analysed by confocal microscopy. Father: 30 years, healthy; Mother 26 years, normal platelet numbers with a decreased mean platelet volume; patient 3: 5 months, requiring blood transfusions, carrying an IVS6+1G>A splice-site mutation in intron 6. Platelet microtubules were challenged with (b) colchicine- (10 µm) or (c) by incubation at 4 C for 3.5 h. (d) Resting platelets of patient 3 were stained for acetylated tubulin (green) and F- actin (red). (e) TEM revealed an increased number and aberrant organization of

17 the microtubules in the WAS patients platelets as compared to healthy controls. Scale bars in a-d represent 3 µm, and in e 1 µm. Supplementary Figure 15 Pfn1 does not directly bind to microtubules. A commercially purchased protein fraction of microtubule associated proteins (MAPF; #MAPF-A, Cytoskeleton Inc.) and platelet lysates of Pfn1 +/+ and Pfn1 -/- platelets were separated by SDS-PAGE, immobilized on a PVDF membrane and probed for tubulin and Pfn1.

18 Supplementary Figure 16 Pfn1 co-immunoprecipitates with WASp in resting or 10 µg ml -1 CRP-activated mouse platelets. WASp was precipitated from sonificated platelet lysates using a mouse anti-wasp antibody. Samples were separated by SDS-PAGE and immunoblotted with a rabbit anti-pfn1 or a rabbit anti-wasp antibody.

19 Supplementary Figure 17 Pfn1 co-localises with microtubules in mouse embryonic fibroblasts and T-cells. (a) Fibroblasts were grown on coverslips or (b) T-cells were allowed to adhere to PLL-coated slides, fixed and stained for F-actin (red), α-tubulin (green), Pfn1 (cyan) and DAPI (blue). Scale bars (lower right in merged images) represent 10 µm for fibroblast and 3 µm for T-cells.

20 Supplementary Figure 18 Normal microtubule organisation in Wip -/- mice. (a) Control and Wip -/- platelets spread (15 min) on fibrinogen were stained for F-actin (red) and α-tubulin (green) and analysed by confocal microscopy. (b) Control and Wip -/- platelets were incubated for 3.5 h at 4 C, fixed on PLL-coated slides and stained for F-actin (red) and α-tubulin (green). Images are representative of at least 3 vs 3 individuals. Scale bars represent 3 µm.

21 Supplementary Figure 19 Western blot images of selected bands displayed in main figures. Boxed areas were cropped for designated figures.

22 Supplementary Figure 20 Western blot images of selected bands displayed in supplementary figures. Boxed areas were cropped for designated figures.