PIP5Kγ-Null Mutation Induces Cytoskeletal Changes within Megakaryocytes.

All eukaryotic cells contain the phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2) that serves multiple roles in signaling cascades critical for actin dynamics. Phosphatidylinositol-4-phosphate 5 kinase (PIP5K) catalyzes the synthesis of PIP2 by phosphorylating PI4P. Although the classical PIP5K isoforms (α, β, and γ) generate the same phospholipid product, the isoforms have different primary structures, are expressed at different levels in various tissues, and localize in different intracellular compartments. Therefore, it is likely that the functions of these isoforms are different as well. PIP5Kγ is unique because it associates directly with talin and consequently localizes in focal adhesion. Given the role of PIP2 in actin dynamics, we generated a murine line containing a null mutation in the PIP5Kγ gene. We found that the targeted disruption of PIP5Kγ results in early prenatal mortality that is associated with pleotropic effects involving the cardiovascular, neurologic, and hematopoietic systems. This early lethality prevented studies of hematopoietic cells derived from the bone marrow or the liver. However, we were able to analyze yolk sac progenitor cells that were treated with thrombopoietin ex vivo, differentiating them into megakaryocytes. After 5 days in culture, many of the non-adherent yolk sac progenitor cells were multinucleated, and approximately 70% expressed CD41 (αIIb), a marker of the megakaryocyte lineage. We then examined their cytoskeletal content and dynamics. Absence of PIP5Kγ had no effect on the quantity of F-actin in unstimulated or thrombin-stimulated cells that were adherent to fibrinogen or were maintained in suspension in the absence of stimulation. Next, we examined membrane dynamics during cell adhesion in real time using spinning disk video confocal microscopy. Wild type megakaryocytes actively formed and contracted lamellipodia and rapidly spread upon the fibrinogen matrix. In contrast, PIP5Kγ-null megakaryocytes continuously extended and retracted membrane blebs, rather than lamellipodia, but eventually spread as much as wild type cells. This observation is consistent with the previous suggestion that PIP2 contributes to the stable association of the membrane with the cytoskeleton. It is also noteworthy that a similar phenotype has been described in cells with defective anchoring of the membrane to the cytoskeleton because of a lack of filamin. Accordingly, our data are consistent with the hypothesis that PIP5Kγ generates compartmentalized pools of PIP2 that contribute to the association of the membrane with the cytoskeleton.