PB0374 - JAM-A regulates zipper-like structures that are essential for maintaining the integrity of megakaryocytes.

Background: Mature megakaryocytes are characterized by an extensive and dense membrane network, called the demarcation membrane system (DMS), which constitutes the membrane reservoir for future platelets. Its unique architecture is tightly controlled in vivo, with disorganization of the DMS leading to defects in thrombopoiesis. While it is established that DMS derives from the plasma membrane invaginations as well as from intracellular sources, the mechanisms by which this architecture is stabilized have received little attention.

Aims: In this study, we characterize zipper-like actin-based structures (ZLS) that appear at sites of plasma membrane invaginations and between adjacent DMS.

Methods: Mice bone marrow was studied by confocal and electron imaging approaches.

Results: Functionally, electron microscopy analysis indicated that ZLSs are sealing junctions from which the extracellular tracers are excluded. Immunostaining experiments demonstrated that JAM-A, a well-known endothelial tight junction regulator, is present in ZLSs and all along the DMS. We used JAM-A knockout mice (JAM-A-/-) to explore its role in DMS organization and thrombopoiesis. JAM-A-/- mice show a 35% decrease in circulating platelet count. In situ, bone marrow JAM-A-/- megakaryocytes are irregularly shaped and cytoplasmic fragments are present in the stroma, pointing to a role of JAM-A in maintaining cell integrity. The DMS is highly distended and is in continuity with the extracellular medium indicating that JAM-A is required to maintain the DMS. Remarkably, JAM-A depletion disrupts ZLS assembly in the plasma membrane invaginations, causing peripheral zone ruptures. Consistently, ex-vivo analysis of bone marrow explant showed an accelerated proplatelet formation in JAM-A-/- megakaryocytes compared to wild type and an increased proportion of proplatelets-extending megakaryocytes.

Conclusion(s): Altogether, we have identified a new structure of the DMS, the ZLS, and underscored its importance in maintenance of the DMS architecture. Furthermore, we propose a role for JAM-A in ZLS formation and MK cell integrity, while preventing early proplatelet release.