The authors have declared that no competing interests exist.
Conceived and designed the experiments: MTH IH AWP. Performed the experiments: MTH MTJVDB. Analyzed the data: MTH MTJVDB. Wrote the paper: MTH MTJVDB IH AWP.
The motor protein myosin Va plays an important role in the trafficking of intracellular vesicles. Mutation of the
Platelet function was studied
Despite the importance of myosin Va in vesicle transport in other cells, our data demonstrate this motor protein has no non-redundant role in the secretion of dense and α-granules or other functional responses in platelets.
The unconventional, non-muscle, class V myosins play an important role in the transport of intracellular vesicles along actin filaments to membrane docking sites
A variety of proteins involved in the regulation of granule transport has been described to interact with myosin Va. In melanocytes, the cargo-carrying C-terminal tail of myosin Va binds to the exophilin melanophilin
The secretion of intracellular granules from platelets is essential in the process of thrombosis. Upon activation, platelets release a wide array of mediators from their dense and α-granules
In the present study, we used a novel targeted
The myosin Va antibody (#3402) was from Cell Signaling Technology (Danvers, MA, USA). The myosin Vb antibody (18), the myosin Vc antibody (Y-19), and the GAPDH antibody (6C5) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The myosin VI antibody (ABT42) was from Millipore (Temecula, CA, USA). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit, anti-mouse and anti-goat secondary antibodies were from Jackson ImmunoResearch Laboratories (Newmarket, UK). FITC-P-selectin and PE-JON/A antibodies were from Emfret Analytics (Eibelstadt, Germany). FITC-LAMP1 (1D4B) antibody was from Abcam (Cambridge, UK). NuPAGE LDS sample buffer was obtained from Invitrogen (Carlsbad, CA, USA). 10× blocking buffer and TRITC-phalloidin were from Sigma-Aldrich (Poole, UK). Fura-PE3 was from Teflabs (Austin, TX, USA). Luciferin-luciferase was from Chronolog (Labmedics, Stockport, UK). AYPGKF-NH2 (PAR4 activating peptide) was from Bachem (Weil-am-Rhein, Germany). CRP (cross-linked collagen-related peptide) was synthesized by Prof Richard Farndale (Department of Biochemistry, University of Cambridge, UK).
A colony of
Washed platelets (2×108/mL) were lysed in NuPAGE LDS sample buffer which was supplemented with 50 mM dithiothreitol. Samples were separated by electrophoresis on 6% Bis-Tris polyacrylamide gels. Proteins were then transferred to polyvinylidene difluoride membranes which were blocked with 1× blocking buffer and probed with specific primary and HRP-conjugated secondary antibodies. Proteins were detected using ECL reagents.
Subcellular morphology of WT and
To determine the dense-granule and α-granule content, total numbers of granules in equivalent-sized fields of view were counted. For each genotype, 10 randomly chosen fields of view were examined. All microscopic images were taken at the same magnification, and the number of cells per field of view was equivalent between WT and
The release of ATP from dense granule release was assessed luminometrically, as previously described
Washed platelets (5×107/mL; 32 µL) were incubated fluorescein isothiocyanate (FITC)-labelled anti-P-selectin antibody, phycoerythrin (PE)-labelled JON/A antibody (4 µL of each), and agonist (4 µL; 1∶10 dilution) for 10 min under non-stirring conditions. Platelets were then fixed with paraformaldehyde (2%). Two-colour analysis was conducted by flow cytometry on a FACSCalibur flow cytometer (BD Biosciences), using CellQuest version 3.1f software (BD Biosciences). The platelet population was identified by forward and side scatter profile.
To assess the timecourse of α-granule secretion, platelets were stimulated for the indicated time with AYPGKF (300 µM) prior to fixation. FITC-labelled anti-P-selectin was added for the final 30 s of stimulation.
Washed platelets (5×107/mL; 36 µL) were incubated FITC-labelled anti-LAMP1 antibody (4 µL), and agonist (4 µL; 1∶10 dilution) for 10 min under non-stirring conditions. Platelets were then fixed with paraformaldehyde (2%). FITC fluorescence was analysed by FACS analysis as above.
Changes in cytosolic [Ca2+]i were measured by spectrofluorometry in washed suspensions of platelets (1×108/mL) loaded with the Ca2+-senstive dye, Fura-PE3, as described previously
Coverslips were coated with fibrinogen (100 µg/ml). Washed platelets (2×107/mL) were dispensed onto the coverslip for 1 hr. Where indicated, platelets were stimulated with AYPGKF (300 µM) for 1 min prior to adhesion. Adherent platelets were fixed with paraformaldehyde (4%), permeabilised with Triton X-100, and stained with TRITC-phalloidin. Adhesion and spreading of platelets was observed by fluorescence microscopy using a cooled CCD camera attached to a Leica DM IRB inverted epifluorescence microscope with a 63× objective. Ten images were taken in different random parts of the coverslip area and analysed using ImageJ software. Platelets were scored as being completely unspread with no filopodia (“no spreading”), having filopodia, having both filopodia and partial lamellipodia (“some spreading”), or having a full lamellipodium (“full spreading”) and expressed as a proportion of the total number of platelets in the image. The proportions of each morphology in each of the ten images were then averaged. This analysis was performed separately on platelets from 3 WT mice and 3
Where presented, mean data are given ± SEM. Statistical significance was determined by 2-way ANOVA with Bonferroni post-test, performing using Prism 4.0 (GraphPad Software). P<0.05 was considered significant.
To evaluate the function of myosin Va in platelets,
(A) Schematic of the targeting vector used to disrupt the
As demonstrated by immunoblotting, myosin Va (207 kDa) was not detected in
Since Rab27 regulates dense granule formation and secretion in platelets
(A) TEM images (4800×, scale bar: 1 µM) show representative images of WT and
Dense granule secretion of ATP, monitored by luminometry, was stimulated by a range of concentrations of the GPVI agonist, collagen-related peptide (CRP) or the thrombin receptor PAR4 agonist, AYPGKF. However, no difference in ATP secretion was observed between wild-type and
Wild-type and
Next, we addressed whether myosin Va has a role in α-granule secretion. By flow cytometric analysis, P-selectin expression on the platelet surface was assessed. P-selectin surface expression induced by various concentrations of CRP and AYPGKF was not significantly affected in
Finally, we investigated whether myosin Va regulates lysosome secretion by assessing agonist-induced surface expression of LAMP1, which correlates with lysosomal enzyme release. Platelet activation induced an increase in surface LAMP1. This was not different between WT and
There are several possible explanations for these data. It is possible that myosin Va genuinely plays no role in granule secretion in platelets, despite a role for Rab27. This could reflect the small size of the platelet, and its extensive plasma membrane-associated target membrane system, the open canalicular system. This is comprised of multiple invaginations of the membrane, forming target sites for fusion of exocytotic vesicles throughout the cell. Effectively, this may mean that the majority of secretory vesicles may already be in a primed and docked position, and that there is no need for myosin-dependent trafficking. It might however be assumed that vesicles require trafficking to platelets within the megakaryocyte, and therefore that myosins may be required for this step. However, data from
On the other hand, it is also possible that other related myosins are expressed or over-expressed in the
Immunoblots showing the expression of myosin Vb, Vc, and VI in lysates from human, wild-type mouse (WT) and
Recently, it has been found that myosin VI regulates fusion pores formed between secretory vesicles and the plasma membrane
It was important also to investigate whether myosin Va plays a role in other platelet responses. Integrin αIIbβ3 activation was assessed by using the activation-specific antibody, Jon/A. CRP or AYPGKF induced integrin αIIbβ3 activation in wild-type and
Wild-type and
Myosin Va transports extensions of the ER into dendritic spines of Purkinje neurons, forming a local Ca2+ store that is required for local Ca2+ release
Wild-type and
Finally, we analysed whether loss of myosin Va affects platelet spreading on a fibrinogen-coated surface.
Wild-type and
We conclude therefore that although myosin Va has been shown to be critical in intracellular actin-dependent vesicle transport in melanocytes and neuroendocrine cells, our data demonstrate that this motor protein is not required for mouse platelet granule secretion and other aspects of mouse platelet function. This contrasts to the important role for Rab27a/b in platelet dense granule biogenesis and secretion
We are grateful for the expert assistance of Elizabeth Aitken in technical support for this work, particularly relating to mouse genotyping. We thank the staff of the Wolfson BioImaging Facility at the University of Bristol for assistance with cell imaging.