Modulation of Cellular NAD Attenuates Cancer-Associated Hypercoagulability and Thrombosis via the Inhibition of Tissue Factor and Formation of Neutrophil Extracellular Traps.

Cancer-associated thrombosis is the second-leading cause of mortality in patients with cancer and presents a poor prognosis, with a lack of effective treatment strategies. NAD(P)H quinone oxidoreductase 1 (NQO1) increases the cellular nicotinamide adenine dinucleotide (NAD) levels by accelerating the oxidation of NADH to NAD, thus playing important roles in cellular homeostasis, energy metabolism, and inflammatory responses. Using a murine orthotopic 4T1 breast cancer model, in which multiple thrombi are generated in the lungs at the late stage of cancer development, we investigated the effects of regulating the cellular NAD levels on cancer-associated thrombosis.

Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins.

Intracellular antibodies have become powerful tools for imaging, modulating and neutralizing endogenous target proteins. Here, we describe an optogenetically activated intracellular antibody (optobody) consisting of split antibody fragments and blue-light inducible heterodimerization domains. We expanded this optobody platform by generating several optobodies from previously developed intracellular antibodies, and demonstrated that photoactivation of gelsolin and β2-adrenergic receptor (β2AR) optobodies suppressed endogenous gelsolin activity and β2AR signaling, respectively.

Noninvasive optical activation of Flp recombinase for genetic manipulation in deep mouse brain regions.

Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode.

BRAF somatic mutation contributes to intrinsic epileptogenicity in pediatric brain tumors.

Pediatric brain tumors are highly associated with epileptic seizures. However, their epileptogenic mechanisms remain unclear. Here, we show that the oncogenic BRAF somatic mutation p.

Cell-matrix adhesion and cell-cell adhesion differentially control basal myosin oscillation and Drosophila egg chamber elongation.

Pulsatile actomyosin contractility, important in tissue morphogenesis, has been studied mainly in apical but less in basal domains. Basal myosin oscillation underlying egg chamber elongation is regulated by both cell-matrix and cell-cell adhesions. However, the mechanism by which these two adhesions govern basal myosin oscillation and tissue elongation is unknown.

PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front.

Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts.

Optogenetic oligomerization of Rab GTPases regulates intracellular membrane trafficking.

Intracellular membrane trafficking, which is involved in diverse cellular processes, is dynamic and difficult to study in a spatiotemporal manner. Here we report an optogenetic strategy, termed light-activated reversible inhibition by assembled trap of intracellular membranes (IM-LARIAT), that uses various Rab GTPases combined with blue-light-induced hetero-interaction between cryptochrome 2 and CIB1. In this system, illumination induces a rapid and reversible intracellular membrane aggregation that disrupts the dynamics and functions of the targeted membrane.

Controlling low rates of cell differentiation through noise and ultrahigh feedback.

Mammalian tissue size is maintained by slow replacement of de-differentiating and dying cells. For adipocytes, key regulators of glucose and lipid metabolism, the renewal rate is only 10% per year. We used computational modeling, quantitative mass spectrometry, and single-cell microscopy to show that cell-to-cell variability, or noise, in protein abundance acts within a network of more than six positive feedbacks to permit pre-adipocytes to differentiate at very low rates.

Consecutive positive feedback loops create a bistable switch that controls preadipocyte-to-adipocyte conversion.

Adipogenesis, or the conversion of proliferating preadipocytes into nondividing adipocytes, is an important part of the vertebrate weight-maintenance program. It is not yet understood how and when an irreversible transition occurs into a distinct state capable of accumulating lipid. Here, we use single-cell fluorescence imaging to show that an all-or-none switch is induced before lipid accumulation occurs.

Parallel adaptive feedback enhances reliability of the Ca2+ signaling system.

Despite large cell-to-cell variations in the concentrations of individual signaling proteins, cells transmit signals correctly. This phenomenon raises the question of what signaling systems do to prevent a predicted high failure rate. Here we combine quantitative modeling, RNA interference, and targeted selective reaction monitoring (SRM) mass spectrometry, and we show for the ubiquitous and fundamental calcium signaling system that cells monitor cytosolic and endoplasmic reticulum (ER) Ca(2+) levels and adjust in parallel the concentrations of the store-operated Ca(2+) influx mediator stromal interaction molecule (STIM), the plasma membrane Ca(2+) pump plasma membrane Ca-ATPase (PMCA), and the ER Ca(2+) pump sarco/ER Ca(2+)-ATPase (SERCA).

Identification of a calmodulin-binding NAC protein as a transcriptional repressor in Arabidopsis.

Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of proteins. However, little is known about how CaM directly regulates transcription. Screening of an Arabidopsis cDNA expression library using horseradish peroxidase-conjugated calmodulin as a probe identified a calmodulin-binding NAC protein (CBNAC).

PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane.

Many signaling, cytoskeletal, and transport proteins have to be localized to the plasma membrane (PM) in order to carry out their function. We surveyed PM-targeting mechanisms by imaging the subcellular localization of 125 fluorescent protein-conjugated Ras, Rab, Arf, and Rho proteins. Out of 48 proteins that were PM-localized, 37 contained clusters of positively charged amino acids.

Rice C2-domain proteins are induced and translocated to the plasma membrane in response to a fungal elicitor.

Hundreds of proteins involved in signaling pathways contain a Ca(2+)-dependent membrane-binding motif called the C2-domain. However, no small C2-domain proteins consisting of a single C2-domain have been reported in animal cells. We have isolated two cDNA clones, OsERG1a and OsERG1b, that encode two small C2-domain proteins of 156 and 159 amino acids, respectively, from a fungal elicitor-treated rice cDNA library.

BWMK1, a rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor.

Mitogen-activated protein kinase (MAPK) cascades are known to transduce plant defense signals, but the downstream components of the MAPK have as yet not been elucidated. Here, we report an MAPK from rice (Oryza sativa), BWMK1, and a transcription factor, OsEREBP1, phosphorylated by the kinase. The MAPK carries a TDY phosphorylation motif instead of the more common TEY motif in its kinase domain and has an unusually extended C-terminal domain that is essential to its kinase activity and translocation to the nucleus.