SUMMARY Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca2+-activated Cl− channels (CaCCs) is linked to Scott syndrome with deficient Ca2+-dependent lipid scrambling. We generated TMEM16F knockout mice that exhibit bleeding defects and protection in an arterial thrombosis model associated with platelet deficiency in Ca2+-dependent phosphatidylserine exposure and procoagulant activity and lack a Ca2+-activated cation current in the platelet precursor megakaryocytes. Heterologous expression of TMEM16F generates a small-conductance Ca2+-activated nonselective cation (SCAN) current with subpicosiemens single-channel conductance rather than a CaCC. TMEM16F-SCAN channels permeate both monovalent and divalent cations, including Ca2+, and exhibit synergistic gating by Ca2+ and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a Ca2+-activated channel permeable to Ca2+ and critical for Ca2+-dependent scramblase activity during blood coagulation.

Microvesicles (MVs) are emerging as a new mechanism of intercellular communication by transferring cellular lipid and protein components to target cells, yet their function in disease is only now being explored. We found that neutrophil-derived MVs were increased in concentration in synovial fluid from rheumatoid arthritis patients compared to paired plasma. Synovial MVs overexpressed the proresolving, anti-inflammatory protein annexin A1 (AnxA1). Mice deficient in TMEM16F, a lipid scramblase required for microvesiculation, exhibited exacerbated cartilage damage when subjected to inflammatory arthritis. To determine the function of MVs in inflammatory arthritis, toward the possibility of MV-based therapeutics, we examined the role of immune cell–derived MVs in rodent models and in human primary chondrocytes. In vitro, exogenous neutrophil-derived AnxA1+ MVs activated anabolic gene expression in chondrocytes, leading to extracellular matrix accumulation and cartilage protection through the reduction in stress-adaptive homeostatic mediators interleukin-8 and prostaglandin E2. In vivo, intra-articular injection of AnxA1+ MV lessened cartilage degradation caused by inflammatory arthritis. Arthritic mice receiving adoptive transfer of whole neutrophils displayed abundant MVs within cartilage matrix and revealed that MVs, but not neutrophils themselves, can penetrate cartilage. Mechanistic studies support a model whereby MV-associated AnxA1 interacts with its receptor FPR2 (formyl peptide receptor 2)/ALX, increasing transforming growth factor–b production by chondrocytes, ultimately leading to cartilage protection. We envisage that MVs, either directly or loaded with therapeutics, can be harnessed as a unique therapeutic strategy for protection in diseases associated with cartilage degeneration.

Counting of transcripts at each DNA template suggested a stochastic initiation mechanism in the experimental system. We found a prototypical activator (human Sp1) regulates transcription by enhancing PIC assembly (presumably by recruiting TFIID). Real-time TFIID binding to DNA was monitored and coupled to transcription detection at the same DNA template for the first time. We also developed methods to detect the production of RNA transcripts in real-time and couple that to the kinetic measurements of RNA polymerase binding at the single-molecule level. using multiple fluorescently labeled General Transcription Factors (GTFs, namely TFIIB TFIID, TFIIE, TFIIF and TFIIH) and Pol II, we are currently investigating the structure of PIC, pathways of its assembly, and the mechanism of transcription modulation by sequence-specific activators and the core promoter DNA elements.