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. 2008 Feb;118(2):812-9.
doi: 10.1172/JCI34239.

Loss of PIP5KIgamma, unlike other PIP5KI isoforms, impairs the integrity of the membrane cytoskeleton in murine megakaryocytes

Yanfeng Wang  1 , Rustem I Litvinov, Xinsheng Chen, Tami L Bach, Lurong Lian, Brian G Petrich, Susan J Monkley, Yasunori Kanaho, David R Critchley, Takehiko Sasaki, Morris J Birnbaum, John W Weisel, John Hartwig, Charles S Abrams
Affiliations

Loss of PIP5KIgamma, unlike other PIP5KI isoforms, impairs the integrity of the membrane cytoskeleton in murine megakaryocytes

Yanfeng Wang et al. J Clin Invest. 2008 Feb.

Erratum in

  • J Clin Invest. 2009 Feb;119(2):421. Kanaho, Yasunori [added]

Abstract

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is an abundant phospholipid that contributes to second messenger formation and has also been shown to contribute to the regulation of cytoskeletal dynamics in all eukaryotic cells. Although the alpha, beta, and gamma isoforms of phosphatidylinositol-4-phosphate-5-kinase I (PIP5KI) all synthesize PIP2, mammalian cells usually contain more than one PIP5KI isoform. This raises the question of whether different isoforms of PIP5KI fulfill different functions. Given the speculated role of PIP(2) in platelet and megakaryocyte actin dynamics, we analyzed murine megakaryocytes lacking individual PIP5KI isoforms. PIP5KIgamma(-/-) megakaryocytes exhibited plasma membrane blebbing accompanied by a decreased association of the membrane with the cytoskeleton. This membrane defect was rescued by adding back wild-type PIP5KIgamma, but not by adding a catalytically inactive mutant or a splice variant lacking the talin-binding motif. Notably, both PIP5KIbeta- and PIP5KIgamma(-/-) cells had impaired PIP(2) synthesis. However, PIP5KIbeta-null cells lacked the membrane-cytoskeleton defect. Furthermore, overexpressing PIP5KIbeta in PIP5KIgamma(-/-) cells failed to revert this defect. Megakaryocytes lacking the PIP5KIgamma-binding partner, talin1, mimicked the membrane-cytoskeleton defect phenotype seen in PIP5KIgamma(-/-) cells. These findings demonstrate a unique role for PIP5KIgamma in the anchoring of the cell membrane to the cytoskeleton in megakaryocytes, probably through a pathway involving talin. These observations further demonstrate that individual PIP5KI isoforms fulfill distinct functions within cells.

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Figures

Figure 1
Figure 1. PIP5KIγ–/– megakaryocytes have defective attachment of the cell membrane to the cytoskeleton.
Yolk sac progenitor cells were differentiated into megakaryocytes by culturing in thrombopoietin for 5 days. The membranes of wild-type (A) and PIP5KIγ–/– (B) megakaryocytes were stained with GFP-PLCδ PH domain, and the cells were layered over immobilized fibrinogen. Shown are confocal images of the plasma membrane morphology of PIP5KIγ+/+ and PIP5KIγ–/– megakaryocytes. Similar results were seen when fluorescent nonspecific lipophilic dye, Vybran DIO, was substituted for GFP-PLCδ PH. Real-time images of cells can also been seen in Supplemental Movies 1 and 2.
Figure 2
Figure 2. Loss of PIP5KIγ disrupts the association of the membrane to the cytoskeleton.
Progenitor cells that were differentiated into megakaryocytes were analyzed for their ability to form membrane tethers by an optical trap pulling on a fibrinogen-coated membrane-attached bead. (A) Data traces of successive force signals produced during repeated contacts of a fibrinogen-coated latex bead with a wild-type megakaryocyte. PIP5KIγ+/+ megakaryocytes possessed a cell membrane firmly bound to the cytoskeleton. At the moment of contact of a bead with a wild-type cell, the bead exerted a small positive compressive force on the cell (left part of upper panel). When the bead attached to the cell was pulled away, the force on the bead increased in the negative direction until the attachment bond was ruptured. At this point, the force rapidly returned to zero. If no attachment occurred, there was no negative rupture force signal. The lower panel shows a tracing derived from a PIP5KIγ–/– megakaryocyte. Initially, the bead freely oscillated and exerted no force signals while it approached the cell with 10-nm steps. After the fibrinogen-coated bead bound to the cell, the optical trap pulled it away. The first downward signal reflects the force needed to tear the membrane part off of the underlying cytoskeleton. Further positive and negative signals reflect cooscillation of the bead and the tethered membrane, which was typical for the PIPKγ-null megakaryocytes. (B) DIC microscopy demonstrated that PIP5KIγ–/– megakaryocytes formed membrane tethers by pulling on a murine fibrinogen-coated bead that was touched to the surface of the cell and then moved apart.
Figure 3
Figure 3. PIP5KIγ, but not other PIP5KI isoforms, is required for stable interaction between the membrane and the cytoskeleton.
(A) The ability of different genotypes of megakaryocytes to form membrane tethers is shown. The total number of interactions analyzed for each cell genotype were: PIP5KIγ–/–, 91; PIP5KIγ+/–, 68; wild-type, 11; PIP5KIβ+/–, 12; and PIP5KIβ–/–, 22. (B) The ability of megakaryocytes derived from adult PIP5KIα-null or control mice to form membrane tethers is shown. For these experiments, the number of interactions analyzed were 345 (PIP5KIα–/–) and 83 (wild-type). (C) Shown is the tether formation in a PIP5KIγ–/– megakaryocyte rescued by infection with retroviruses that induced expression of GFP fused to either wild-type PIP5KIγ (n = 151), a catalytically inactive mutant PIP5KIγ D316A R447A K448A (PIP5KIγ KD, n = 126), the PIP5KIγ splice variant (PIP5KIγ Δ636-661, n = 132) lacking the terminal 26–amino acid talin binding domain, or PIP5KIβ (n = 71). The control bar on the left (no rescue) shows the tether formation in PIP5KIγ–/– megakaryocytes infected with empty retrovirus (n = 15). The statistical analysis was performed using the χ2 test.
Figure 4
Figure 4. Loss of either PIP5KIβ or PIP5KIγ impairs PIP2 synthesis.
PIP5KIβ–/– and PIP5KIγ–/– progenitor cells were differentiated into megakaryocytes and incubated for 2 hours with free 32P prior to stimulation with thrombin. Cells were lysed, fractionated by thin-layer chromatography, and analyzed for their relative PIP2 concentrations. Data was normalized to the concentration of PA. The graph shows the mean ± SEM for 6 experiments.
Figure 5
Figure 5. Talin1-null megakaryocytes mimic cells lacking PIP5KIγ.
Megakaryocytes were derived from the femurs of adult wild-type mice (C57BL/6) or from conditionally targeted talin1 mice with (Tln1fl/fl;PF4Cre+) or without (Tln1fl/fl;PF4Cre–) a Cre transgene. (A) Immunoblot showing level of talin and actin expression in megakaryocytes of different genotypes. (B) DIC images of wild-type (C57BL/6) and talin1-null (Tln1fl/fl;PF4Cre+) megakaryocytes demonstrate that loss of talin1 results in the formation of membrane blebs. (C) The ability of different megakaryocytes to form membrane tethers is shown. For these experiments, the number of cells analyzed was C57BL/6 (n = 66), Tln1fl/fl;PF4Cre– (n = 227), and Tln1fl/fl;PF4Cre+ (n = 178). The results show that the stable anchoring of the cell membrane to the cytoskeleton correlates with talin levels.
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