TY - JOUR T1 - A Key Cytoskeletal Regulator of Ubiquitination Amplifies TGFβ Signaling During Mouse Developmental Vascular Patterning JF - bioRxiv DO - 10.1101/055129 SP - 055129 AU - Ronak Shetty AU - Divyesh Joshi AU - Mamta Jain AU - Madavan Vasudevan AU - Jasper Chrysolite Paul AU - Ganesh Bhat AU - Poulomi Banerjee AU - Takaya Abe AU - Hiroshi Kiyonari AU - K. Vijayraghavan AU - Maneesha S. Inamdar Y1 - 2016/01/01 UR - http://biorxiv.org/content/early/2016/05/24/055129.abstract N2 - Vascular development involves de novo formation of a capillary plexus, which is then pruned and remodeled by angiogenic events. Cytoskeletal remodeling and directional endothelial migration are essential for developmental and pathological angiogenesis. Smad-dependent TGFβ signaling controls vascular patterning and is negatively regulated by microtubules. Here we show that a positive regulator of TGFβ signaling is essential for developmental vascular patterning and microtubule stability. Rudhira/BCAS3 is known to bind microtubules and to play a nodal role in cytoskeletal remodeling and directional endothelial cell (EC) migration in vitro. We demonstrate that the molecular and cellular function of Rudhira is deployed at critical steps in vascular patterning. We generated the first floxed mice for rudhira and find that global or endothelial knockout of rudhira results in mid-gestation lethality due to aberrant embryonic and extra-embryonic vessel patterning and defective cardiac morphogenesis. Rudhira null yolk sac ECs show random and retarded migration. Yolk sac transcriptome analysis revealed key mediators of angiogenic processes and TGFβ receptor signaling were perturbed in rudhira null mutants. Molecular and biochemical analyses showed that rudhira depletion reduced microtubule stability but increased expression of pathway inhibitors leading to high levels of SMAD2/3 ubiquitination and reduced activation. These effects were not rescued by exogenous TGFβ. However, TGFβ treatment of wild type ECs increased Rudhira expression. Further, exogenous Rudhira, which promotes directional cell migration, caused increased SMAD2/3 nuclear translocation and reduced inhibitor levels. Therefore, we propose that Rudhira and TGFβ signaling are mutually dependent. Rudhira has a dual function in promoting TGFβ signaling, possibly by sequestering microtubules and simultaneously preventing SMAD2/3 ubiquitination to permit EC migration and vascular patterning. TGFβ signaling and aberrant human Rudhira (Breast Cancer Amplified Sequence 3, BCAS3) expression are both associated with tumour metastasis. Our study identifies a cytoskeletal, cell type-specific modulator of TGFβ signaling important in development and cancer. ER -