Integrative proteomic and cytological analysis of the effects of extracellular Ca(2+) influx on Pinus bungeana pollen tube development

Ca (2+) is an essential ion in the control of pollen germination and tube growth. However, the control of pollen tube development by Ca (2+) signaling and its interactions with cytoskeletal components, energy-providing pathways, and cell-expansion machinery remain elusive. Here, we used nifedipine (Nif) to study Ca (2+) functions in differential protein expression and other cellular processes in Pinus bungeana pollen tube growth. Proteomics analysis indicated that 50 proteins showed differential expression with varying doses of Nif. Thirty-four of these were homologous to previously reported proteins and were classified into different functional categories closely related to tip-growth machinery. Blocking the L-type Ca (2+) channel with Nif in the pollen tube membrane induced several early alterations within a short time, including a reduction of extracellular Ca (2+) influx and a subsequently dramatic decrease in cytosolic free Ca (2+) concentration ([Ca (2+)] c), concomitant with ultrastructural abnormalities and changes in the abundance of proteins involved in energy production and signaling. Secondary alterations included actin filament depolymerization, disrupted patterns of endocytosis/exocytosis, and cell wall remodeling, along with changes in the proteins involved in these processes. These results suggested that extracellular Ca (2+) influx was necessary for the maintenance of the typical tip-focused [Ca (2+)] c gradient in the P. bungeana pollen tube, and that reduced adenosine triphosphate production (ATP), depolymerization of the cytoskeleton, and abnormal endocytosis/exocytosis, together with enhanced rigidity of cell walls, were responsible for the growth arrest observed in pollen tubes treated with Nif.     

Alterations in the actin cytoskeleton of pollen tubes are induced by the self-incompatibility reaction in Papaver rhoeas

Self-incompatibility (SI) is a genetically controlled process used to prevent self-pollination. In Papaver rhoeas, the induction of SI is triggered by a Ca(2)+-dependent signaling pathway that results in the rapid and S allele-specific inhibition of pollen tube tip growth. Tip growth of cells is dependent on a functioning actin cytoskeleton. We have investigated the effect of self-incompatibility (S) proteins on the actin cytoskeleton in poppy pollen tubes. Here, we report that the actin cytoskeleton of incompatible pollen tubes is rapidly and dramatically rearranged during the SI response, not only in our in vitro SI system but also in vivo. We demonstrate that nonspecific inhibition of growth does not result in similar actin rearrangements. Because the SI-induced alterations are not observed if growth stops, this clearly demonstrates that these alterations are triggered by the SI signaling cascade rather than merely resulting from the consequent inhibition of growth. We establish a detailed time course of events and discuss the mechanisms that might be involved. Our data strongly implicate a role for the actin cytoskeleton as a target for signaling pathways involved in the SI response of P. rhoeas.     

The Actin Cytoskeleton and Signaling Network during Pollen Tube Tip Growth

The organization and dynamics of the actin cytoskeleton play key roles in many aspects of plant cell development. The actin cytoskeleton responds to internal developmental cues and en-vironmental signals and is involved in cell division, subcellular organelle movement, cell polarity and polar cell growth. The tip-growing pollen tubes provide an ideal model system to investigate fundamental mechanisms of underlying polarized cell growth. In this system, most signaling cascades required for tip growth, such as Ca~(2+)-, small GTPases- and lipid-mediated signaling have been found to be involved in transmitting signals to a large group of actin-binding proteins. These actin-binding proteins subsequently regulate the structure of the actin network, as well as the rapid turnover of actin filaments (F-actin), thereby eventually controlling tip growth. The actin cytoskeleton acts as an integrator in which multiple signaling pathways converge, providing a general growth and regulatory mechanism that applies not only for tip growth but also for polarized diffuse growth in plants.     

Differential display proteomic analysis of Picea meyeri pollen germination and pollen-tube growth after inhibition of actin polymerization by latrunculin B

To investigate roles of the actin cytoskeleton in growth of the pollen tube of Picea meyeri, we used the actin polymerization inhibitor latrunculin B (LATB) under quantitatively controlled conditions. At low concentrations, LATB inhibited polymerization of the actin cytoskeleton in the growing pollen tube, which rapidly inhibited tip growth. The proteomic approach was used to analyse protein expression-profile changes during pollen germination and subsequent pollen-tube development with disturbed organization of the actin cytoskeleton. Two-dimensional electrophoresis and staining with Coomassie Brilliant Blue revealed nearly 600 protein spots. A total of 84 of these were differentially displayed at different hours with varying doses of LATB, and 53 upregulated or downregulated proteins were identified by mass spectrometry. These proteins were grouped into distinct functional categories including signalling, actin cytoskeleton organization, cell expansion and carbohydrate metabolism. Moreover, actin disruption affected the morphology of Golgi stacks, mitochondria and amyloplasts, along with a differential expression of proteins involved in their functions. These findings provide new insights into the multifaceted mechanism of actin cytoskeleton functions and its interaction with signalling, cell-expansion machinery and energy-providing pathways.     

Evidence for copurification of micronuclei in sucrose density gradient-enriched plasma membranes from cell lines

Sucrose density gradient-enriched membrane preparations and membrane fraction enrichment through affinity purification techniques are commonly used in proteomic analysis. However, published proteomic profiles characterized by the above methods show the presence of nuclear proteins in addition to membrane proteins. While shuttling of nuclear proteins across cellular compartments and their transient residency at membrane interfaces could explain some of these observations, the presence of nuclear proteins in proteomic profiles generated with crude and enriched membranes could be the result of nonspecific contamination of nuclear debris during cell fractionation procedures. We hypothesized that micronuclei arising from the genomic instability inherent to cancer cells may copurify with plasma membrane fractions on sucrose gradients. Using sucrose gradient-enriched plasma membranes from breast cancer cell lines derived from the MCF-7 cell line, we provide experimental evidence to indicate that micronuclei are present in fresh preparations of plasma membranes. The origin of these micronuclei was traced to budding of nuclei in intact cells. Furthermore, mass spectrometric analysis confirmed the presence of nuclear proteins as well as membrane and associated signaling proteins in sucrose gradient-enriched preparations.     

Mitochondrial matrix phosphoproteome: effect of extra mitochondrial calcium

Post-translational modification of mitochondrial proteins by phosphorylation or dephosphorylation plays an essential role in numerous cell signaling pathways involved in regulating energy metabolism and in mitochondrion-induced apoptosis. Here we present a phosphoproteomic screen of the mitochondrial matrix proteins and begin to establish the protein phosphorylations acutely associated with calcium ions (Ca(2+)) signaling in porcine heart mitochondria. Forty-five phosphorylated proteins were detected by gel electrophoresis-mass spectrometry of Pro-Q Diamond staining, while many more Pro-Q Diamond-stained proteins evaded mass spectrometry detection. Time-dependent (32)P incorporation in intact mitochondria confirmed the extensive matrix protein phosphoryation and revealed the dynamic nature of this process. Classes of proteins that were detected included all of the mitochondrial respiratory chain complexes, as well as enzymes involved in intermediary metabolism, such as pyruvate dehydrogenase (PDH), citrate synthase, and acyl-CoA dehydrogenases. These data demonstrate that the phosphoproteome of the mitochondrial matrix is extensive and dynamic. Ca(2+) has previously been shown to activate various dehydrogenases, promote the generation of reactive oxygen species (ROS), and initiate apoptosis via cytochrome c release. To evaluate the Ca(2+) signaling network, the effects of a Ca(2+) challenge sufficient to release cytochrome c were evaluated on the mitochondrial phosphoproteome. Novel Ca(2+)-induced dephosphorylation was observed in manganese superoxide dismutase (MnSOD) as well as the previously characterized PDH. A Ca(2+) dose-dependent dephosphorylation of MnSOD was associated with an approximately 2-fold maximum increase in activity; neither the dephosphorylation nor activity changes were induced by ROS production in the absence of Ca(2+). These data demonstrate the use of a phosphoproteome screen in determining mitochondrial signaling pathways and reveal new pathways for Ca(2+) modification of mitochondrial function at the level of MnSOD.     

Quantitative shotgun proteomic analysis of cold-stressed mature rice anthers

Rice crops are vulnerable to low temperatures. During development, the reproductive stage is particularly sensitive to cold exposure, which causes abnormal pollen development and a high degree of male sterility. In this study, shotgun proteomic analysis was used to analyze rice anthers containing pollen grains from a cold-tolerant variety, Dianxi 4. Protein expression was compared between normal anthers and anthers exposed to cold temperatures at the young microspore stage. In total, 3835 non-redundant proteins were identified in the rice anther. Of these, 441 proteins were differentially expressed between normal and cold-treated anthers. Pollen allergens, ATP synthase, actin, profilin, and β-expansin proteins were highly abundant, reflecting anther development, pollen germination, and pollen tube elongation. Starch and sucrose metabolic proteins such as α-amylase precursor and 4-α-glucanotransferase exhibited reduced expression after cold exposure. Among the proteins that exhibited increased expression after cold exposure, C2 domain proteins, and GRPs were identified as candidate signaling factors for mediation of the cold tolerance response. Through high-throughput proteomic analysis we were able to reveal proteomic changes against cold stress and suggest two signaling factors as the candidate genes.     

Signal-mediated depolymerization of actin in pollen during the self-incompatibility response

Signal perception and the integration of signals into networks that effect cellular changes is essential for all cells. The self-incompatibility (SI) response in field poppy pollen triggers a Ca(2+)-dependent signaling cascade that results in the inhibition of incompatible pollen. SI also stimulates dramatic alterations in the actin cytoskeleton. By measuring the amount of filamentous (F-) actin in pollen before and during the SI response, we demonstrate that SI stimulates a rapid and large reduction in F-actin level that is sustained for at least 1 h. This represents quantitative evidence for stimulus-mediated depolymerization of F-actin in plant cells by a defined biological stimulus. Surprisingly, there are remarkably few examples of sustained reductions in F-actin levels stimulated by a biologically relevant ligand. Actin depolymerization also was achieved in pollen by treatments that increase cytosolic free Ca(2+) artificially, providing evidence that actin is a target for the Ca(2+) signals triggered by the SI response. By determining the cellular concentrations and binding constants for native profilin from poppy pollen, we show that profilin has Ca(2+)-dependent monomeric actin-sequestering activity. Although profilin is likely to contribute to stimulus-mediated actin depolymerization, our data suggest a role for additional actin binding proteins. We propose that Ca(2+)-mediated depolymerization of F-actin may be a mechanism whereby SI-induced tip growth inhibition is achieved.     

Proteomic analysis of tomato (Lycopersicon esculentum) pollen

In flowering plants, pollen grains are produced in the anther and released to the external environment with the primary function of delivering sperm cells to the female gametophyte. This study was conducted to identify proteins in tomato pollen and to analyse their roles in relation to pollen function. Tomato is an important crop which is grown worldwide and is an excellent experimental system. Proteins were extracted from pollen, separated by two-dimensional gel electrophoresis (2-DE), and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting. Of the 960 spots observed on Colloidal Coomassie Blue (CCB)-stained 2-DE gels, 190 were selected for analysis. Of these, 158 spots, representing 133 distinct proteins, were identified by searching the NCBInr and Expressed Sequence Tag databases. The identified proteins were classified based on designated functions and the majority included those involved in defence mechanisms, energy conversions, protein synthesis and processing, cytoskeleton formation, Ca(2+) signalling, and as allergens. A number of proteins in tomato pollen were similar to those reported in the pollen of other species; however, several additional proteins with roles in defence mechanisms, metabolic processes, and hormone signalling were identified. The potential roles of the identified proteins in the survival strategy of the small, independent, two-celled pollen grain of tomato, and subsequently in pollen germination and tube growth are discussed.     

Calcium-dependent protein kinase isoforms in Petunia have distinct functions in pollen tube growth, including regulating polarity

Calcium is a key regulator of pollen tube growth, but little is known concerning the downstream components of the signaling pathways involved. We identified two pollen-expressed calmodulin-like domain protein kinases from Petunia inflata, CALMODULIN-LIKE DOMAIN PROTEIN KINASE1 (Pi CDPK1) and Pi CDPK2. Transient overexpression or expression of catalytically modified Pi CDPK1 disrupted pollen tube growth polarity, whereas expression of Pi CDPK2 constructs inhibited tube growth but not polarity. Pi CDPK1 exhibited plasma membrane localization most likely mediated by acylation, and we present evidence that suggests this localization is critical to the biological function of this kinase. Pi CDPK2 substantially localized to as yet unidentified internal membrane compartments, and this localization was again, at least partially, mediated by acylation. In contrast with Pi CDPK1, altering the localization of Pi CDPK2 did not noticeably alter the effect of overexpressing this isoform on pollen tube growth. Ca(2+) requirements for Pi CDPK1 activation correlated closely with Ca(2+) concentrations measured in the growth zone at the pollen tube apex. Interestingly, loss of polarity associated with overexpression of Pi CDPK1 was associated with elevated cytosolic Ca(2+) throughout the bulging tube tip, suggesting that Pi CDPK1 may participate in maintaining Ca(2+) homeostasis. These results are discussed in relation to previous models for Ca(2+) regulation of pollen tube growth.     

Amino acid-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor are mediated by a phospholipase C/inositol 1,4,5-trisphosphate-independent pathway that requires G12, Rho, filamin-A, and the actin cytoskeleton

The G protein-coupled Ca(2+)-sensing receptor (CaR) is an allosteric protein that responds to two different agonists, Ca(2+) and aromatic amino acids, with the production of sinusoidal or transient oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). Here, we examined whether these differing patterns of [Ca(2+)](i) oscillations produced by the CaR are mediated by separate signal transduction pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of inositol 1,4,5-trisphosphate in single cells, we found that stimulation of CaR by an increase in the extracellular Ca(2+) concentration ([Ca(2+)](o)) leads to periodic synthesis of inositol 1,4,5-trisphosphate, whereas l-phenylalanine stimulation of the CaR does not induce any detectable change in the level this second messenger. Furthermore, we identified a novel pathway that mediates transient [Ca(2+)](i) oscillations produced by the CaR in response to l-phenylalanine, which requires the organization of the actin cytoskeleton and involves the small GTPase Rho, heterotrimeric proteins of the G(12) subfamily, the C-terminal region of the CaR, and the scaffolding protein filamin-A. Our model envisages that Ca(2+) or amino acids stabilize unique CaR conformations that favor coupling to different G proteins and subsequent activation of distinct downstream signaling pathways.     

Signals and targets of the self-incompatibility response in pollen of Papaver rhoeas

Self-incompatibility (SI) in Papaver rhoeas involves an allele-specific recognition between stigmatic S-proteins and pollen, resulting in inhibition of incompatible pollen. A picture of some of the signalling events and mechanisms involved in this specific inhibition of pollen tube growth is beginning to be built up. This highly specific response triggers a Ca(2+)-dependent signalling cascade in incompatible pollen when a stigmatic S-protein interacts with it. Rapid increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) can now be attributed (at least in part) to Ca(2+) influx. The rapid loss of the pollen apical Ca(2+) gradient within approximately 1-2 min is accompanied by the inhibition of pollen tube tip growth. Concomitant with this time-frame, hyper-phosphorylation of p26, a soluble pollen phosphoprotein is detected. Characterization of p26 reveals that it is a soluble inorganic pyrophosphatase, which suggests a possible direct functional role in pollen tube growth. Slightly later, a putative MAP kinase (p52) is thought to be activated. Finally, preliminary evidence that programmed cell death (PCD) may be triggered in this response is described. A key target for these signals, the actin cytoskeleton, has also been identified. In this article the current understanding of some of the components of this signalling cascade and how they are beginning to throw some light on possible mechanisms involved in this SI-induced inhibition of pollen tube growth, is discussed.     

Proteomic analysis of vascular endothelial cells in response to laminar shear stress

Isotope-coded affinity tags (cICAT) coupled with mass spectrometric analysis is one of the leading technologies for quantitative proteomic profiling and protein quantification. We performed proteomic analysis of bovine aortic endothelial cells (BAEC) in response to laminar shear stress using cICAT labeling coupled with LC-MS/MS. Protein expressions in BAEC under 15 dynes/cm2 of shear stress for 10 min, 3 h, and 6 h were compared with matched stationary controls. Analysis of each sample produced 1800-2400 proteins at >or=75% confidence level. We found 142, 213, and 186 candidate proteins that were up- or down-regulated by at least two-fold after 10 min, 3 h, and 6 h of shear stress, respectively. Some of these proteins have known cellular functions and they encompass many signaling pathways. The signaling pathways that respond to shear stress include those of integrins, G-protein-coupled receptors, glutamate receptors, PI3K/AKT, apoptosis, Notch and cAMP-mediated signaling pathways. The validity of the mass spectrometric analysis was also confirmed by Western blot and confocal immunofluorescence microscopy. The present quantitative proteomic analysis suggests novel potential regulatory mechanisms in vascular endothelial cells in response to shear stress. These results provide preliminary footprints for further studies on the signaling mechanisms induced by shear stress.     

Calcium signaling dysfunction in heart disease

In the heart, Ca(2+) is crucial for the regulation of contraction and intracellular signaling, processes, which are vital to the functioning of the healthy heart. Ca(2+) -activated signaling pathways must function against a background of large, rapid, and tightly regulated changes in intracellular free Ca(2+) concentrations during each contraction and relaxation cycle. This review highlights a number of proteins that regulate signaling Ca(2+) in both normal and pathological conditions including cardiac hypertrophy and heart failure, and discusses how these pathways are not regulated by the marked elevation in free intracellular calcium ([Ca(2+) ](i)) during contraction but require smaller sustained increases in Ca(2+) concentration. In addition, we present published evidence that the pool of Ca(2+) that regulates signaling is compartmentalized into distinct cellular microdomains and is thus distinct from that regulating contraction.     

The actin cytoskeleton is a target of the self-incompatibility response in Papaver rhoeas

The integration of signals received by a cell, and their transduction to targets, is essential for all cellular responses. The cytoskeleton has been identified as a major target of signalling cascades in both animal and plant cells. Self-incompatibility (SI) in Papaver rhoeas involves an allele-specific recognition between stigmatic S-proteins and pollen, resulting in the inhibition of incompatible pollen. This highly specific response triggers a Ca(2+)-dependent signalling cascade in incompatible pollen when a stigmatic S-protein interacts with it. It has been demonstrated recently that SI induces dramatic alterations in the organization of the pollen actin cytoskeleton. This implicates the actin cytoskeleton as a key target for the SI-stimulated signals. The cytological alterations to the actin cytoskeleton that are triggered in response to SI are described here and there seem to be several stages that are distinguishable temporally. Evidence was obtained that F-actin depolymerization is also stimulated. The current understanding that the actin cytoskeleton is a target for the signals triggered by the SI response is discussed. It is suggested that these F-actin alterations may be Ca(2+)-mediated and that this could be a mechanism whereby SI-induced tip growth inhibition is achieved. The potential for actin-binding proteins to act as key mediators of this response is discussed and the mechanisms that may be responsible for effecting these changes are described. In particular, the parallels between sustained actin rearrangements during SI and in apoptosis of animal cells are considered.     

Integrin-mediated mechanotransduction in renal vascular smooth muscle cells: activation of calcium sparks

Integrins are transmembrane heterodimeric proteins that link extracellular matrix (ECM) to cytoskeleton and have been shown to function as mechanotransducers in nonmuscle cells. Synthetic integrin-binding peptide triggers Ca(2+) mobilization and contraction in vascular smooth muscle cells (VSMCs) of rat afferent arteriole, indicating that interactions between the ECM and integrins modulate vascular tone. To examine whether integrins transduce extracellular mechanical stress into intracellular Ca(2+) signaling events in VSMCs, unidirectional mechanical force was applied to freshly isolated renal VSMCs through paramagnetic beads coated with fibronectin (natural ligand of alpha(5)beta(1)-integrin in VSMCs). Pulling of fibronectin-coated beads with an electromagnet triggered Ca(2+) sparks, followed by global Ca(2+) mobilization. Paramagnetic beads coated with low-density lipoprotein, whose receptors are not linked to cytoskeleton, were minimally effective in triggering Ca(2+) sparks and global Ca(2+) mobilization. Preincubation with ryanodine, cytochalasin-D, or colchicine substantially reduced the occurrence of Ca(2+) sparks triggered by fibronectin-coated beads. Binding of VSMCs with antibodies specific to the extracellular domains of alpha(5-) and beta(1)-integrins triggered Ca(2+) sparks simulating the effects of fibronectin-coated beads. Preincubation of microperfused afferent arterioles with ryanodine or integrin-specific binding peptide inhibited pressure-induced myogenic constriction. In conclusion, integrins transduce mechanical force into intracellular Ca(2+) signaling events in renal VSMCs. Integrin-mediated mechanotransduction is probably involved in myogenic response of afferent arterioles.     

The mammalian Sec6/8 complex interacts with Ca(2+) signaling complexes and regulates their activity

The localization of various Ca(2+) transport and signaling proteins in secretory cells is highly restricted, resulting in polarized agonist-stimulated Ca(2+) waves. In the present work, we examined the possible roles of the Sec6/8 complex or the exocyst in polarized Ca(2+) signaling in pancreatic acinar cells. Immunolocalization by confocal microscopy showed that the Sec6/8 complex is excluded from tight junctions and secretory granules in these cells. The Sec6/8 complex was found in at least two cellular compartments, part of the complex showed similar, but not identical, localization with the Golgi apparatus and part of the complex associated with Ca(2+) signaling proteins next to the plasma membrane at the apical pole. Accordingly, immunoprecipitation (IP) of Sec8 did not coimmunoprecipitate betaCOP, Golgi 58K protein, or mannosidase II, all Golgi-resident proteins. By contrast, IP of Sec8 coimmunoprecipitates Sec6, type 3 inositol 1,4,5-trisphosphate receptors (IP(3)R3), and the Gbetagamma subunit of G proteins from pancreatic acinar cell extracts. Furthermore, the anti-Sec8 antibodies coimmunoprecipitate actin, Sec6, the plasma membrane Ca(2+) pump, the G protein subunits Galphaq and Gbetagamma, the beta1 isoform of phospholipase C, and the ER resident IP(3)R1 from brain microsomal extracts. Antibodies against the various signaling and Ca(2+) transport proteins coimmunoprecipitate Sec8 and the other signaling proteins. Dissociation of actin filaments in the immunoprecipitate had no effect on the interaction between Sec6 and Sec8, but released the actin and dissociated the interaction between the Sec6/8 complex and Ca(2+) signaling proteins. Hence, the interaction between the Sec6/8 and Ca(2+) signaling complexes is likely mediated by the actin cytoskeleton. The anti-Sec6 and anti-Sec8 antibodies inhibited Ca(2+) signaling at a step upstream of Ca(2+) release by IP(3). Disruption of the actin cytoskeleton with latrunculin B in intact cells resulted in partial translocation of Sec6 and Sec8 from membranes to the cytosol and interfered with propagation of agonist-evoked Ca(2+) waves. Our results suggest that the Sec6/8 complex has multiple roles in secretory cells including governing the polarized expression of Ca(2+) signaling complexes and regulation of their activity.