Dataset Information

Localization Microscopy of Actin Cytoskeleton in Human Platelets.

ABSTRACT: Here, we measure the actin cytoskeleton arrangement of different morphological states of human platelets using a new protocol for photo-switching of rhodamine class fluorophores. A new medium composition was established for imaging the cytoskeleton using Alexa Fluor 488 conjugated to phalloidin. Morphological states of platelets bound to a glass substrate are visualized and quantified by two-dimensional localization microscopy at nanoscopic resolution. Marker-less drift correction yields localization of individual Alexa 488 conjugated to phalloidin with a positional accuracy of 12 nm.

SUBMITTER: Mayr S

PROVIDER: S-EPMC5979344 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Localization Microscopy of Actin Cytoskeleton in Human Platelets.

Mayr Sandra S Hauser Fabian F Peterbauer Anja A Tauscher Andreas A Naderer Christoph C Axmann Markus M Plochberger Birgit B Jacak Jaroslaw J

International journal of molecular sciences 20180411 4

Here, we measure the actin cytoskeleton arrangement of different morphological states of human platelets using a new protocol for photo-switching of rhodamine class fluorophores. A new medium composition was established for imaging the cytoskeleton using Alexa Fluor 488 conjugated to phalloidin. Morphological states of platelets bound to a glass substrate are visualized and quantified by two-dimensional localization microscopy at nanoscopic resolution. Marker-less drift correction yields localiz ...[more]

PMID: 29641438

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Project description:Thrombospondin-1 (TSP-1) is an adhesive glycoprotein which, when secreted from alpha-granules of activated platelets, can bind to the cell surface and participate in platelet aggregate formation. In this study, we show that thrombin activation leads to the rapid and specific association of a large amount of secreted alpha-granular TSP-1 with the actin cytoskeleton. This cytoskeletal association of TSP-1 was correlated with platelet secretion, but not aggregation, and was inhibited by cytochalasin D, an inhibitor of actin polymerization. Association of TSP-1 with the actin cytoskeleton was mediated by membrane receptors, as shown by using MAII, a TSP-1-specific monoclonal antibody that inhibited both TSP-1 surface binding to activated platelets and cytoskeletal association. TSP-1 and its potential membrane receptors, e.g. alphaIIbbeta3 integrin, CD36 and CD47, concomitantly associated with the actin cytoskeleton. However, studies on platelets from a patient with type I Glanzmann's thrombasthenia lacking alphaIIbbeta3 and another with barely detectable CD36 showed normal TSP-1 surface expression and association with the actin cytoskeleton. Likewise, no involvement of CD47 in TSP-1 association with the actin cytoskeleton could be inferred from experiments with control platelets using the function-blocking anti-CD47 antibody B6H12. Finally, assembly of signalling complexes, as observed through translocation of tyrosine-phosphorylated proteins and kinases to the actin cytoskeleton, was found to occur in concert with cytoskeletal association of TSP-1, in control platelets as well as in thrombasthenic and CD36-deficient platelets. Our results imply a role for the actin cytoskeleton in the membrane-surface expression process of TSP-1 molecules and suggest a possible coupling of TSP-1 receptors to signalling events occurring independently of alphaIIbbeta3 or CD36. These results provide new insights into the link between surface-bound TSP-1 and the contractile actin microfilament system which may promote platelet aggregate cohesion.

Dataset Information

Localization Microscopy of Actin Cytoskeleton in Human Platelets.

Publications

Localization Microscopy of Actin Cytoskeleton in Human Platelets.

Similar Datasets

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