There may be redundancy of function Title Loaded From File between this motor protein and myosin Va in the control of granule secretion. This may be more important in mouse platelets than in human platelets, as mRNA analysis indicates a greater abundance of myo6 mRNA in mouse platelets [14]. The possible role of myosin VI in mouse platelet exocytosis deserves further attention.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 [26]. We tested therefore whether myosin Va also transports Ca2+ stores into platelets during thrombopoiesis. Mouse platelets were loaded with the Ca2+-sensitive dye, FuraPE3. Rapid depletion of the intracellular Ca2+ stores by ionomycin produced a transient increase in [Ca2+]i that was not significantly different in Myo5a2/2 platelets (Fig. 6A). Furthermore, Ca2+ signals in response to a range of concentrations of CRP were not significantly altered (Fig. 6B). These data suggest that myosin Va is not required to transport intracellular Ca2+ stores into platelets, since the stores are equivalently loaded with releasable calcium, and is not involved in agonist-induced Ca2+ signalling in platelets. Finally, we analysed whether loss of myosin Va affects platelet spreading on a fibrinogen-coated surface. Myo5a2/2 platelets did not show a difference in spreading on this surface whether in presence or absence of stimulation with AYPGKF (Fig. 7A). Morphological analysis also did not reveal any difference in subtype fractions (no spreading, filopodial, some spreading or full spreading) between myosin Va-deficient and wild-type platelets (Fig. 7B). 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 [9?1], and suggests that Rab27 proteins act through effectors other than myosin Va. One such effector is Munc13-4, which is essential for platelet dense granule secretion [27?8]. However, since Munc134-deficient platelets have normal granule numbers [27], there is likely to be another Rab27 effector that is involved in granule biogenesis that remains to be described.AcknowledgmentsWe 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.Myo5a2/2 platelets show normal integrin aIIbb3 activation, Ca2+ signalling and spreadingIt was important also to investigate whether myosin Va plays a role in other platelet responses. Integrin aIIbb3 activation was assessed by using the activation-specific antibody, Jon/A. CRP or AYPGKF induced integrin aIIbb3 activation in wild-type and Myo5a2/2 platelets with no Title Loaded From File significant differences over a range of concentrations of agonists (Fig. 5).Author ContributionsConceived and designed the experiments: MTH IH AWP. Performed the experiments: MTH MTJVDB. Analyzed the data: MTH MTJVDB. Wrote the paper: MTH MTJVDB IH AWP.
Penile cancer affects predominantly men aged between 50 and 70 years [1?]. Penile cancer is associated with several established risk factors and associated di.There may be redundancy of function between this motor protein and myosin Va in the control of granule secretion. This may be more important in mouse platelets than in human platelets, as mRNA analysis indicates a greater abundance of myo6 mRNA in mouse platelets [14]. The possible role of myosin VI in mouse platelet exocytosis deserves further attention.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 [26]. We tested therefore whether myosin Va also transports Ca2+ stores into platelets during thrombopoiesis. Mouse platelets were loaded with the Ca2+-sensitive dye, FuraPE3. Rapid depletion of the intracellular Ca2+ stores by ionomycin produced a transient increase in [Ca2+]i that was not significantly different in Myo5a2/2 platelets (Fig. 6A). Furthermore, Ca2+ signals in response to a range of concentrations of CRP were not significantly altered (Fig. 6B). These data suggest that myosin Va is not required to transport intracellular Ca2+ stores into platelets, since the stores are equivalently loaded with releasable calcium, and is not involved in agonist-induced Ca2+ signalling in platelets. Finally, we analysed whether loss of myosin Va affects platelet spreading on a fibrinogen-coated surface. Myo5a2/2 platelets did not show a difference in spreading on this surface whether in presence or absence of stimulation with AYPGKF (Fig. 7A). Morphological analysis also did not reveal any difference in subtype fractions (no spreading, filopodial, some spreading or full spreading) between myosin Va-deficient and wild-type platelets (Fig. 7B). 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 [9?1], and suggests that Rab27 proteins act through effectors other than myosin Va. One such effector is Munc13-4, which is essential for platelet dense granule secretion [27?8]. However, since Munc134-deficient platelets have normal granule numbers [27], there is likely to be another Rab27 effector that is involved in granule biogenesis that remains to be described.AcknowledgmentsWe 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.Myo5a2/2 platelets show normal integrin aIIbb3 activation, Ca2+ signalling and spreadingIt was important also to investigate whether myosin Va plays a role in other platelet responses. Integrin aIIbb3 activation was assessed by using the activation-specific antibody, Jon/A. CRP or AYPGKF induced integrin aIIbb3 activation in wild-type and Myo5a2/2 platelets with no significant differences over a range of concentrations of agonists (Fig. 5).Author ContributionsConceived and designed the experiments: MTH IH AWP. Performed the experiments: MTH MTJVDB. Analyzed the data: MTH MTJVDB. Wrote the paper: MTH MTJVDB IH AWP.
Penile cancer affects predominantly men aged between 50 and 70 years [1?]. Penile cancer is associated with several established risk factors and associated di.