Decoding information in cell shape.

Shape is an indicator of cell health. But how is the information in shape decoded? We hypothesize that decoding occurs by modulation of signaling through changes in plasma membrane curvature. Using analytical approaches and numerical simulations, we studied how elongation of cell shape affects plasma membrane signaling. Mathematical analyses reveal transient accumulation of activated receptors at regions of higher curvature with increasing cell eccentricity. This distribution of activated receptors is periodic, following the Mathieu function, and it arises from local imbalance between reaction and diffusion of soluble ligands and receptors in the plane of the membrane. Numerical simulations show that transient microdomains of activated receptors amplify signals to downstream protein kinases. For growth factor receptor pathways, increasing cell eccentricity elevates the levels of activated cytoplasmic Src and nuclear MAPK1,2. These predictions were experimentally validated by changing cellular eccentricity, showing that shape is a locus of retrievable information storage in cells.

Multiple functions of phospholipase Cß1 at a glance.

Phospholipase Cß (PLCß) is the main effector of the Gq family of heterotrimeric G proteins that transduces signals from hormones and neurotransmitters into Ca2+ signals. While PLCß is critical for Ca2+ responses, recent studies have suggested that PLCß has additional roles independent of its lipase activity. These novel functions are carried out by a cytosolic population of PLCß that binds and inhibits the component 3 promoter of RNA-induced silencing complex (C3PO) to impact cytosolic RNA populations. Additionally, cytosolic PLCß binds to stress granule proteins, keeping them dispersed and thus inhibiting stress granule formation. Upon activation of the Gα subunit of Gq (Gαq), cytosolic PLCß relocalizes to the membrane, releasing C3PO and stress granule proteins, which in turn promotes activation of C3PO and RNA processing, as well as sequestration of specific transcripts into newly formed stress granules. As highlighted in this Cell Science at a Glance and the accompanying poster, the link between Gαq signaling, increased intracellular Ca2+ and changes in RNA processing impacts neuronal cell differentiation and may also affect neuronal development and dysfunction.

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. After describing the key features of synaptic plasticity, we summarize the general role of calcium in cell function and the signaling pathways responsible for intracellular increase in calcium levels. We then present findings showing that increases in intracellular calcium levels cause neurites to contract and break synaptic connections by changes in membrane tension.

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. However, the mechanistic link between these functions is not clear. Here, we found that deforming caveolae by subjecting cells to mild osmotic stress (150-300 mOsm) changes levels of GAPDH, Hsp90, and Ras only when Cav1/cavin-1 levels are reduced, suggesting a link between caveola deformation and global protein expression. We show that this link may be due to relocalization of cavin-1 to the nucleus upon caveola deformation. Cavin-1 relocalization is also seen when Cav1-Gαq contacts change upon stimulation. Furthermore, Cav1 and cavin-1 levels have been shown to have profound effects on cytosolic RNA levels, which in turn impact the ability of cells to form stress granules and RNA-processing bodies (p-bodies) which sequester and degrade mRNAs, respectively. Our studies here using a cavin-1-knockout cell line indicate adaptive changes in cytosolic RNA levels but a reduced ability to form stress granules. Taken together, our findings suggest that caveolae, through release of cavin-1, communicate extracellular cues to the cell interior to impact transcriptional and translational.

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. We find that IQGAP1 and PIPKIγ interact on both the plasma membrane and in cytosol. Epidermal growth factor (EGF) stimulation, which can initiate cytoskeletal changes, drives the movement of the cytosolic pool toward the plasma membrane to promote cytoskeletal changes. We also observe that a significant population of cytosolic IQGAP1-PIPKIγ complexes localize to early endosomes, and in some instances form aggregated clusters which become highly mobile upon EGF stimulation. Our imaging studies show that PIPKIγ and PI3K bind simultaneously to IQGAP1, which may accelerate conversion of PI4P to PI(3,4,5)P3 that is required for cytoskeletal changes. Additionally, we find that IQGAP1 is responsible for PIPKIγ association with two proteins associated with cytoskeletal changes, talin and Cdc42, during EGF stimulation. These results directly show that IQGAP1 provides a physical link between phosphoinositides (through PIPKIγ), focal adhesion formation (through talin), and cytoskeletal reorganization (through Cdc42) upon EGF stimulation. Taken together, our results support the importance of IQGAP1 in regulating cell migration by linking phosphoinositide lipid signaling with cytoskeletal reorganization.

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/PLCß 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. Following the intracellular domain of eGFP-APP, we find that Gαq stimulation increases aggregation as followed by number and brightness (N&B) analysis of single molecule fluorescence time series. Additionally, we show that APP aggregation is affected by changes in the levels of PLCß1 and its cytosolic binding partners. Our studies show the neurotransmitter activation of Gαq/PLCß reduces translation of APP and increases aggregation of its intracellular domain. These studies better establish a link between APP production and complexation and Gαq stimulation.

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. Mitochondrial ATP synthase was validated in cultured cells lysates and in live intact cells. Our studies show sixty percent inhibition of ATP synthase by 260 µM phomoxanthone A.

Stimulation of the Gαq/phospholipase Cß1 signaling pathway returns differentiated cells to a stem-like state.

Phospholipase Cß1 is activated by Gαq to generate calcium signals in response to hormones and neurotransmitters. Besides carrying out this plasma membrane function, PLCß1 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 PLCß1 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. In this state, PC12 cells have a spherical morphology, resume proliferation, and express the stem cell transcription factors nanog and Oct4. Similar changes are seen when C3PO is down-regulated. This return to a stem-like state is accompanied by shifts in multiple miR populations. Surprisingly, de-differentiation can be induced by extended stimulation of Gαq where cells return to a spherical morphology and levels of specific miRs return to their undifferentiated values. In complementary studies, we followed the real-time hydrolysis of a fluorescent-tagged miR in cells where PLCß1 or C3PO were down-regulated in PC12 cells and find substantial differences in miR processing in the undifferentiated and differentiated states. Taken together, our studies suggest that PLCß1, through its ability to regulate C3PO and endogenous miR populations, mediates the differentiation of two types of cultured neuronal cells.

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. Re-Track is fast, platform independent, and user friendly, and it can be used to follow biological events such as changes in neuronal connections, tip-growing cells such as moss, adaptive migration of cells, and similar behavior in non-biological systems.

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. In muscle cells, GPCR-Gαq signals are enhanced when these proteins are localized in caveolae membrane domains whose curved structure can flatten with stretch. When we statically stretch rat aortic smooth muscle A10 cells by 1-5%, cellular calcium appears unperturbed as indicated by a calcium indicator. However, when we activate the bradykinin type 2 receptor (B2R)/Gαq pathway, we observe a loss in calcium that appears to be mediated through perturbations in calcium-activated stretch receptors. In contrast, if we apply oscillating stretch, calcium levels are enhanced. We tested whether the observed changes in B2R-Gαq calcium signals were caused by stretch-induced disruption of caveolae using a combination of silencing RNA technology and growth conditions. We find that stretch changes the ability of monoclonal caveolin antibodies to bind caveolae indicating a change in configuration of the domains. This change is seen by the inability of cells to survive stretch cycles when the level of caveolae is significantly reduced. Our studies show that the effect of calcium signals by mechanical stretch is mediated by the type of stretch and the amount of caveolae.

Phospholipase Cß1 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 Cß (PLCß), which generates calcium signals from sensory stimuli. We found that in most cancerous cell lines there is positive correlation between PLCß1 levels and cell proliferation. In cells of neuronal lineage, however, reducing PLCß1 levels increases the rate of proliferation. Using a combination of biochemical and biophysical methods, we find that, in the G1 phase, a cytosolic population of PLCß1 associates with cyclin-dependent kinase 16 (CDK16), a neuron-specific enzyme that is activated by cyclin Y to inactivate the antioncogenic protein p27Kip1. Binding of PLCß1 directly inhibits CDK16 activity and in turn reduces the ability of cells to enter the S phase. Activation of Gαq by carbachol causes movement of PLCß from the cytosol to the plasma membrane, reducing its association with CDK16. Similarly, the overexpression of activated Gαq moves PLCß1 to the membrane, reverses G1 arrest, and promotes proliferation, thereby connecting external stimuli with cell proliferation. Our results present a model in which the transient high expression of PLCß1 that occurs at the onset of differentiation arrests cells in the G1 phase through its association with CDK16 and allows CDK16 to transition to its postmitotic function of neurite outgrowth and trafficking of synaptic vesicles. The novel role of PLCß1 in neuronal cell proliferation offers a unique interaction that can be manipulated to guide cells into a neuronal phenotype or to develop therapies for neuroblastomas.-Garwain, O., Valla, K., Scarlata, S. Phospholipase Cß1 regulates proliferation of neuronal cells.

Super-resolution Visualization of Caveola Deformation in Response to Osmotic Stress.

Caveolae are protein-dense plasma membrane domains structurally composed of caveolin-1 or -3 along with other proteins. Our previous studies have shown that caveolae enhance calcium signals generated through the Gαq/phospholipase Cß signaling pathway and that subjecting cells to hypo-osmotic stress reverses this enhancement. In this study, we have used super-resolution fluorescence microscopy supplemented by fluorescence correlation studies to determine the structural factors that underlie this behavior. We find similar and significant population of Gαq and one of its receptors, bradykinin type 2 receptor (B2R), as well as a significant population of Gαi and its coupled ß2-adrenergic receptor (ßAR), are localized to caveola domains. Although mild osmotic stress deforms caveolae and alters interactions between the caveolae and these proteins, the general structure and the localization of caveola components remain largely unchanged. This deformation eliminates the ability of caveolae to stabilize calcium signals mediated through Gαq-B2R, but does not affect cAMP signals mediated through Gαi and ßAR. Structurally, we find that mild osmotic stress corresponding roughly to a pressure of 3.82 newtons/m2 increases the domain diameter by ∼30% and increases the fluorescence intensity in the center of the domain mouth suggesting a flattening of the invagination. Approximate calculations show that caveolae in muscle tissue have the strength to handle the stress of muscle movement.

Nitrosative stress uncovers potent ß2-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 ß-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). Control dilation to isoproterenol (EC50: 10-7 mol/l) became biphasic as a function of concentration after 2 min of exposure to SNP (10-4 M), with increased potency at picomolar dilation uncovered and decreased efficacy at the micromolar dilation. Control dilation to curcumin was likewise altered after SNP, but only the increased potency at a low dose was uncovered, whereas micromolar dilation was eliminated. The picomolar dilations were blocked by the potent BAR-2 inverse agonist carazolol (10-9 mol/l). Dynamin inhibition with dynasore mimicked this effect, suggesting that SNP preexposure prevented BAR agonist internalization. Using HeLa cells transfected with BAR-2 tagged with monomeric red fluorescent protein, exposure to 10-8-10-6 mol/l curcumin resulted in internalization and colocalization of BAR-2 and curcumin (FRET) that was prevented by oxidative stress (10-3 mol/l CoCl2), supporting that stress prevented internalization of the BAR agonist with the micromolar agonist. This study presents novel data supporting that distinct pools of BARs are differentially available after inflammatory insult. NEW & NOTEWORTHY Preexposure to an oxidative/nitrosative proinflammatory insult provides a "protective preconditioning" against future oxidative damage. We examined immediate vasoactive and molecular consequences of a brief preexposure via ß-adrenergic receptor signaling in small arterioles. Blocked receptor internalization with elevated reactive oxygen levels coincides with a significant and unexpected vasodilation to ß-adrenergic agonists at picomolar doses.

Phospholipase Cß-TRAX Association Is Required for PC12 Cell Differentiation.

When treated with nerve growth factor, PC12 cells will differentiate over the course of several days. Here, we have followed changes during differentiation in the cellular levels of phosphoinositide-specific phospholipase Cß (PLCß) and its activator, Gαq, which together mediate Ca2+ release. We also followed changes in the level of the novel PLCß binding partner TRAX (translin-associated factor X), which promotes RNA-induced gene silencing. We find that the level of PLCß increases 4-fold within 24 h, whereas Gαq increases only 1.4-fold, and this increase occurs ∼24 h later than PLCß. Alternately, the level of TRAX remains constant over the 72 h tested. When PLCß1 or TRAX is down-regulated, differentiation does not occur. The impact of PLCß on differentiation appears independent of Gαq as down-regulating Gαq at constant PLCß does not affect differentiation. Förster resonance energy transfer studies after PLCß association with its partners indicate that PLCß induced soon after nerve growth factor treatment associates with TRAX rather than Gαq Functional measurements of Ca2+ signals to assess the activity of PLCß-Gαq complexes and measurements of the reversal of siRNA(GAPDH) to assess the activity of PLCß-TRAX complexes additionally suggest that the newly synthesized PLCß associates with TRAX to impact RNA-induced silencing. Taken together, our studies show that PLCß, through its ability to bind TRAX and reverse RNA silencing of specific genes, plays a key role in switching PC12 cells to their differentiated state.

RNA-induced silencing attenuates G protein-mediated calcium signals.

Phospholipase Cß (PLCß) is activated by G protein subunits in response to environmental stimuli to increase intracellular calcium. In cells, a significant portion of PLCß is cytosolic, where it binds a protein complex required for efficient RNA-induced silencing called C3PO (component 3 promoter of RISC). Binding between C3PO and PLCß raises the possibility that RNA silencing activity can affect the ability of PLCß to mediate calcium signals. By use of human and rat neuronal cell lines (SK-N-SH and PC12), we show that overexpression of one of the main components of C3PO diminishes Ca(2+) release in response to Gαq/PLCß stimulation by 30 to 40%. In untransfected SK-N-SH or PC12 cells, the introduction of siRNA(GAPDH) [small interfering RNA(glyceraldehyde 3-phosphate dehydrogenase)] reduces PLCß-mediated calcium signals by ∼30%, but addition of siRNA(Hsp90) (heat shock protein 90) had little effect. Fluorescence imaging studies suggest an increase in PLCß-C3PO association in cells treated with siRNA(GAPDH) but not siRNA(Hsp90). Taken together, our studies raise the possibility that Ca(2+) responses to extracellular stimuli can be modulated by components of the RNA silencing machinery.-Philip, F., Sahu, S., Golebiewska, U., Scarlata, S. RNA-induced silencing attenuates G protein-mediated calcium signals.

Osmotic Stress Reduces Ca2+ Signals through Deformation of Caveolae.

Caveolae are membrane invaginations that can sequester various signaling proteins. Caveolae have been shown to provide mechanical strength to cells by flattening to accommodate increased volume when cells are subjected to hypo-osmotic stress. We have previously found that caveolin, the main structural component of caveolae, specifically binds Gαq and stabilizes its activation state resulting in an enhanced Ca(2+) signal upon activation. Here, we show that osmotic stress caused by decreasing the osmolarity in half reversibly changes the configuration of caveolae without releasing a significant portion of caveolin molecules. This change in configuration due to flattening leads to a loss in Cav1-Gαq association. This loss in Gαq/Cav1 association due to osmotic stress results in a significant reduction of Gαq/phospholipase Cß-mediated Ca(2+) signals. This reduced Ca(2+) response is also seen when caveolae are reduced by treatment with siRNA(Cav1) or by dissolving them by methyl-ß-cyclodextran. No change in Ca(2+) release with osmotic swelling can be seen when growth factor pathways are activated. Taken together, these results connect the mechanical deformation of caveolae to Gαq-mediated Ca(2+) signals.

Hydrolysis rates of different small interfering RNAs (siRNAs) by the RNA silencing promoter complex, C3PO, determines their regulation by phospholipase Cß.

C3PO plays a key role in promoting RNA-induced gene silencing. C3PO consists of two subunits of the endonuclease translin-associated factor X (TRAX) and six subunits of the nucleotide-binding protein translin. We have found that TRAX binds strongly to phospholipase Cß (PLCß), which transmits G protein signals from many hormones and sensory inputs. The association between PLCß and TRAX is thought to underlie the ability of PLCß to reverse gene silencing by small interfering RNAs. However, this reversal only occurs for some genes (e.g. GAPDH and LDH) but not others (e.g. Hsp90 and cyclophilin A). To understand this specificity, we carried out studies using fluorescence-based methods. In cells, we find that PLCß, TRAX, and their complexes are identically distributed through the cytosol suggesting that selectivity is not due to large scale sequestration of either the free or complexed proteins. Using purified proteins, we find that PLCß binds ∼5-fold more weakly to translin than to TRAX but ∼2-fold more strongly to C3PO. PLCß does not alter TRAX-translin assembly to C3PO, and brightness studies suggest one PLCß binds to one C3PO octamer without a change in the number of TRAX/translin molecules suggesting that PLCß binds to an external site. Functionally, we find that C3PO hydrolyzes siRNA(GAPDH) at a faster rate than siRNA(Hsp90). However, when PLCß is bound to C3PO, the hydrolysis rate of siRNA(GAPDH) becomes comparable with siRNA(Hsp90). Our results show that the selectivity of PLCß toward certain genes lies in the rate at which the RNA is hydrolyzed by C3PO.

Defining the oligomerization state of γ-synuclein in solution and in cells.

γ-Synuclein is expressed at high levels in neuronal cells and in multiple invasive cancers. Like its family member α-synuclein, γ-synuclein is thought to be natively unfolded but does not readily form fibrils. The function of γ-synuclein is unknown, but we have found that it interacts strongly with the enzyme phospholipase Cß (PLCß), altering its interaction with G proteins. As a first step in determining its role, we have characterized its oligomerization using fluorescence homotransfer, photon-counting histogram analysis, and native gel electrophoresis. We found that when its expressed in Escherichia coli and purified, γ-synuclein appears monomeric on chromatographs under denaturing conditions, but under native conditions, it appears as oligomers of varying sizes. We followed the monomer-to-tetramer association by labeling the protein with fluorescein and following the concentration-dependent loss in fluorescence anisotropy resulting from fluorescence homotransfer. We also performed photon-counting histogram analysis at increasing concentrations of fluorescein-labeled γ-synuclein and found concentration-dependent oligomerization. Addition of PLCß2, a strong γ-synuclein binding partner whose cellular expression is correlated with γ-synuclein, results in disruption of γ-synuclein oligomers. Similarly, its binding to lipid membranes promotes the monomer form. When we exogenously express γ-synuclein or microinject purified protein into cells, the protein appears monomeric. Our studies show that even though purified γ-synuclein form oligomers, when binding partners are present, as in cells, it dissociates to a monomer to bind these partners, which in turn may modify protein function and integrity.

Linking alpha-synuclein properties with oxidation: a hypothesis on a mechanism underling cellular aggregation.

α-Synuclein is a small, natively unstructured protein with propensity to aggregate. α-Synuclein fibrils are major components of Lewy bodies that are hallmarks of many neurodegenerative diseases. The solution properties and aggregation behavior of α-synuclein has been well characterized, but despite numerous studies that address the role of α-synuclein in cells, a clear physiological function of this protein remains a mystery. Over a hundred review articles of α-synuclein have been written in the last decade, making it difficult to list all of the important studies that have added to our insight of α-synuclein physiology. Instead, we briefly review the status of α-synuclein research and propose a model based on the idea that α-synuclein may not have an intrinsic activity in cells but rather, it modifies the function of a group of protein partners that in turn affect cell processes. We propose that it is the loss of its cellular partners under oxidative conditions that promotes α-synuclein aggregation accelerating neuronal death.

Unraveling Hidden Cell Signaling Pathways Using Biophysical Methods: Application to the Gαq/Phospholipase Cß 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/PLCß signaling pathway is activated by agents that mediate allergic responses, neurotransmission, and heart rate, as well as other functions that are critical for survival. This Review describes the factors that must be considered in delineating signaling pathways and describes the novel translational role that we have uncovered for this signaling pathway.