Persistent Metabolic Changes Are Induced by 24 h Low-Dose Lead (Pb) Exposure in Zebrafish Embryos.

Lead (Pb) is a heavy metal associated with a range of toxic effects. Relatively few studies attempt to understand the impact of lead on development from a mechanistic perspective. (zebrafish) embryos are a model organism for studying the developmental consequences of exposure to chemical agents.

Hypoosmotic stress shifts transcription of circadian genes.

Cells respond to hypoosmotic stress by initial swelling followed by intracellular increases in the number of osmolytes and initiation of gene transcription that allow cells to adapt to the stress. Here, we have studied the genes that change expression under mild hypoosmotic stress for 12 and 24 h in rat cultured smooth muscle cells (WKO-3M22). We find shifts in the transcription of many genes, several of which are associated with circadian rhythm, such as per1, nr1d1, per2, dbp, and Ciart.

The scaffolding protein IQGAP1 enhances EGFR signaling by promoting oligomerization and preventing degradation.

IQGAP1 is a large, multi-domain scaffold that connects and modulates different signaling networks including the one initiated by epidermal growth factor (EGF). In this study, we have used live cell fluorescence imaging methods along with other biochemical techniques to follow the mechanisms used by IQGAP1 to enhance EGF signaling. We show that IQGAP1 enhances EGF signaling by promoting the oligomerization of its receptor, EGFR, upon EGF addition along with concurrent IQGAP oligomerization.

Human ESCRT-I and ALIX function as scaffolding helical filaments .

Unlabelled: The Endosomal Sorting Complex Required for Transport (ESCRT) is an evolutionarily conserved machinery that performs reverse-topology membrane scission in cells universally required from cytokinesis to budding of enveloped viruses. Upstream acting ESCRT-I and ALIX control these events and link recruitment of viral and cellular partners to late-acting ESCRT-III CHMP4 through incompletely understood mechanisms. Using structure-function analyses combined with super-resolution imaging, we show that ESCRT-I and ALIX function as distinct helical filaments .

PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells.

The Gα/phospholipase C-β (PLCβ) signaling system mediates calcium responses to a variety of hormones and neurotransmitters. Recent studies suggest that PLCβ1 expression plays a role in the differentiation of two types of cultured neuronal cells (PC12 and SK-N-SH) through a mechanism independent of Gα. Here, we show that, similar to that observed in PC12 and SK-N-SH cells, PLCβ1 expression increases when human NT2 cells are induced to differentiate either through cytosine-β-D-arabinofuranoside or retinoic acid.

The role of calcium in neuronal membrane tension and synaptic plasticity.

Calcium is a primary second messenger that plays a role in cellular functions including growth, movement and responses to drugs. The role that calcium plays in mediating communication between neurons by synaptic vesicle release is well established. This review focuses on the dependence of the physical properties of neuronal plasma membranes on calcium levels.

Unraveling Hidden Cell Signaling Pathways Using Biophysical Methods: Application to the Gαq/Phospholipase Cβ Signaling System.

The success of pharmaceutical therapies relies on how well cells respond to a particular drug, but accurately predicting responses can be difficult due to the complex and numerous potential molecular interactions that are possible in cells, and the responses of individuals can be variable due to cryptic and unexpected interactions. With the advancement of proteomics and fluorescence imaging methods, it is now possible to elucidate novel secondary signaling pathways and predict unexpected responses that might otherwise be missed, allowing for the development of better therapeutics. The Gαq/PLCβ signaling pathway is activated by agents that mediate allergic responses, neurotransmission, and heart rate, as well as other functions that are critical for survival.

Multiple functions of phospholipase Cβ1 at a glance.

Phospholipase Cβ (PLCβ) is the main effector of the Gq family of heterotrimeric G proteins that transduces signals from hormones and neurotransmitters into Ca2+ signals. While PLCβ is critical for Ca2+ responses, recent studies have suggested that PLCβ has additional roles independent of its lipase activity. These novel functions are carried out by a cytosolic population of PLCβ that binds and inhibits the component 3 promoter of RNA-induced silencing complex (C3PO) to impact cytosolic RNA populations.

Phomoxanthone A Targets ATP Synthase.

Phomoxanthone A is a naturally occurring molecule and a powerful anti-cancer agent, although its mechanism of action is unknown. To facilitate the determination of its biological target(s), we used affinity-based labelling using a phomoxanthone A probe. Labelled proteins were pulled down, subjected to chemoproteomics analysis using LC-MS/MS and ATP synthase was identified as a likely target.

Live Cell Fluorescence Imaging Shows Neurotransmitter Activation Promotes Aggregation of the Intracellular Domain of Amyloid Precursor Protein.

Amyloid precursor protein (APP) is a major contributor to the pathology of Alzheimer's and other neurodegenerative diseases through the accumulation of extracellular plaques. Here, we have studied changes in APP translation and aggregation of the APP intracellular domain when the Gαq/PLCβ signaling system is activated by neurotransmitters. Using RT-PCR and a molecular beacon that follows APP mRNA in live cells, we find that Gαq activation sequesters APP mRNA similar to the stress granule response found in heat shock and hypo-osmotic shock thereby shutting down the production of APP.

Activation of Gαq sequesters specific transcripts into Ago2 particles.

The Gαq/phospholipase Cβ1 (PLCβ1) signaling system mediates calcium responses from hormones and neurotransmitters. While PLCβ1 functions on the plasma membrane, there is an atypical cytosolic population that binds Argonaute 2 (Ago2) and other proteins associated with stress granules preventing their aggregation. Activation of Gαq relocalizes cytosolic PLCβ1 to the membrane, releasing bound proteins, promoting the formation of stress granules.

Deformation of caveolae impacts global transcription and translation processes through relocalization of cavin-1.

Caveolae are invaginated membrane domains that provide mechanical strength to cells in addition to being focal points for the localization of signaling molecules. Caveolae are formed through the aggregation of caveolin-1 or -3 (Cav1/3), membrane proteins that assemble into multifunctional complexes with the help of caveola-associated protein cavin-1. In addition to its role in the formation of caveolae, cavin-1, also called polymerase I and transcript release factor, is further known to promote ribosomal RNA transcription in the nucleus.

IQGAP1 scaffolding links phosphoinositide kinases to cytoskeletal reorganization.

IQGAP1 is a multidomain scaffold protein that coordinates the direction and impact of multiple signaling pathways by scaffolding its various binding partners. However, the spatial and temporal resolution of IQGAP1 scaffolding remains unclear. Here, we use fluorescence imaging and correlation methods that allow for real-time live-cell changes in IQGAP1 localization and complex formation during signaling.

Stimulation of phospholipase Cβ1 by Gα promotes the assembly of stress granule proteins.

During adverse conditions, mammalian cells suppress protein production by sequestering the translational machinery in membrane-less organelles known as stress granules. Here, we found that activation of the G protein subunit Gα promoted the formation of particles that contained stress granule proteins through a mechanism linked to a cytosolic fraction of phospholipase Cβ1 (PLCβ1). In experiments with PC12 and A10 cells, we showed that under basal conditions, cytosolic PLCβ1 bound to stress granule–associated proteins, including PABPC1, eIF5A, and Ago2.

Stimulation of the Gαq/phospholipase Cβ1 signaling pathway returns differentiated cells to a stem-like state.

Phospholipase Cβ1 is activated by Gαq to generate calcium signals in response to hormones and neurotransmitters. Besides carrying out this plasma membrane function, PLCβ1 has a cytosolic population that helps to drive the differentiation of PC12 cells by inhibiting a nuclease that promotes RNA-induced silencing (C3PO). Here, we show that down-regulating PLCβ1 or reducing its cytosolic population by activating Gαq to localize it to the plasma membrane returns differentiated PC12 and SK-N-SH cells to an undifferentiated state.

The Gαq/phospholipase Cβ signaling system represses tau aggregation.

Alzheimer's disease is typified by calcium dysfunction and neurofibrillary tangles of tau aggregates along with mitotic proteins. Using PC12 cells as a model system, we determined whether the Gαq/PLCβ/ calcium signaling pathway impacts the manifestation of Alzheimer's disease. Down-regulating PLCβ significantly increases tau protein expression and causes a large increase in tau aggregation.

Re-track: Software to analyze the retraction and protrusion velocities of neurites, filopodia and other structures.

We have developed new software, Re-track, that will quantify the rates of retraction and protrusion of structures emanating from the central core of a cell, such as neurites or filopodia. Re-Track, uses time-lapse images of cells in TIFF format and calculates the velocity of retraction or protrusion of a selected structure. The software uses a flexible moving boundary and has the ability to correct this boundary throughout analysis.

Regulation of bifunctional proteins in cells: Lessons from the phospholipase Cβ/G protein pathway.

Some proteins can serve multiple functions depending on different cellular conditions. An example of a bifunctional protein is inositide-specific mammalian phospholipase Cβ (PLCβ). PLCβ is activated by G proteins in response to hormones and neurotransmitters to increase intracellular calcium.

Gαq-mediated calcium dynamics and membrane tension modulate neurite plasticity.

The formation and disruption of synaptic connections during development are a fundamental step in neural circuit formation. Subneuronal structures such as neurites are known to be sensitive to the level of spontaneous neuronal activity, but the specifics of how neurotransmitter-induced calcium activity regulates neurite homeostasis are not yet fully understood. In response to stimulation by neurotransmitters such as acetylcholine, calcium responses in cells are mediated by the Gαq/phospholipase Cβ (PLCβ)/phosphatidylinositol 4,5-bisphosphate (PI(4,5)P) signaling pathway.

The role of phospholipase Cβ on the plasma membrane and in the cytosol: How modular domains enable novel functions.

Phospholipase Cβ (PLCβ) is a signaling enzyme activated by G proteins to generate calcium signals. The catalytic core of PLCβ is surrounded by modular domains that mediate the interaction of the enzyme with known protein partners on the plasma membrane. The C-terminal region PLCβ contains a novel coiled-coil domain that is required for Gαq binding and activation.

Mechanical Stretch Redefines Membrane Gαq-Calcium Signaling Complexes.

Muscle cells are routinely subjected to mechanical stretch but the impact of stretch on the organization of membrane domains is unknown. In this study, we characterize the effect of stretch on GPCR-Gαq protein signaling. Activation of this pathway leads to an increase in intracellular calcium.

Nitrosative stress uncovers potent β-adrenergic receptor-linked vasodilation further enhanced by blockade of clathrin endosome formation.

This study investigated the effect of sodium nitroprusside (SNP) preexposure on vasodilation via the β-adrenergic receptor (BAR) system. SNP was used as a nitrosative/oxidative proinflammatory insult. Small arterioles were visualized by intravital microscopy in the hamster cheek pouch tissue (isoflurane, n = 45).

Phospholipase Cβ1 regulates proliferation of neuronal cells.

Cells have developed lineage-specific mechanisms to control proliferation and drive morphologic changes upon differentiation. A hallmark of differentiation is the assembly of signaling molecules that transduce extracellular signals, such as the production of the G protein-regulated enzyme phospholipase Cβ (PLCβ), which generates calcium signals from sensory stimuli. We found that in most cancerous cell lines there is positive correlation between PLCβ1 levels and cell proliferation.