Grb14 inhibits FGF receptor signaling through the regulation of PLCγ recruitment and activation

To decipher the mechanism involved in Grb14 binding to the activated fibroblast growth factor receptor (FGFR), we used the bioluminescence resonance energy transfer (BRET) technique and the Xenopus oocyte model. We showed that Grb14 was recruited to FGFR1 into a trimeric complex containing also phospholipase C gamma (PLCγ). The presence of Grb14 altered FGF-induced PLCγ phosphorylation and activation. Grb14-FGFR interaction involved the Grb14-SH2 domain and the FGFR pY766 residue, which is the PLCγ binding site. Our data led to a molecular model whereby Grb14 binding to the phosphorylated FGFR induces a conformational change that unmasks a PLCγ binding motif on Grb14, allowing trapping and inactivation of PLCγ.

Structured summary

MINT-8019680: Grb14 (uniprotkb:O88900) physically interacts (MI:0915) with FGFR1 (uniprotkb:P11362) by anti tag coimmunoprecipitation (MI:0007)MINT-8019693, MINT-8019727: Grb14 (uniprotkb:O88900) physically interacts (MI:0915) with FGFR1 (uniprotkb:P11362) by bioluminescence resonance energy transfer (MI:0012)MINT-8019714, MINT-8019746: PLC gamma1 (uniprotkb:P19174) physically interacts (MI:0915) with FGFR1 (uniprotkb:P11362) by bioluminescence resonance energy transfer (MI:0012)     

Differential transgene expression patterns in Alzheimer mouse models revealed by novel human amyloid precursor protein‐specific antibodies

Alzheimer's disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aβ deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aβ pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aβ deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age‐related and brain region‐specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aβ in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP‐transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP‐transgenic mouse and one APP‐transgenic rat model. We observed remarkable differences in expression levels and brain region‐specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP‐transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals.     

The Sept4 septin locus is required for sperm terminal differentiation in mice

The murine septin4 gene (Sept4) has been implicated in diverse cellular functions, including cytokinesis, apoptosis, and tumor suppression. Here, we investigated the function of Sept4 proteins during mouse development by creating a targeted deletion of the Sept4 genomic locus. Sept4 mutant mice are viable but male sterile due to immotile and structurally defective sperm. During spermatogenesis, Sept4 proteins were essential for proper mitochondrial architecture and establishment of the annulus, a ring-like structure in the tail region of sperm. In addition, Sept4 mutant sperm showed defects in the elimination of residual cytoplasm during sperm maturation and had increased staining for the caspase inhibitor XIAP. This is consistent with a role of the proapoptotic Sept4 protein ARTS in promoting caspase-mediated removal of cytoplasm via inhibition of XIAP. Our results indicate that Sept4 proteins play distinct but evolutionarily conserved functions in different cellular compartments.     

The Nod factor-independent symbiotic signaling pathway: development of Agrobacterium rhizogenes-mediated transformation for the legume Aeschynomene indica

The nitrogen-fixing symbiosis between Aeschynomene indica and photosynthetic bradyrhizobia is the only legume-rhizobium association described to date that does not require lipochito-oligosaccharide Nod factors (NF). To assist in deciphering the molecular basis of this NF-independent interaction, we have developed a protocol for Agrobacterium rhizogenes-mediated transformation of A. indica. The cotransformation frequency (79%), the nodulation efficiency of transgenic roots (90%), and the expression pattern of the 35S Cauliflower mosaic virus promoter in transgenic nodules were all comparable to those obtained for model legumes. We have made use of this tool to monitor the heterologous spatio-temporal expression of the pMtENOD11-β-glucuronidase fusion, a widely used molecular reporter for rhizobial infection and nodulation in both legumes and actinorhizal plants. While MtENOD11 promoter activation was not observed in A. indica roots prior to nodulation, strong reporter-gene expression was observed in the invaded cells of young nodules and in the cell layers bordering the central zone of older nodules. We conclude that pMtENOD11 expression can be used as an infection-related marker in A. indica and that Agrobacterium rhizogenes-mediated root transformation of Aeschynomene spp. will be an invaluable tool for determining the molecular basis of the NF-independent symbiosis.     

Chemical analysis and immunolocalisation of lignin and suberin in endodermal and hypodermal/rhizodermal cell walls of developing maize (Zea mays L.) primary roots

The composition of suberin and lignin in endodermal cell walls (ECWs) and in rhizodermal/hypodermal cell walls (RHCWs) of developing primary maize (Zea mays L.) roots was analysed after depolymerisation of enzymatically isolated cell wall material. Absolute suberin amounts related to root length significantly increased from primary ECWs (Casparian strips) to secondary ECWs (suberin lamella). During further maturation of the endodermis, reaching the final tertiary developmental state characterised by the deposition of lignified secondary cell walls (u-shaped cell wall deposits), suberin amounts remained constant. Absolute amounts of lignin related to root length constantly increased throughout the change from primary to tertiary ECWs. The suberin of Casparian strips contained high amounts of carboxylic and 2-hydroxy acids, and differed substantially from the suberin of secondary and tertiary ECWs, which was dominated by high contents of ω-hydroxycarboxylic and 1,ω-dicarboxylic acids. Furthermore, the chain-length distribution of suberin monomers in primary ECWs ranged from C₁₆ to C₂₄, whereas in secondary and tertiary ECWs a shift towards higher chain lengths (C₁₆ to C₂₈) was observed. The lignin composition of Casparian strips (primary ECWs) showed a high syringyl content and was similar to lignin in secondary cell walls of the tertiary ECWs, whereas lignin in secondary ECWs contained higher amounts of p-hydroxyphenyl units. The suberin and lignin compositions of RHCWs rarely changed with increasing root age. However, compared to the suberin in ECWs, where C₁₆ and C₁₈ were the most prominent chain lengths, the suberin of RHCWs was dominated by the higher chain lengths (C₂₄ and C₂₆). The composition of RHCW lignin was similar to that of secondary-ECW lignin. Using lignin-specific antibodies, lignin epitopes were indeed found to be located in the Casparian strip. Surprisingly, the mature suberin layers of tertiary ECWs contained comparable amounts of lignin-like epitopes. Received: 19 August 1998 / Accepted: 3 February 1999     

Regulation of water flux through trunks, branches, and leaves in trees of a lowland tropical forest

We studied regulation of whole-tree water use in individuals of five diverse canopy tree species growing in a Panamanian seasonal forest. A construction crane equipped with a gondola was used to access the upper crowns and points along the branches and trunks of the study trees for making concurrent measurements of sap flow at the whole-tree and branch levels, and vapor phase conductances and water status at the leaf level. These measurements were integrated to assess physiological regulation of water use from the whole-tree to the single-leaf scale. Whole-tree water use ranged from 379 kg day⁻¹ in a 35 m-tall Anacardium excelsum tree to 46 kg day⁻¹ in an 18 m-tall Cecropia longipes tree. The dependence of whole-tree and branch sap velocity and sap flow on sapwood area was essentially identical in the five trees studied. However, large differences in transpiration per unit leaf area (E) among individuals and among branches on the same individual were observed. These differences were substantially reduced when E was normalized by the corresponding branch leaf area:sapwood area ratio (LA/SA). Variation in stomatal conductance (g _s) and crown conductance (g _c), a total vapor phase conductance that includes stomatal and boundary layer components, was closely associated with variation in the leaf area-specific total hydraulic conductance of the soil/leaf pathway (G _t). Vapor phase conductance in all five trees responded similarly to variation in G _t. Large diurnal variations in G _t were associated with diurnal variation in exchange of water between the transpiration stream and internal stem storage compartments. Differences in stomatal regulation of transpiration on a leaf area basis appeared to be governed largely by tree size and hydraulic architectural features rather than physiological differences in the responsiveness of stomata. We suggest that reliance on measurements gathered at a single scale or inadequate range of scale may result in misleading conclusions concerning physiological differences in regulation of transpiration. Received: 1 October 1997 / Accepted: 6 March 1998     

Prioritizing road mitigation using ecologically based land-use planning

Wildlife–vehicle collisions (WVCs) are a critical threat to biodiversity and human safety. To implement WVC mitigation measures where most needed, we need to link models predicting the probability of the presence of species, at large spatial scales, with the likelihood of occurring collisions along roads. Here, we propose a framework for the prioritization of road sections for implementing mitigation measures using ecologically based information. Within this framework, we first model the likelihood of WVC occurrence for focal species in road vicinity areas. We then use spatial prioritization tools to select road sections that entail a higher probability of WVC across the focal species using user-defined criteria for weighting species records. We applied this framework to Mato Grosso do Sul state (Brazil), using systematic information on WVC collected over 3 years along ca. 2000 km of roads. We focused on the WVC involving three large mammals commonly road-killed therein, which represent a threat to humans when involved in WVC: the lowland tapir Tapirus terrestris, giant anteater Myrmecophaga tridactyla and capybara Hydrochoerus hydrochaeris. We were able to identify road sections (<10% of the road network) that should be prioritized for implementing mitigation actions, which could significantly reduce the number of WVCs. However, the large extent of the road network classified as a priority for mitigation suggests the need to engage the public and private sectors in the early stages of the decision-making process, in order to reach a consensus on the prioritization. Our framework may improve the environmental licensing process, namely by guiding where mitigation measures should be implemented first.     

MIM through MOM: the awakening of Bax and Bak pores

The mechanism whereby mitochondrial DNA (mtDNA) is released into the cytosol and activates the cGAS/STING inflammatory pathway during Bax/Bax‐mediated apoptosis is unknown. In this issue, Riley et al ( 2018 ) report that widening of Bax and Bak pores on the mitochondrial outer membrane (MOM) during apoptosis allows the extrusion of the mitochondrial inner membrane (MIM) into the cytosol and its permeabilization to release mtDNA independently of caspases. In this scenario, Bax and Bak emerge as key modulators of the apoptotic immunogenic response.     

Regulation of colony morphology and biofilm formation in Shewanella algae

Bacterial colony morphology can reflect different physiological stages such as virulence or biofilm formation. In this work we used transposon mutagenesis to identify genes that alter colony morphology and cause differential Congo Red (CR) and Brilliant Blue G (BBG) binding in Shewanella algae, a marine indigenous bacterium and occasional human pathogen. Microscopic analysis of colonies formed by the wild-type strain S. algae CECT 5071 and three transposon integration mutants representing the diversity of colony morphotypes showed production of biofilm extracellular polymeric substances (EPS) and distinctive morphological alterations. Electrophoretic and chemical analyses of extracted EPS showed differential patterns between strains, although the targets of CR and BBG binding remain to be identified. Galactose and galactosamine were the preponderant sugars in the colony biofilm EPS of S. algae. Surface-associated biofilm formation of transposon integration mutants was not directly correlated with a distinct colony morphotype. The hybrid sensor histidine kinase BarA abrogated surface-associated biofilm formation. Ectopic expression of the kinase and mutants in the phosphorelay cascade partially recovered biofilm formation. Altogether, this work provides the basic analysis to subsequently address the complex and intertwined networks regulating colony morphology and biofilm formation in this poorly understood species.