PIP5KIγ Knockout Megakaryocytes Have Defects in Their Cytoskeleton & Demarcation Membrane System, yet Form Proplatlets & Platelets.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a critical component of intracellular signaling cascades as well as actin dynamics. It is synthesized by the phosphorylation of PI4P by one of three isoforms of PIP5KI (α,β, or γ). We found that the targeted disruption of PIP5KIγ results in early prenatal mortality that prevented studies of bone marrow or liver hematopoietic cells. However, we were able to analyze yolk sac progenitor cells differentiated ex vivo into megakaryocytes. Imaging in the electron microscope showed that PIP5KIγ-null megakaryocytes have normal architectural appearance of their microtubules, and formed proplatelets and platelets. This demonstrates that PIP5KIγ is not essential for the development of megakaryocytes, proplatelets, or platelets. PIP5KIγ knockout megakaryocytes had normal basal levels of PIP2, but decreased synthesis following stimulation with thrombin. Using spinning disk video confocal microscopy, we found that wild type megakaryocytes actively formed and contracted lamellipodia, and rapidly spread upon a fibrinogen matrix. In contrast, PIP5KIγ-null megakaryocytes continuously and slowly extended and retracted membrane blebs, rather than lamellipodia. We analyzed whether PIP5KIγ-null megakaryocytes have a defect in their ability to anchor their membranes to the cytoskeleton accounting for the blebbing phenotype by using laser tweezers to pull the cell membrane apart from the cytoskeleton. Fibrinogen-coated beads bound both wild type and PIP5KIγ-null megakaryocytes. Yet wild type cells had rigid membranes that resisted stretching by trapped beads that were pulled by laser tweezers. In contrast, the majority of PIP5KIγ-null megakaryocytes had flexible membranes that were easily stretched by pulling on the fibrinogen-coated beads, and ultimately allowed membrane tethers to form. We found that the membrane phenotype was completely reverted by adding back wild type PIP5KIγ, but not by a catalytically inactive PIP5KIγ mutant. This shows that the lipid kinase activity of PIP5KIγ regulates the membrane phenotype. Surprisingly, PIP5KIβ-null megakaryocytes have at least as great of a defect in PIP2 synthesis but lack this membrane abnormality. Furthermore, overexpression of PIP5KIβ in PIP5KIγ-null megakaryocytes fails to rescue the cytoskeletal defect. Adding back a splice variant of PIP5KIγ that lacks the binding site for talin, also fails to revert this cytoskeletal defect present in PIP5KIγ-null cells. This suggests that talin binding and PIP2 synthesized specifically by the PIP5KIγ isoform are required for the cytoskeletal organization of megakaryocytes. Given the proposed role of PIP2 on the regulation of the demarcation membrane system (DMS), we expressed GFP-fused to β3-integrin and followed its trafficking from the DMS to the surface membrane. GFP-β3 situated on the DMS externalized to the cellular membrane within minutes of plating a wild type megakaryocyte upon immobilized fibrinogen. In megakaryocytes lacking PIP5KIγ, GFP-β3 situated on the DMS poorly externalized, and was barely detectable on the cell membrane 1 hour after adhering to immobilized fibrinogen. This shows that trafficking of αIIbβ3 from the DMS to the cell membrane requires PIP5KIγ. Together, this demonstrates that spatially restricted production of PIP2 by PIP5KIγ is required to preserve the integrity of the membrane cytoskeleton, and that PIP2 synthesized by PIP5KIβ in megakaryocytes can not contribute to this process.