CLE-CLAVATA1 peptide-receptor signaling module regulates the expansion of plant root systems in a nitrogen-dependent manner

Morphological plasticity of root systems is critically important for plant survival because it allows plants to optimize their capacity to take up water and nutrients from the soil environment. Here we show that a signaling module composed of nitrogen (N)-responsive CLE (CLAVATA3/ESR-related) peptides and the CLAVATA1 (CLV1) leucine-rich repeat receptor-like kinase is expressed in the root vasculature in Arabidopsis thaliana and plays a crucial role in regulating the expansion of the root system under N-deficient conditions. CLE1, -3, -4, and -7 were induced by N deficiency in roots, predominantly expressed in root pericycle cells, and their overexpression repressed the growth of lateral root primordia and their emergence from the primary root. In contrast, clv1 mutants showed progressive outgrowth of lateral root primordia into lateral roots under N-deficient conditions. The clv1 phenotype was reverted by introducing a CLV1 promoter-driven CLV1:GFP construct producing CLV1:GFP fusion proteins in phloem companion cells of roots. The overaccumulation of CLE2, -3, -4, and -7 in clv1 mutants suggested the amplitude of the CLE peptide signals being feedback-regulated by CLV1. When CLE3 was overexpressed under its own promoter in wild-type plants, the length of lateral roots was negatively correlated with increasing CLE3 mRNA levels; however, this inhibitory action of CLE3 was abrogated in the clv1 mutant background. Our findings identify the N-responsive CLE-CLV1 signaling module as an essential mechanism restrictively controlling the expansion of the lateral root system in N-deficient environments.Living organisms have developed dynamic strategies to explore nutrients in the environment. Morphological plasticity of plant roots and microorganisms is often compared with foraging behavior of animals. Plant roots are highly dynamic systems because they can modify their structure to reach nutrient resources in soil and optimize their nutrient uptake capacities. This strategy appears to be associated with morphological adaptation, because plants are sessile in nature and nutrient availabilities in soil are often altered by surrounding biotic and abiotic factors and climate changes. Morphological modifications of plant root systems are particularly prominent when they grow in soil environments with unbalanced nutrient availabilities (1–4). Among the essential elements required for plant growth, nitrogen (N) has a particularly strong effect on root development (1–6). Lateral roots can be developed in N-rich soil patches where adequate amounts of nitrate (NO₃⁻) or ammonium (NH₄⁺) are available, whereas this local outgrowth of lateral roots is restricted in N-deficient patches (7–9). In addition to these local N responses, lateral root growth is stimulated in response to mild N deficiency and suppressed under excess N supply by systemic plant signals carrying information on the nutritional status of distant plant organs (4, 10–13). These morphological responses are important for plant fitness and N acquisition, despite the cost for structuring the root system architecture (2, 6). However, lateral root growth is not sustained when plants are deprived of N for an extended period (4). Under such severe circumstances, the development of new lateral roots should rather be restricted to prevent the risk of extending roots into N-poor environments. Economizing the cost for root development appears to be an important morphological strategy for plant survival.To modify root traits in response to changing N availabilities, plants use various types of signaling molecules including hormones and small RNAs (10, 13–17). In particular, auxin signaling proteins and auxin transporters have been proven essential for lateral root development in response to local nitrate supplies (10, 14–17). These proteins are involved in increasing auxin sensitivity or auxin accumulation at lateral root initials or lateral root tips exposed to NO₃⁻, and the NRT1.1 nitrate transporter has been suggested to play a key role in NO₃⁻ sensing (8, 17, 18). In addition, mutations of the nitrate transporter NRT2.1 have been shown to repress or stimulate lateral root initiation depending on N conditions and sucrose supply (12, 19). Thus, N-dependent root development is apparently under control of complex mechanisms, although its signaling components have remained largely unidentified. In this study, we have identified several homologs of the CLE (CLAVATA3/ESR-related) gene family (20–24) to be up-regulated by N deficiency and involved in this yet unresolved regulatory mechanism. CLAVATA3 (CLV3) is known as a signaling peptide that binds to the CLAVATA1 (CLV1) leucine-rich repeat receptor-like kinase (LRR-RLK) and controls stem cell differentiation in the shoot apical meristem (25–32). CLE-receptor signaling modules are also known to control meristem function in the primary and lateral roots (33–35). The N-responsive CLE peptides described in the present study belong to the group of CLE peptides with the highest sequence similarity to CLAVATA3 (CLV3) (21–23) and may partly substitute for CLV3 in the shoot apical meristem (31, 36, 37). Our present findings indicate that the N-responsive CLE peptides and CLV1 are signaling components required for translating an N-deficient nutritional status into a morphological response inhibiting the outgrowth of lateral root primordia in Arabidopsis. The present study demonstrates a unique function of the CLE-CLV1 signaling module in roots and provides new insights into signaling mechanisms regulating the expansion of the plant root system in N-deficient environments.     

Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling

The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF–CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.The RAF serine/threonine protein kinase is a component of the three-tiered MAPK signaling pathway that regulates cell growth and many other essential biological processes (1, 2). In normal cells, extracellular mitogenic stimuli are transmitted to the nucleus via the receptor–RAS–RAF–MAPK cascade (2). Abnormal activation of this pathway is a central event in many human cancers and results from activating mutations in BRAF itself or in upstream factors (such as the RAS genes) (3, 4). As the RAS proteins so far are intractable pharmacologic targets (5), attention has shifted to development of small-molecule RAF inhibitors (RAFis) as antitumor therapeutic agents (6). To date, RAFi clinical efficacy has been demonstrated only for BRAF^V600E melanoma (6–8). In tumors with WT BRAF or mutant RAS, most RAFis paradoxically promote growth, at times malignant in nature (6). Moreover, BRAF mutant melanomas that are initially sensitive to RAFi rapidly become resistant by using a variety of compensatory mechanisms including RAF isoform switching and activation of other pathways including RTKs, RAS, or PI3K (9). In addition, some RAFis (e.g., vemurafenib) accelerate the occurrence of secondary squamous cell carcinomas (10).Several lines of investigation suggest that multimer formation plays an important role in RAF activation and tumor responses to RAFi (11–16). RAF dimerization-mediated signaling was first suggested by the observation that artificial dimerization activates RAF (17, 18). Next, immunoprecipitation (IP) suggested that formation of homo- and heterotypic RAF “dimers” is associated with active RAS (15, 16, 19). X-ray crystallography of the BRAF catalytic domain (CatB) identified critical residues postulated to enable CatB–CatB dimer formation (11). Mutations of these residues (e.g., R509H in BRAF and, equivalently, R401H in CRAF) profoundly diminished dimerization and kinase activity of RAF (11–13). Recently, dimerization also was implicated in RAFi-mediated activation of RAF in BRAF^WT tumors (12, 13), in acquired resistance of BRAF^V600E tumors to RAFi (14), and in the development of RAFi-induced secondary squamous carcinomas (10).Although these studies implicate RAF multimer formation in the regulation of signaling in some circumstances, they do not provide direct characterization of the nature of these multimers, nor the information about their intracellular locations. This is in a large part because of the lack of techniques with sufficient spatial and stoichiometric (protein counting) resolution to visualize RAF multimers inside an intact cell. For example, X-ray crystallography studies used purified and truncated RAF proteins (11, 20). IP measures protein–protein interactions but does not provide stoichiometric or cellular localization information of the protein complexes (12, 13).The foregoing studies underscore the need for better tools to study the RAF complexes involved in cell signaling. In the present work, we show that recently introduced single-molecule superresolution imaging techniques such as photoactivated localization microscopy (PALM) (21, 22) can be used for direct, quantitative analysis of RAF multimer formation inside an intact mammalian cell. We first demonstrate that PALM provides sufficient spatial and stoichiometric resolution to distinguish artificial protein dimers and higher-order multimers from monomers when using a suitable fluorescent probe and combined with quantitative spatial analysis. We then apply the quantitative PALM imaging approach to study CRAF multimerization under resting and various activating conditions, including the presence of active mutant RAS, N-terminal truncation, and artificial membrane localization. Our results clearly indicate the formation and significance of RAF dimers in cell signaling. The biological and therapeutic implications of these results are discussed.     

BMP signaling is required for the generation of primordial germ cells in an insect

Two modes of germ cell formation are known in animals. Specification through maternally inherited germ plasm occurs in many well-characterized model organisms, but most animals lack germ plasm by morphological and functional criteria. The only known alternative mechanism is induction, experimentally described only in mice, which specify germ cells through bone morphogenetic protein (BMP) signal-mediated induction of a subpopulation of mesodermal cells. Until this report, no experimental evidence of an inductive germ cell signal for specification has been available outside of vertebrates. Here we provide functional genetic experimental evidence consistent with a role for BMP signaling in germ cell formation in a basally branching insect. We show that primordial germ cells of the cricket Gryllus bimaculatus transduce BMP signals and require BMP pathway activity for their formation. Moreover, increased BMP activity leads to ectopic and supernumerary germ cells. Given the commonality of BMP signaling in mouse and cricket germ cell induction, we suggest that BMP-based germ cell formation may be a shared ancestral mechanism in animals.There are two well-characterized modes of animal germ cell specification. In the inheritance mode, observed in Drosophila melanogaster, Caenorhabditis elegans, and Xenopus laevis, maternally provided cytoplasmic determinants (germ plasm) specify a subset of early embryonic cells as germ cells. In contrast, mice specify their germ line through the induction mode, in which a zygotic cell–cell signaling mechanism specifies germ cells later in development. We previously hypothesized that the inductive mode was ancestral among metazoans and that the inheritance mode had evolved independently in multiple derived lineages (1, 2). Consistent with this hypothesis, multiple basally branching insects do not segregate maternally provided germ plasm, unlike the relatively derived Drosophila model (3, 4). However, experimental evidence for the inductive mode was available only for salamanders (5, 6) and mice (7–10), and to date, inferences of induction outside of vertebrates have been based on gene expression and cytological data (1, 11–16).Because Drosophila is highly derived with respect to many aspects of development (17), we examined germ cell development in the cricket Gryllus bimaculatus, a basally branching insect that may shed light on putative ancestral mechanisms of specifying germ cells. We previously showed that unlike Drosophila, Gryllus primordial germ cell (PGC) specification requires zygotic mechanisms rather than germ plasm or the oskar germ-line determinant (4, 18). However, the signals that might induce PGC formation in Gryllus remained unknown. Because mammals require the highly conserved bone morphogenetic protein (BMP) pathway to specify PGCs (8–10, 19, 20), we investigated BMP signaling as a candidate for regulating inductive germ cell specification in Gryllus.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Galactose metabolic genes in yeast respond to a ratio of galactose and glucose

Natural environments are filled with multiple, often competing, signals. In contrast, biological systems are often studied in “well-controlled” environments where only a single input is varied, potentially missing important interactions between signals. Catabolite repression of galactose by glucose is one of the best-studied eukaryotic signal integration systems. In this system, it is believed that galactose metabolic (GAL) genes are induced only when glucose levels drop below a threshold. In contrast, we show that GAL gene induction occurs at a constant external galactose:glucose ratio across a wide range of sugar concentrations. We systematically perturbed the components of the canonical galactose/glucose signaling pathways and found that these components do not account for ratio sensing. Instead we provide evidence that ratio sensing occurs upstream of the canonical signaling pathway and results from the competitive binding of the two sugars to hexose transporters. We show that a mutant that behaves as the classical model expects (i.e., cannot use galactose above a glucose threshold) has a fitness disadvantage compared with wild type. A number of common biological signaling motifs can give rise to ratio sensing, typically through negative interactions between opposing signaling molecules. We therefore suspect that this previously unidentified nutrient sensing paradigm may be common and overlooked in biology.The ability to integrate multiple cues about nutrient availability from the environment and coordinate uptake, metabolism, and regulatory networks is a major determinant of microbial cell fitness (1–3). The energy and building blocks needed for growth can come from many different sources, leading to a complex combinatorial signal integration problem. In an environment that contains a mixture of sugars, such as glucose and galactose, microbial cells regulate their response according to a carbon hierarchy mediated by catabolite repression. Galactose metabolic genes (GAL genes) are induced to a significant degree only after glucose-based catabolite repression is relieved, resulting in a lag in growth at the point of glucose exhaustion while GAL pathway proteins are produced (1–6). Recent studies of sugar integration in bacteria (7, 8) suggested that in these organisms the combinatorial response results from the multiplication of individual responses to different sugars.The response of Saccharomyces cerevisiae to galactose is one of the best-studied eukaryotic signaling pathways (1, 4–6, 9–12). The GAL response has become a canonical example for combinatorial signal integration based on a genetic switch (10–13). All GAL genes are induced by the activator Gal4p in response to galactose (14) but repressed by Mig1p when glucose is present (15). The inhibition of GAL genes by glucose is thought to occur at a threshold concentration, with signal integration occurring at promoters (11). These conclusions rest on a limited sampling of combinations of concentrations of glucose and galactose (SI Appendix, Fig. S6) (16–21). Our goal, therefore, was to use modern high-throughput techniques that allow us to characterize the GAL genes’ metabolic response in detail.