CCM2-CCM3 interaction stabilizes their protein expression and permits endothelial network formation.
Bottom Line: Mutations in the essential adaptor proteins CCM2 or CCM3 lead to cerebral cavernous malformations (CCM), vascular lesions that most frequently occur in the brain and are strongly associated with hemorrhagic stroke, seizures, and other neurological disorders.CCM2 binds CCM3, but the molecular basis of this interaction, and its functional significance, have not been elucidated.However, CCM3 expression in the absence of CCM2 is sufficient to support normal cell growth, revealing complex-independent roles for CCM3.
Affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520.Show MeSH
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Mentions: Loss of CCM proteins has previously been shown to impair the ability of endothelial cells to assemble into vessel-like tubular networks resembling capillaries in vitro (Borikova et al., 2010; Wüstehube et al., 2010). We therefore used our knockdown and reconstitution system to investigate the importance of the CCM2–CCM3 interaction in this process. In keeping with previous reports (Jones et al., 1998), stimulating confluent EA.hy926 cell cultures with 2.5% ethanol for 3 h followed by plating on growth factor–reduced Matrigel in low serum (0.5% FBS) medium for 20 h resulted in the assembly of cells into branched networks. The short time course, coupled with the use of low serum and growth factor–reduced Matrigel, means there is little to no cell proliferation during the assay, minimizing the potential effect of altered growth rates on tube formation. As expected, both parental and scramble control knockdown EA.hy926 cells formed striking networks (Fig. 6 A). In addition to inspection of networks, we quantified the percentage of cells in networks (defined as linear or branched assemblies of at least five cells) across multiple fields and at least three independent experiments (see Materials and methods for details). This revealed that >90% of parental and control cells were in networks (Fig. 6 B). Consistent with previous work (Borikova et al., 2010), knockdown of CCM2 or CCM3 severely perturbed network formation, resulting in very few cells (<20%) being present in networks, and the very few networks present were generally very short (Fig. 6, A and B).
Affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520.