Phase separation of an actin nucleator by junctional microtubules regulates epithelial function

Liquid-liquid phase separation (LLPS) is involved in various dynamic biological phenomena. In epithelial cells, dynamic regulation of junctional actin filaments tethered to the apical junctional complex (AJC) is critical for maintaining internal homeostasis against external perturbations; however, the role of LLPS in this process remains unknown. Here, after identifying a multifunctional actin nucleator, cordon bleu (Cobl), as an AJC-enriched microtubule-associated protein, we conducted comprehensive in vitro and in vivo analyses. We found that apical microtubules promoted LLPS of Cobl at the AJC, and Cobl actin assembly activity increased upon LLPS. Thus, microtubules spatiotemporally regulated junctional actin assembly for epithelial morphogenesis and paracellular barriers. Collectively, these findings established that LLPS of the actin nucleator Cobl mediated dynamic microtubule-actin cross-talk in junctions, which fine-tuned the epithelial barrier

Membrane-deformation ability of ANKHD1 is involved in the early endosome enlargement

Ankyrin-repeat domains (ARDs) are conserved in large numbers of proteins. ARDs are composed of various numbers of ankyrin repeats (ANKs). ARDs often adopt curved structures reminiscent of the Bin-Amphiphysin-Rvs (BAR) domain, which is the dimeric scaffold for membrane tubulation. BAR domains sometimes have amphipathic helices for membrane tubulation and vesiculation. However, it is unclear whether ARD-containing proteins exhibit similar membrane deformation properties. We found that the ARD of ankyrin repeat and KH domain-containing protein 1 (ANKHD1) dimerizes and deforms membranes into tubules and vesicles. Among 25 ANKs of ANKHD1, the first 15 ANKs can form a dimer, and the latter 10 ANKs enable membrane tubulation and vesiculation through an adjacent amphipathic helix and a predicted curved structure with a positively charged surface, analogous to BAR domains. Knockdown and localization of ANKHD1 suggested its involvement in the negative regulation of early endosome enlargement owing to its membrane vesiculation

TRPV4 channel activity is modulated by direct interaction of the ankyrin domain to PI(4,5)P2

[プレスリリース]バイオサイエンス研究科分子医学細胞生物学研究室の末次志郎教授らの研究グループが生理的に重要なイオンを運ぶ通り道TRPV4の新たな制御機構を解明（2014/09/29）Mutations in the ankyrin repeat domain (ARD) of ​TRPV4 are responsible for several channelopathies, including Charcot–Marie–Tooth disease type 2C and congenital distal and scapuloperoneal spinal muscular atrophy. However, the molecular pathogenesis mediated by these mutations remains elusive, mainly due to limited understanding of the ​TRPV4 ARD function. Here we show that phosphoinositide binding to the ​TRPV4 ARD leads to suppression of the channel activity. Among the phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) most potently binds to the ​TRPV4 ARD. The crystal structure of the ​TRPV4 ARD in complex with ​inositol-1,4,5-trisphosphate, the head-group of PI(4,5)P2, and the molecular-dynamics simulations revealed the PI(4,5)P2-binding amino-acid residues. The ​TRPV4 channel activities were increased by titration or hydrolysis of membrane PI(4,5)P2. Notably, disease-associated ​TRPV4 mutations that cause a gain-of-function phenotype abolished PI(4,5)P2 binding and PI(4,5)P2 sensitivity. These findings identify ​TRPV4 ARD as a lipid-binding domain in which interactions with PI(4,5)P2 normalize the channel activity in ​TRPV4

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by in vivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior