CIP4-Dependent Defects in Platelet Biogenesis Involve Changes in Membrane Rigidity Independently of WASP

Abstract 705

Using the Src kinase Lyn as bait in yeast two-hybrid screen, we isolated Cdc42-interacting protein 4 (CIP4). CIP4 is an F-BAR protein implicated in membrane curvature, membrane tubulation, and vesicle formation. CIP4 contains an SH3 domain that interacts with Wiskott-Aldrich Syndrome protein (WASP). We have reported thrombocytopenia in CIP4 knockout (KO) male mice, which was of similar severity as observed in WASP knockout male mice. Because a cardinal feature of Wiskott-Aldrich Syndrome is thrombocytopenia, we hypothesized that the CIP4-dependent thrombocytopenia occurs through the same mechanism as that in WASP-dependent thrombocytopenia. Interestingly, we have just generated a CIP4, WASP double knockout that displays a more severe thrombocytopenia. Neither CFU-MK progenitor studies nor ploidy studies showed a difference between CIP4 KO, WASP KO, double KO, and Wild Type (WT) mice. Thus, we reasoned that the defect lies in the structural production of platelets. We first studied where the proteins were localized and performed immunofluorescence staining and confocal microscopy analysis in cultured mouse megakaryocytes. We found a diffuse staining of CIP4 throughout the cytosol, the nucleus and proplatelets, with accumulation at the plasma membrane. In mouse megakaryocytes WASP staining was diffuse. In the human megakaryocytic CHRF-288-11 cell line, CIP4 staining was also diffuse. Because F-BAR proteins are also known to interact through their SH3 domain with members of the dynamin family, we investigated if CIP4 interacted with dynamin in the megakaryocytic lineage. While we saw diffuse staining for dynamin 3 in mouse megakaryocytes, we found co-localization of dynamin 3 with CIP4 at the cell periphery after exposure to known proplatelet-inducing agents such as phorbol ester (PMA) or fibronectin. We also performed functional assays in CIP4 KO mice. Since we did not find a difference in megakaryocytic progenitor numbers or in ploidy between the CIP4 knockout and the wild type mice, we studied later steps in platelet biogenesis. Preliminary studies were not indicative for decreased platelet lifespan in vivo in CIP4 KO mice. We counted the proplatelet formation in bone marrow-derived megakaryocytes in culture and found that CIP4 knockout megakaryocytes had decreased proplatelet formation compared with wild type (WT: 10.1 ± 1.6%, CIP4 KO: 4.5 ± 1%, p=0.04). We confirmed these findings in the CHRF-288-11 cell line with lentivirally mediated shRNA knockdown of CIP4 by counting the proplatelet-like protrusion formation in response to PMA (control: 15.4± 1.7%, CIP4 knockdown: 8.9 ± 1%, p=0.03). Because of the known direct interaction of CIP4 with the plasma membrane, we hypothesized that CIP4 deficit leads to changes in plasma membrane rigidity. By measuring fluorescence anisotropy, we confirmed decreased plasma membrane fluidity in CHRF-288-11 cells with CIP4 knockdown in response to agents that promote proplatelet-like protrusion formation, PMA and fibronectin. When using PMA, membrane fluidity increased by 4% or more in control samples (similar to the change obtained with known membrane perturbing agent ethanol 10 percent v/v), but did not increase in cells with CIP4 knockdown or N-WASP knockdown. Even more, plasma membrane rigidity increased by 3 ± 1.4% in CHRF-288-11 cells with CIP4 knockdown. Preliminary results showed the same loss of membrane fluidity in splenic B lymphocytes from CIP4 KO mice isolated (a 5% membrane fluidity increase in WT vs. none in CIP4 KO B lymphocytes in response to PMA). After exposure to fibronectin, membrane fluidity increased by 6.4 ± 1.3% in control CHRF-288-11 cells, whereas it did not increase in cells with CIP4 knockdown, and it increased in cells with N-WASP knockdown but only by 3 ± 0.9%. Interestingly, while CHRF-288-11 cells with knockdown of WASP still showed loss of plasma membrane fluidity in response to PMA (increased only by 1.8 ± 0.7%), this was not seen with fibronectin stimulation, where the membrane fluidity increase (5 ± 0.5%) did not appear statistically different from the response in control cells.

Altogether our results point to decreased membrane fluidity in CIP4 deficient cells and they suggest that other pathways besides WASP pathways are involved in CIP4 dependent platelet biogenesis.