Defective angiogenesis underlies over 50 malignant, ischemic and inflammatory disorders yet long-term therapeutic applications inevitably fail, thus highlighting the need for greater understanding of the vast crosstalk and compensatory mechanisms. Based on proteomic profiling of angiogenic endothelial components, here we report βIV-spectrin, a non-erythrocytic cytoskeletal protein, as a critical regulator of sprouting angiogenesis. Early loss of endothelial-specific βIV-spectrin promotes embryonic lethality in mice due to hypervascularization and hemorrhagic defects whereas neonatal depletion yields higher vascular density and tip cell populations in developing retina. During sprouting, βIV-spectrin expresses in stalk cells to inhibit their tip cell potential by enhancing VEGFR2 turnover in a manner independent of most cell-fate determining mechanisms. Rather, βIV-spectrin recruits CaMKII to the plasma membrane to directly phosphorylate VEGFR2 at Ser984, a previously undefined phosphoregulatory site that strongly induces VEGFR2 internalization and degradation. These findings support a distinct spectrin-based mechanism of tip-stalk cell specification during vascular development.

Drp1 is a key regulator of mitochondrial fission, a large cytoplasmic GTPase recruited to the mitochondrial surface via transmembrane adaptors to initiate scission. While Brownian motion likely accounts for the local interactions between Drp1 and the mitochondrial adaptors, how this essential enzyme is targeted from more distal regions like the cell periphery remains unknown. Based on proteomic interactome screening and cell-based studies, we report that GIPC mediates the actin-based retrograde transport of Drp1 towards the perinuclear mitochondria to enhance fission. Drp1 interacts with GIPC through its atypical C-terminal PDZ-binding motif. Loss of this interaction abrogates Drp1 retrograde transport resulting in cytoplasmic mislocalization and reduced fission despite retaining normal intrinsic GTPase activity. Functionally, we demonstrate that GIPC potentiates the Drp1-driven proliferative and migratory capacity in cancer cells. Together, these findings establish a direct molecular link between altered GIPC expression and Drp1 function in cancer progression and metabolic disorders.

βIV-spectrin is a membrane cytoskeletal protein with specialized roles in the nervous system and heart. Recent evidence also indicates a fundamental role for βIV-spectrin in angiogenesis as its endothelial-specific gene deletion in mice enhances embryonic lethality due to hypervascularization and hemorrhagic defects. During early vascular sprouting, βIV-spectrin is believed to inhibit tip cell sprouting in favor of the stalk cell phenotype by mediating VEGFR2 internalization and degradation. Despite these essential roles, mechanisms governing βIV-spectrin expression remain unknown. Here we identify bone morphogenetic protein 9 (BMP9) as a major inducer of βIV-spectrin gene expression in the vascular system. We show that BMP9 signals through the ALK1/Smad1 pathway to induce βIV-spectrin expression, which then recruits CaMKII to the cell membrane to induce phosphorylation-dependent VEGFR2 turnover. Although BMP9 signaling promotes stalk cell behavior through activation of hallmark stalk cell genes ID-1/3 and Hes-1 and Notch signaling crosstalk, we find that βIV-spectrin acts upstream of these pathways as loss of βIV-spectrin in neonate mice leads to retinal hypervascularization due to excessive VEGFR2 levels, increased tip cell populations, and strong Notch inhibition irrespective of BMP9 treatment. These findings demonstrate βIV-spectrin as a BMP9 gene target critical for tip/stalk cell selection during nascent vessel sprouting.

Dynamic changes in the endoplasmic reticulum (ER) morphology are central to maintaining cellular homeostasis. Microtubules (MT) facilitate the continuous remodeling of the ER network into sheets and tubules by coordinating with many ER-shaping protein complexes, although how this process is controlled by extracellular signals remains unknown. Here we report that TAK1, a kinase responsive to numerous growth factors and cytokines including TGF-β and TNF-α, triggers ER tubulation by activating αTAT1, an MT-acetylating enzyme that enhances ER-sliding. We show that this TAK1/αTAT-dependent ER remodeling promotes cell survival by actively downregulating BOK, an ER membrane-associated proapoptotic effector. While BOK is normally protected from degradation when complexed with IP3R, it is rapidly degraded upon their dissociation during the ER sheets-to-tubules conversion. These findings demonstrate a distinct mechanism of ligand-induced ER remodeling and suggest that the TAK1/αTAT pathway may be a key target in ER stress and dysfunction.

Mitochondrial remodeling is a fundamental process underlying cellular respiration and metabolism. Here we report TAK1 as a direct regulator of mitochondrial fusion. TAK1 is activated by a variety of mitogenic factors, cytokines and environmental stimuli, which we find induces rapid fragmentation through Mfn2 inactivation. TAK1 phosphorylates Mfn2 at Ser249, which inhibits the binding of GTP required for Mfn trans-dimerization and mitochondrial membrane fusion. Accordingly, expression of Mfn2-S249 phosphomimetics (Mfn2-E/D) constitutively promote fission whereas alanine mutant (Mfn2-A) yields hyperfused mitochondria and increased bioenergetics in cells. In mice, Mfn2-E knock-in yields embryonic lethality in homozygotes whereas heterozygotes are viable but exhibit increased visceral fat accumulation despite normal body weight and cognitive/motor functions compared to wildtype and Mfn2-A mice. Mature white adipocytes isolated from mutant mice reveal cell-autonomous TAK1-related effects on mitochondrial remodeling and lipid metabolism. These results identify Mfn2-S249 as a dynamic phosphoregulatory switch of mitochondrial fusion during development and energy homeostasis.