Multistep Signaling Research Articles

Multistep phosphorelay in fungi: the enigma of multiple signals and a limited number of signaling pathways

Current knowledge of fungal ecology depends on the understanding of the perception and processing of biotic and abiotic factors facilitating environmental interactions. Ongoing research regarding fungal signaling mechanisms has not only increased our appreciation for multistep phosphorelay signaling systems as an asset, but also sheds light on the role that these upgrades of the simple two-component signaling systems play in fungal biology, stress response, differentiation processes, metabolism, and pathogenicity. In contrast to two-component signaling systems, multistep phosphorelay systems comprise a phosphotransfer protein enabling modular phosphate transfer from the sensor hybrid histidine kinase to the response regulator protein. Histidine kinases were found to perceive a lot of environmental signals and integrate them into a number of signaling pathways. The major question is how multistep phosphorelay signal transduction takes place in detail, since most fungi possess only one single histidine-containing phosphotransfer protein enabling phosphate transfer. One single phosphotransfer protein routes various signals in a specific way and coordinates the phosphates precisely to different target locations within the cell. This mini review opens the door and presents three hypotheses as the basis for further investigations to unravel the complex signaling mechanisms in fungal multistep phosphorelay systems. Scaffold proteins, frequency-based signaling, and alternative splicing are discussed as putative candidates to implement fungal signaling in multistep phosphorelay systems having only one phosphotransfer protein for coordinated phosphate transfer.

Non-contact remote estimation of cardiovascular parameters

Abstract Cardiovascular disease is a serious threat to human health. It is crucial to monitor the cardiovascular parameters reliably and conveniently. Non-contact measurement has been widely studied. However, there are some inevitable factors that limit the use of the platform and even lead to inaccuracy estimation. Hence, a novel non-contact method that estimates cardiovascular parameters under ambient light is proposed. The most suitable region of interest (ROI) is determined by a colormap, which is a map consists of Fast Fourier Transform (FFT) peak amplitude of every pixel. Comparisons suggest that the region includes cheeks and nose is the most appropriate, followed by the forehead. To remove the motion artifacts caused by body movement, face detection and tracking algorithms are performed. Further, a multi-step signal process that combines independent component analysis (ICA) and wavelet de-noising is utilized to extract clean signal from the noise-corrupted raw waveform. Moreover, various distance between subject and camera and the change of ambient light intensity are considered, where statistics results reveal that this non-contact methodology is blind to these factors. Compared with the gold standard pulse oximeter, the proposed method shows a high accuracy even in the presence of motion artifacts.

Turning Nonspecific Interference into Signal Amplification: Covalent Biosensing Nanoassembly Enabled by Metal-Catalyzed Cross-Coupling.

In this work, a new method of protein detection in complicated samples is proposed. This method employs probe-target recognition to induce cross-linking among the probe, the target, and the nonspecific proteins in the complicated sample as a means to convert interference into effective signal amplification. This also eliminates the necessity of multistep signal amplification in a separate solution system. On the basis of this strategy, a simple and robust assay for the activity of serum cathepsin B is established. Peptide probes immobilized on a sensing slide can recognize cathepsin B, and this can induce thiol-alkyne covalent coupling between the probe and cathepsin B. Meanwhile, applying electrochemical potential scanning to this sensing surface, Cu binding fragments of the probe peptide can be released into the solution phase to act as an electrochemical catalyst for oxidative dityrosine cross-linking among all proteins including the captured cathepsin B and the nonspecific proteins. A continuous nanoassembly covalently anchored on the sensing surface can gradually form, allowing violent detergent rinsing to remove residual interference. Using this method, not only sensitivity in the picomolar range can be achieved for serum analysis, the results of the analysis can also reliably discriminate benign and cancerous ovarian conditions. These results may suggest prospective application of this method in early screening of cancer in the future.

Evolution of a multi-step phosphorelay signal transduction system in Ensifer: recruitment of the sigma factor RpoN and a novel enhancer-binding protein triggers acid-activated gene expression.

Most Ensifer strains are comparatively acid sensitive, compromising their persistence in low pH soils. In the acid-tolerant strain Ensifer medicae WSM419, the acid-activated expression of lpiA is essential for enhancing survival in lethal acidic conditions. Here we characterise a multi-step phosphorelay signal transduction pathway consisting of TcsA, TcrA, FsrR, RpoN and its cognate enhancer-binding protein EbpA, which is required for the induction of lpiA and the downstream acvB gene. The fsrR, tcrA, tcsA and rpoN genes were constitutively expressed, whereas lpiA and acvB were strongly acid-induced. RACE mapping revealed that lpiA/acvB were co-transcribed as an operon from an RpoN promoter. In most Ensifer species, lpiA/acvB is located on the chromosome and the sequence upstream of lpiA lacks an RpoN-binding site. Nearly all Ensifer meliloti strains completely lack ebpA, tcrA, tcsA and fsrR regulatory loci. In contrast, E. medicae strains have lpiA/acvB and ebpA/tcrA/tcsA/fsrR co-located on the pSymA megaplasmid, with lpiA/acvB expression coupled to an RpoN promoter. Here we provide a model for the expression of lpiA/acvB in E. medicae. This unique acid-activated regulatory system provides insights into an evolutionary process which may assist the adaptation of E. medicae to acidic environmental niches.

GATA Factor-Dependent Positive-Feedback Circuit Controls Acute Myeloid Leukemia Cell Proliferation: Mechanisms and Targets for Therapeutic Intervention

Abstract Recent studies have established a relationship between GATA-2, a master regulator of hematopoiesis, and acute myeloid leukemia (AML). Loss-of-function mutations and elevated GATA2 expression are implicated in AML. While inhibitory GATA2 mutations occur in patients with primary immunodeficiency, which progresses to AML, GATA2 can be overexpressed in AML patients with poor prognosis. Many questions remain unanswered regarding mechanisms by which GATA2 deregulation results in AML. We reported that the Ras-p38α pathway induces multi-site GATA-2 phosphorylation in proerythroblasts, and Ser192 is required for the phosphorylation. Because RAS is mutated in 20% of AML patients, and elevated GATA2 expression correlates with poor prognosis, we tested whether this pathway functions in AML. GATA-2 was phosphorylated in the steady-state in AML cell lines, and oncogenic Ras(G12V) expression induced GATA-2 hyperphosphorylation in a p38/ERK-dependent manner. GATA-2 contains a sequence that conforms to the MAPK docking site termed "DEF motif". Mutation of S192 or the DEF motif abrogated Ras(G12V)-mediated GATA-2 hyperphosphorylation. We analyzed function using a Mouse Aortic Endothelial (MAE) cell line, in which GATA-2 and Ras(G12V) synergistically induce expression of specific GATA-2 target genes, e.g. Hdc. The DEF mutation abrogated GATA-2 hyperphosphorylation by Ras(G12V) and attenuated GATA-2 activity to induce Hdc expression in the presence of Ras(G12V) in MAE cells (50% of control, p < 0.05). GATA-2 hyperphosphorylation was also induced by expression of constitutively-active (ca) p38α and caMEK-1. caMEK-1-induced hyperphosphorylation was suppressed by the p38 inhibitor, SB203580 (p < 0.05) while the MEK inhibitor U0126 did not influence cap38α-induced hyperphosphorylation. The phosphatase inhibitor okadaic acid induced GATA-2 hyperphosphorylation, and p38 was more important than ERK in this context. These data support a model in which a hierarchical network of kinases controls GATA-2 phosphorylation and function in AML. Phospho-proteomics is being used to establish interconnectivity between network components and temporal aspects of the multi-step signaling mechanism. ChIP-Seq analysis in Kasumi-3 AML cells revealed GATA-2 occupancy at IL1B and CXCL2. IL1B and CXCR2, encoding the CXCL2 receptor, are implicated in AML. GATA-2 downregulation decreased expression of these genes (IL1B: 81.5% decrease, p < 0.001, CXCL2: 73% decrease, p < 0.001). Inhibition of ERK or p38 attenuated GATA-2 phosphorylation, decreased GATA-2 occupancy at GATA2, IL1B, and CXCL2, and lowered mRNA expression of these genes. To determine the consequences of the reduced occupancy, FAIRE was used to quantitate accessibility at the occupancy sites. Under conditions of reduced GATA-2 occupancy, accessibility decreased 50-65% at the GATA2 -77 enhancer and GATA-2 occupancy sites of IL1B and CXCL2 (p < 0.05). IL-1β and CXCL2 stimulated GATA-2 phosphorylation via p38 and ERK activation in Kasumi-1 and primary AML cells and increased GATA-2 chromatin occupancy and target gene expression. These results reveal a Ras-p38/ERK-GATA-2-IL-1β/CXCL2 axis that constitutes a positive-feedback circuit. We tested whether this circuit impacts Kasumi-1 cell proliferation. GATA2 downregulation reduced the proliferative rate (p < 0.001), which was partially rescued with recombinant CXCL2 (p < 0.01). To determine whether the signal-dependent mechanism can be extrapolated to human AML patients, we analyzed AML datasets from TCGA and GEO. GATA2 and CXCL2 expression was elevated in AML (p < 0.01). GATA2, IL1B, and CXCL2 mRNA levels correlated in M5 AML (GATA2 vs. IL1B: p=0.0015, GATA2 vs. CXCL2: p=0.0046, IL1B vs. CXCL2: p=0.000001). Among the patients with normal karyotype AML, high GATA2 and CXCL2 expression correlated with significantly reduced survival (p=0.003). In aggregate, our results establish a Ras-p38/ERK-GATA-2-IL-1β/CXCL2 axis in AML. It is attractive to propose that the GATA-2-dependent positive-feedback circuit is deployed upon ectopic elevation of GATA-2 activity in certain AML contexts and may harbor potential therapeutic targets. Given the profound capacity of this mechanism to alter GATA-2 activity, it is crucial to consider the GATA-2 activation state, rather than mRNA or total GATA-2 protein levels in clinical scenarios. Disclosures No relevant conflicts of interest to declare.

Silicon phthalocyanine-decorated TiO2 mesocrystal coupled with multifunctional all-carbon structure for multistep cascade signal amplifier in photoelectrochemical immunoassay

Herein, a new photoelectrochemical sensor for aflatoxin B1 (AFB1) detection was established based on the all-carbon nanohybrids as transducer scaffold accompanied with quasi-octahedral TiO2 mesocrystal (QOTM) absorbed dentritic Si phthalocyanines (SiPcs) as the efficient bioprobe. Specifically, the all-carbon nanocomposites exhibited fine photoelectric properties due to the photo-generated electrons of carbon quantum dots could be rapidly accepted by conductive carbon nanohorns, which evidently expedited the electron transport, and the nanohybrids were first employed as affinity support to anchor AFB1 antibody. The dentritic SiPcs sensitized QOTM presented better photocurrent response and stability in comparison with QOTM alone due to impressive light-harvesting capability of the dentritic SiPcs. Under light irradiation, the photo-induced electrons of SiPcs migrated to the QOTM, and successively transferred to carbon quantum dots as electron relay mediator then via carbon nanohorns to the electrode surface based on suitable band alignment. This hierarchical charge transport cascade paradigm efficiently impeded the charge recombination, resulting in noticeably increased photocurrent. In this competitive immunosensor, the AFB1 standards compete with the labeled AFB1, leading to photocurrent decreased with the increasing target concentration in a wide linear range of 10−6–102ng/mL. The newly developed methodology provided a versatile approach for ultrasensitive detection of small molecules.

Correlation analysis based MIMO neuro-fuzzy Hammerstein model with noises

A novel identification algorithm for neuro-fuzzy based MIMO Hammerstein system with noises by using the correlation analysis method is presented in this paper. A special test signal that contains independent separable signals and uniformly random multi-step signal is adopted to identify the MIMO Hammerstein system, resulting in the identification problem of the linear model separated from that of nonlinear part. As a result, it can circumvent the problem of initialization and convergence of the model parameters encountered by the existing iterative algorithms used for identification of MIMO Hammerstein model. Moreover, least square method based parameter identification algorithms of dynamic linear part and static nonlinear part are proposed to avoid the influence of noise. Examples are used to illustrate the effectiveness of the proposed method.

SFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance.

Cancer is a disease of aging, and aged cancer patients have a poorer prognosis. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumor progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression1–4 we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. We find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signaling cascade in melanoma cells that results in a decrease in β-catenin and MITF, and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to ROS-induced DNA damage, rendering them more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumor progression, offering new paradigms for the design of therapy for the elderly.

A New Class of Targetable, Genetically Encoded Single-Color Biosensors for Multiplexed Monitoring of Phosphoinositides

Phosphoinositides are involved in numerous signaling processes, even though they represent only a small fraction of all phospholipids in cellular membranes. Phosphoinositide species can be interconverted reversibly, allowing them to serve as second messengers to activate signaling downstream or as membrane anchors by recruiting specific proteins. Many membrane proteins, especially ion channels, are regulated through direct interactions with specific phosphoinositide species. Several genetically encodable biosensors have been engineered to observe phosphoinositide signaling in living cells. These biosensors signal the binding of specific binding domains to phosphoinositides by either translocation of fluorescence or changes in FRET efficiency. However, monitoring further parameter or multiple phosphoinositide species simultaneously, especially in different cellular compartments, still poses a challenge. This hinders a full understanding of the role that phosphoinositides play in multi-step signaling pathways. In order to facilitate multiplexed imaging, we tested different strategies for developing single-color intensity based sensors and identified a generalizable unimolecular sensor design for phosphoinositides based on the sensor InPAkt. Targeting green and red fluorescent sensors for PI(4,5)P2, PI(3,4)P2 and PI(3,4,5)P3 to different plasma membrane compartments, we were able to investigate phosphoinositide dynamics and additional parameters simultaneously during Gq and growth factor signaling.

Monotonicity of Kinetic Proofreading

Abstract: This manuscript studies the monotonicity of multi-step ligand-receptor signalling motifs. Monotonicity with respect to parameters and state perturbations allow not only to exclude periodic solutions, but also to easily bound the responses in cases of bounded perturbations. In classical coordinates, multi-step ligand-receptor signalling motifs are known not to be monotone. However, a generic coordinate transform allows for deriving conditions on the kinetic rate constants such that the signal is monotonously affected by perturbations to any one of the kinetic rate constants. The result is illustrated at the hand of a model of kinetic proofreading.

The identification of neuro-fuzzy based MIMO Hammerstein model with separable input signals

A novel identification method of neuro-fuzzy based MIMO Hammerstein model by using the correlation analysis method is presented in this paper. A special test signal that contains independent separable signals and uniformly random multi-step signal is adopted to identify the MIMO Hammerstein process, resulting in the identification problem of the linear model separated from that of nonlinear part. As a result, the identification of the dynamic linear element can be separated from the static nonlinear element without any redundant adjustable parameters. Moreover, it can circumvent the problem of initialization and convergence of the model parameters discussed in the existing iterative algorithms used for identification of MIMO Hammerstein model. Examples are used to illustrate the effectiveness of the proposed method.

Exploring molecular recognition mechanisms in the multistep phosphorelay signaling in plants: crystal structure of His phosphotransmitter AHP2 and modeling of its interaction with sensor histidine kinase CKI1

Exploring molecular recognition mechanisms in the multistep phosphorelay signaling in plants: crystal structure of His phosphotransmitter AHP2 and modeling of its interaction with sensor histidine kinase CKI1

Multistep current signal in protein translocation through graphene nanopores.

In nanopore sensing experiments, the properties of molecules are probed by the variation of ionic currents flowing through the nanopore. In this context, the electronic properties and the single-layer thickness of graphene constitute a major advantage for molecule characterization. Here we analyze the translocation pathway of the thioredoxin protein across a graphene nanopore, and the related ionic currents, by integrating two nonequilibrium molecular dynamics methods with a bioinformatic structural analysis. To obtain a qualitative picture of the translocation process and to identify salient features we performed unsupervised structural clustering on translocation conformations. This allowed us to identify some specific and robust translocation intermediates, characterized by significantly different ionic current flows. We found that the ion current strictly anticorrelates with the amount of pore occupancy by thioredoxin residues, providing a putative explanation of the multilevel current scenario observed in recently published translocation experiments.

Applicability of an automatic surface detection approach to micro-pulse photon-counting lidar altimetry data: implications for canopy height retrieval from future ICESat-2 data

We develop and validate an automated approach to determine canopy height, an important metric for global biomass assessments, from micro-pulse photon-counting lidar data collected over forested ecosystems. Such a lidar system is planned to be launched aboard the National Aeronautics and Space Administration’s follow-on Ice, Cloud and land Elevation Satellite mission (ICESat-2) in 2017. For algorithm development purposes in preparation for the mission, the ICESat-2 project team produced simulated ICESat-2 data sets from airborne observations of a commercial micro-pulse lidar instrument (developed by Sigma Space Corporation) over two forests in the eastern USA. The technique derived in this article is based on a multi-step mathematical and statistical signal extraction process which is applied to the simulated ICESat-2 data set. First, ground and canopy surfaces are approximately extracted using the statistical information derived from the histogram of elevations for accumulated photons in 100 footprints. Second, a signal probability metric is generated to help identify the location of ground, canopy-top, and volume-scattered photons. According to the signal probability metric, the ground surface is recovered by locating the lowermost high-photon density clusters in each simulated ICESat-2 footprint. Thereafter, canopy surface is retrieved by finding the elevation at which the 95th percentile of the above-ground photons exists. The remaining noise is reduced by cubic spline interpolation in an iterative manner. We validate the results of the analysis against the full-resolution airborne photon-counting lidar data, digital terrain models (DTMs), and canopy height models (CHMs) for the study areas. With ground surface residuals ranging from 0.2 to 0.5 m and canopy height residuals ranging from 1.6 to 2.2 m, our results indicate that the algorithm performs very well over forested ecosystems of canopy closure of as much as 80%. Given the method’s success in the challenging case of canopy height determination, it is readily applicable to retrieval of land ice and sea ice surfaces from micro-pulse lidar altimeter data. These results will advance data processing and analysis methods to help maximize the ability of the ICESat-2 mission to meet its science objectives.

Multi-step mode signal conversion algorithms in hall sensor devices

This paper presents a numerical and experimental based data analysis of electromagnetic noise immunity in magnetic field measurement devices on Hall sensor transducers. Relevance of this problem takes place in sensor networks with autonomous low-powered sources, magnetic field mapping devices for charged particle accelerators fusion reactors, etc. The proposed algorithm is based on averaging of several step by step signals, especially, acquired by three consecutive measurements in time interval: (t + dt), (t), (t – dt). In (t + dt) and (t – dt) time intervals a Hall sensor current pulse magnitude and directions are the same. Contrary to these, in (t) time interval the Hall sensor current has the opposite direction. It is shown that the efficiency of triple step measurements is three times higher than in double step measurement. The integral performance criterion concept of the signal converter effectiveness is introduced. The performance criterion factor is defined as a ratio of signal converter electromagnetic noise immunity to its energy consumption. A signal transducer optimized to noise immunity study, its operating modes and Hall sensor parameters are described. Both calculation and experiment results show that the performance criterion factor in triple step measurements is twice higher than in double step measurements.

Cytokinins résumé: their signaling and role in programmed cell death in plants.

Cytokinins (CKs) are a large group of plant hormones which play a crucial role in many physiological processes in plants. One of the interesting functions of CKs is the control of programmed cell death (PCD). It seems that all CKs-dependent phenomena including PCD are accompanied by special multi-step phosphorelay signaling pathway. This pathway consists of three elements: histidine kinase receptors (HKs), histidine phosphotransfer proteins (HPs) and response regulators (RRs). This review shows the résumé of the latest knowledge about CKs signaling pathways in many physiological processes in plants with special attention paid to PCD process.

Ultrasensitive response motifs: basic amplifiers in molecular signalling networks

Multi-component signal transduction pathways and gene regulatory circuits underpin integrated cellular responses to perturbations. A recurring set of network motifs serve as the basic building blocks of these molecular signalling networks. This review focuses on ultrasensitive response motifs (URMs) that amplify small percentage changes in the input signal into larger percentage changes in the output response. URMs generally possess a sigmoid input–output relationship that is steeper than the Michaelis–Menten type of response and is often approximated by the Hill function. Six types of URMs can be commonly found in intracellular molecular networks and each has a distinct kinetic mechanism for signal amplification. These URMs are: (i) positive cooperative binding, (ii) homo-multimerization, (iii) multistep signalling, (iv) molecular titration, (v) zero-order covalent modification cycle and (vi) positive feedback. Multiple URMs can be combined to generate highly switch-like responses. Serving as basic signal amplifiers, these URMs are essential for molecular circuits to produce complex nonlinear dynamics, including multistability, robust adaptation and oscillation. These dynamic properties are in turn responsible for higher-level cellular behaviours, such as cell fate determination, homeostasis and biological rhythm.

Hyperactivated JNK Is a Therapeutic Target in pVHL-Deficient Renal Cell Carcinoma

Clear cell renal cell carcinomas (RCC), the major histologic subtype of RCC accounting for more than 80% of cases, are typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Although accumulation of hypoxia-inducible factor alpha (HIF-α) is the most well-studied effect of VHL inactivation, direct inhibition of HIFα or restoration of wild-type pVHL protein expression has not proved readily feasible, given the limitations associated with pharmacologic targeting of transcription factors (i.e., HIF-α) and gene replacement therapy of tumor suppressor genes (i.e., VHL). Here, we have established that phosphorylated c-Jun, a substrate of the c-Jun-NH(2)-kinase (JNK), is selectively activated in clear cell RCC patient specimens. Using multiple isogenic cell lines, we show that HIF-α-independent JNK hyperactivation is unique to the pVHL-deficient state. Importantly, pVHL-deficient RCCs are dependent upon JNK activity for in vitro and in vivo growth. A multistep signaling pathway that links pVHL loss to JNK activation involves the formation of a CARD9/BCL10/TRAF6 complex as a proximal signal to sequentially stimulate TAK1 (MAPKKK), MKK4 (MAPKK), and JNK (MAPK). JNK stimulates c-Jun phosphorylation, activation, and dimerization with c-Fos to form a transcriptionally competent AP1 complex that drives transcription of the Twist gene and induces epithelial-mesenchymal transition. Thus, JNK represents a novel molecular target that is selectively activated in and drives the growth of pVHL-deficient clear cell RCCs. These findings can serve as the preclinical foundation for directed efforts to characterize potent pharmacologic inhibitors of the JNK pathway for clinical translation.

Identification of five B-type response regulators as members of a multistep phosphorelay system interacting with histidine-containing phosphotransfer partners of Populus osmosensor

BackgroundIn plants, the multistep phosphorelay signaling pathway mediates responses to environmental factors and plant hormones. This system is composed of three successive partners: hybrid Histidine-aspartate Kinases (HKs), Histidine-containing Phosphotransfer proteins (HPts), and Response Regulators (RRs). Among the third partners, B-type RR family members are the final output elements of the pathway; they act as transcription factors and clearly play a pivotal role in the early response to cytokinin in Arabidopsis. While interactions studies between partners belonging to the multistep phosphorelay system are mainly focused on protagonists involved in cytokinin or ethylene pathways, very few reports are available concerning partners of osmotic stress signaling pathway.ResultsIn Populus, we identified eight B-type RR proteins, RR12-16, 19, 21 and 22 in the Dorskamp genotype. To assess HPt/B-type RR interactions and consequently determine potential third partners in the osmosensing multistep phosphorelay system, we performed global yeast two-hybrid (Y2H) assays in combination with Bimolecular Fluorescence Complementation (BiFC) assays in plant cells. We found that all B-type RRs are able to interact with HPt predominant partners (HPt2, 7 and 9) of HK1, which is putatively involved in the osmosensing pathway. However, different profiles of interaction are observed depending on the studied HPt. HPt/RR interactions displayed a nuclear localization, while the nuclear and cytosolic localization of HPt and nuclear localization of RR proteins were validated. Although the nuclear localization of HPt/RR interaction was expected, this work constitutes the first evidence of such an interaction in plants. Furthermore, the pertinence of this partnership is reinforced by highlighting a co-expression of B-type RR transcripts and the other partners (HK1 and HPts) belonging to a potential osmosensing pathway.ConclusionBased on the interaction studies between identified B-type RR and HPt proteins, and the co-expression analysis of transcripts of these potential partners in poplar organs, our results favor the model that RR12, 13, 14, 16 and 19 are able to interact with the main partners of HK1, HPt2, 7 and 9, and this HPt/RR interaction occurs within the nucleus. On the whole, the five B-type RRs of interest could be third protagonists putatively involved in the osmosensing signaling pathway in Populus.

Stimulus-dependent phosphorylation of profilin-1 in angiogenesis

Angiogenesis, the formation of new blood vessels, is fundamental to development and post-injury tissue repair. Vascular endothelial growth factor (VEGF)-A guides and enhances endothelial cell migration to initiate angiogenesis. Profilin-1 (Pfn-1) is an actin-binding protein that enhances actin filament formation and cell migration, but stimulus-dependent regulation of Pfn-1 has not been observed. Here, we show that VEGF-A-inducible phosphorylation of Pfn-1 at Tyr 129 is critical for endothelial cell migration and angiogenesis. Chemotactic activation of VEGF receptor kinase-2 (VEGFR2) and Src induces Pfn-1 phosphorylation in the cell leading edge, promoting Pfn-1 binding to actin and actin polymerization. Conditional endothelial knock-in of phosphorylation-deficient Pfn1(Y129F) in mice reveals that Pfn-1 phosphorylation is critical for angiogenesis in response to wounding and ischaemic injury, but not for developmental angiogenesis. Thus, VEGFR2/Src-mediated phosphorylation of Pfn-1 bypasses canonical, multistep intracellular signalling events to initiate endothelial cell migration and angiogenesis, and might serve as a selective therapeutic target for anti-angiogenic therapy.