Assembly and Function of the Regulator of G protein Signaling 14 (RGS14)·H-Ras Signaling Complex in Live Cells Are Regulated by Gαi1 and Gαi-linked G Protein-coupled Receptors

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gα_i/o-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive Gα_i1/3-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive Gα_i1-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive Gα_i1-GDP but not active Gα_i1-GTP. Active H-Ras(G/V) interacts with a native RGS14·Gα_i1 complex in brain lysates, and co-expression of RGS14 and Gα_i1 in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the Gα_i-linked α_2A-adrenergic receptor induces a conformational change in the Gα_i1·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive Gα_i1-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.     

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gα_i/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gα_i1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gα_i1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gα_o-AlF₄⁻. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gα_i1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gα_o-AlF₄⁻ and an AlF₄⁻-insensitive mutant (G42R) of Gα_i1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gα_i1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gα_o-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.     

RGS14 is a multifunctional scaffold that integrates G protein and Ras/Raf MAPkinase signalling pathways

MAPkinase signalling is essential for cell growth, differentiation and cell physiology. G proteins and tyrosine kinase receptors each modulate MAPkinase signalling through distinct pathways. We report here that RGS14 is an integrator of G protein and MAPKinase signalling pathways. RGS14 contains a GPR/GoLoco (GL) domain that forms a stable complex with inactive Giα1/3–GDP, and a tandem (R1, R2) Ras binding domain (RBD). We find that RGS14 binds and regulates the subcellular localization and activities of H-Ras and Raf kinases in cells. Activated H-Ras binds RGS14 at the R1 RBD to form a stable complex at cell membranes. RGS14 also co-localizes with and forms a complex with Raf kinases in cells. The regulatory region of Raf-1 binds the RBD region of RGS14, and H-Ras and Raf each facilitate one another's binding to RGS14. RGS14 selectively inhibits PDGF-, but not EGF- or serum-stimulated Erk phosphorylation. This inhibition is dependent on H-Ras binding to RGS14 and is reversed by co-expression of Giα1, which binds and recruits RGS14 to the plasma membrane. Giα1 binding to RGS14 inhibits Raf binding, indicating that Giα1 and Raf binding to RGS14 are mutually exclusive. Taken together, these findings indicate that RGS14 is a newly appreciated integrator of G protein and Ras/Raf signalling pathways.     

Calcium signalling in pollen of Papaver rhoeas undergoing the self-incompatibility (SI) response

Self-incompatibility (SI) is a genetically controlled system used by many flowering plants to prevent self-pollination. We established, using calcium imaging, that the SI response in Papaver rhoeas L. (poppy) pollen involves a Ca²⁺-mediated intracellular signalling pathway. Here we review what is known about the signalling components and cascades implicated in the SI response in poppy pollen. We present some studies using calcium green (CG-1) that show SI-induced alterations in CG-1 fluorescence and localization. We have begun to examine potential sources of Ca²⁺ involved in the responses induced by SI. This work presents preliminary data showing that influx of extracellular Ca²⁺ at the ”shank” of the pollen tube is possible. This is the first evidence suggesting that influx at this localization may play a role in the SI response. We also describe preliminary studies that begin to investigate whether the phosphoinositide signalling pathway is implicated in the SI response. Received: 12 December 2000 / Revision Accepted: 22 June 2001     

Rearrangement of Actin Microfilaments in Plant Root Hairs Responding to Rhizobium etli Nodulation Signals

The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.     

Calmodulin is uniformly distributed during cell division in living stamen hair cells ofTradescantia virginiana

Summary Because the activity of calmodulin (CaM) may be dependent upon its structural distribution, we have examined its spatial localization in living cells. We have focused on cell division and cell plate formation, where conventional immunofluorescence studies report that CaM is specifically associated with microtubules (MTs) of the spindle and the phragmoplast. In dividing stamen hair cells ofTradescantia virginiana that were injected with fluorescently labeled CaM and examined by confocal laser scanning microscopy (CLSM), we found that the labeled protein is uniformly distributed throughout the cell and is not localized with the phragmoplast MTs or any other obvious structure. To explore why these images from live cells differ from those prepared by immunolabeling, we investigated the fate of CaM during fixation and compared it with the localization of fixable dextran and tubulin. The results show that fixation causes severe changes in cell morphology and in the distribution of CaM and dextran in three quarters of the cells. Conversely, injected rhodamine-tubulin did not show redistribution after fixation. We conclude that in the live cell, CaM is largely uniformly distributed throughout the cytoplasm, and secondly that conventional chemical fixation does not preserve CaM, and probably many other soluble proteins, in its in vivo distribution. The role postulated for CaM in mitosis, solely based on indirect immunofluorescence microscopy, has to be re-evaluated.Abbreviations BSA bovine serum albumin - CaM calmodulin - CLSM confocal laser scanning microscopy - Cy3 indocarbocyanine - EDTA ethylenediamine-tetraacetic acid - EGTA ethylene glycol bis (-aminoethyl ether)-N,N,NN-tetraacetic acid - FITC fluoresceinisothiocyanate - IAF 5-iodoacetamido-fluorescein - MT microtubule - PBS phosphate-buffered saline - TBS Tris-buffered saline     

Confocal fluorescence microscopy of plant cells

Summary The confocal laser scanning microscope (CLSM) has become a vital instrument for the examination of subcellular structure, especially in fluorescently stained cells. Because of its ability to markedly reduce out-of-focus flare, when compared to the conventional wide-field fluorescence microscope, the CLSM provides a substantial improvement in resolution along the z axis and permits optical sectioning of cells. These developments have been particularly helpful for the investigation of plant cells and tissues, which because of their shape, size, and optical properties have been difficult to analyze at high resolution by conventional means. We review the contribution that the CLSM has made to the study of plant cells. We first consider the principle of operation of the CLSM, including a discussion of image processing, and of lasers and appropriate fluorescent dyes. We then summarize several studies of both fixed and live plant cells in which the instrument has provided new or much clearer information about cellular substructure than has been possible heretofore. Attention is given to the visualization of different components, including especially the cytoskeleton, endomembranes, nuclear components, and relevant ions, and their changes in relationship to physiological and developmental processes. We conclude with an effort to anticipate advances in technology that will improve and extend the performance of the CLSM. In addition to the usual bibliography, we provide internet addresses for information about the CLSM.     

THE FEEDING MECHANISM OF AVIAN MALARIAL PARASITES

Electron microscope studies of the erythrocytic forms, including gametocytes and asexual schizonts, of the protozoa Plasmodium fallax, P. lophurae, and P. cathemerium, have revealed a "cytostome," a specialized organelle of the pellicular membrane which is active in the ingestion of host cell cytoplasm. In material fixed in glutaraldehyde and postfixed in OsO₄, the cytostome appears in face view as a pore limited by two dense circular membranes and having an inside diameter of approximately 190 mµ. In cross-section, the cytostome is a cavity bounded on each side by two dense segments corresponding to the two dense circles observed in face view; its base consists of a single unit membrane. In the process of feeding, the cytostome cavity enlarges by expansion of its membrane, permitting a large quantity of red cell cytoplasm to come into contact with the cytostome wall. Subsequent digestion of erythrocyte cytoplasm occurs exclusively in food vacuoles which emanate from the cytostome invagination. As digestion progresses, the food vacuoles initially stain more densely and there is a marked build-up of hemozoin granules. In the final stage of digestion, a single membrane surrounds a cluster of residual pigment particles and very little of the original host cell cytoplasm remains. The cytostome in exoerythrocytic stages of P. fallax has been observed only in merozoites and does not seem to play the same role in the feeding mechanism.     

MICROTUBULES AND EARLY STAGES OF CELL-PLATE FORMATION IN THE ENDOSPERM OF HAEMANTHUS KATHERINAE BAKER

A fine structure study of the phragmoplast and developing cell plate has been made on glutaraldehyde-osmium tetroxide-fixed, dividing, cultured cells of the liquid endosperm of Haemanthus katherinae Baker. The phragmoplast arises between the telophase nuclei, usually in association with a remnant strand of spindle elements, and consists of an accumulation of microtubules oriented at right angles to the plane of the future cell plate. The microtubules, which are 200–240 A in diameter, occur in small clusters spaced at approximately 0.2–0.3 µ intervals along the plate. Short interconnections interpreted as "cross-bridges" have been observed between individual microtubules. Within each cluster there is an electron-opaque zone about 0.3 µ in width which can be attributed in part to an overlap of microtubules from both sides of the plate and in part to a local accumulation of an amorphous electron-opaque material. During development these dense zones become aligned in a plane which itself defines the plane of the plate. Vesicles, commonly observed in long files, are derived from a cytoplasmic matrix rich in elements of the endoplasmic reticulum and sparse in dictyosomes. They aggregate between the clusters of microtubules and eventually coalesce to form the cell plate.     

An atypical crista resembling a "tight junction" in bean root mitochondria

The conformation and structure of an atypical crista found in a small percentage of the mitochondria in root tip cells of Phaseolus vulgaris L. have been studied electron microscopically in material fixed in glutaraldehyde followed by osmium tetroxide. In its transformation into an atypical crista, a normal crista elongates, broadens, and flattens, and the inner leaflets of its apposed unit membranes appear to fuse in a manner analogous to the formation of "tight junctions" between certain animal cells. The result is a large platelike, quintuple-layered structure, 240–260 A thick, whose long axis parallels that of the mitochondrion. The outer layers of the "plate," bordering on the mitochondrial matrix, are thickened and exhibit striking patterns in the micrographs. The structure of the plate is compared with that previously described for tight junctions between animal cells.