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Abnormal subcellular distribution of myosin and talin in Wistar Furth rat platelets
T I Pestina1, C W Jackson, P E Stenberg
1Department of Hematology/Oncology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
Abstract:
The roles of most cytoskeletal proteins in platelet formation and function remain largely undefined. We earlier detected megakaryocyte membrane blebbing and a unique antigenic determinant associated with a missense mutation in the cytoskeletal protein, talin, in an animal model of hereditary macrothrombocytopenia, the Wistar Furth (WF) rat, which led us to examine the distribution of talin and other cytoskeletal proteins in resting normal and WF rat platelets. In contrast to the conclusions of an earlier ultrastructural analysis, our biochemical and ultrastructural immunogold studies indicate a significant membrane-association of talin in both resting normal and WF rat platelets as found earlier for rat megakaryocytes. Talin was associated with plasma membranes, membranes of the surface-connected canalicular system, and with alpha-granule membranes of both normal and WF rat platelets, but as in WF megakaryocytes, talin was absent from the large membrane complexes of WF platelets. An even more striking difference was seen in the distribution of myosin in subcellular fractions of normal and WF rat platelets separated in density gradients, in which the proportion of myosin in the least dense WF rat platelet membrane fraction was one half that in the same normal platelet fraction. This difference was balanced by a fourfold increase in myosin in the most dense WF rat subcellular fraction, which is highly enriched for alpha-granules. These results support our hypothesis that the platelet abnormalities of the WF rat are related to defects in the megakaryocyte-platelet cytoskeleton.
Insights
Cytoskeletal protein defects in Wistar Furth (WF) rats cause hereditary macrothrombocytopenia. Talin and myosin distribution differs in WF platelets, suggesting a megakaryocyte-platelet cytoskeleton defect.
Area of Science:
- Hematology
- Cell Biology
- Biochemistry
Background:
- The function of cytoskeletal proteins in platelet formation is not well understood.
- Hereditary macrothrombocytopenia in Wistar Furth (WF) rats is linked to a mutation in the cytoskeletal protein talin.
- Previous studies suggested limited membrane association of talin in platelets.
Purpose of the Study:
- To investigate the distribution of talin and myosin in normal and WF rat platelets.
- To determine the role of cytoskeletal proteins in the platelet abnormalities observed in WF rats.
Main Methods:
- Biochemical analysis of subcellular fractions from normal and WF rat platelets.
- Ultrastructural immunogold labeling to localize talin and myosin.
- Density gradient centrifugation to separate platelet components.
Main Results:
- Talin is significantly associated with platelet membranes, including plasma membranes, the surface-connected canalicular system, and alpha-granule membranes in both normal and WF rats.
- Talin is notably absent from large membrane complexes in WF platelets.
- Myosin distribution is altered in WF platelets, with reduced presence in lighter membrane fractions and increased presence in denser, alpha-granule-enriched fractions.
Conclusions:
- The findings challenge previous assumptions about talin's membrane association in platelets.
- Altered distribution of talin and myosin in WF rat platelets supports the hypothesis of a megakaryocyte-platelet cytoskeleton defect.
- These cytoskeletal abnormalities are likely responsible for the hereditary macrothrombocytopenia in WF rats.