Structure of the RZZ complex and molecular basis of Spindly‐driven corona assembly at human kinetochores

In metazoans, a ≈1 megadalton (MDa) multiprotein complex comprising the dynein–dynactin adaptor Spindly and the ROD–Zwilch–ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high‐resolution cryo‐EM structure that captures the essential features of the RZZ complex, including a farnesyl‐binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ–Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation‐dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long‐sought‐for molecular basis of corona assembly on metazoan kinetochores.     

Activation of pregnane X receptor disrupts glucocorticoid and mineralocorticoid homeostasis

The pregnane X receptor (PXR) was isolated as a xenobiotic receptor that regulates responses to various xenobiotic agents. In this study, we show that PXR plays an important endobiotic role in adrenal steroid homeostasis. Activation of PXR by genetic (transgene) or pharmacological (ligand, such as rifampicin) markedly increased plasma concentrations of corticosterone and aldosterone, the respective primary glucocorticoid and mineralocorticoid in rodents. The increased levels of corticosterone and aldosterone were associated with activation of adrenal steroidogenic enzymes, including cytochrome P450 (CYP)11a1, CYP11b1, CYP11b2, and 3beta-hydroxysteroid dehydrogenase. The PXR-activating transgenic mice also exhibited hypertrophy of the adrenal cortex, loss of glucocorticoid circadian rhythm, and lack of glucocorticoid responses to psychogenic stress. Interestingly, the transgenic mice had normal pituitary secretion of ACTH and the corticosterone-suppressing effect of dexamethasone was intact, suggesting a functional hypothalamus-pituitary-adrenal axis despite a severe disruption of adrenal steroid homeostasis. The ACTH-independent hypercortisolism in the PXR-activating transgenic mice is reminiscent of the pseudo-Cushing's syndrome in patients. The glucocorticoid effect appears to be PXR specific, as the activation of constitutive androstane receptor in transgenic mice had little effect. We propose that PXR is a potential endocrine disrupting factor that may have broad implications in steroid homeostasis and drug-hormone interactions.     

Relationships between serpentine soils and vegetation in a xeric inner-Alpine environment

Aims

In serpentinitic areas non-endemic plants suffer from the serpentine syndrome, due to high Ni and Mg concentrations, low nutrients and Ca/Mg ratio. We evaluated the environment-soil-vegetation relationships in a xeric inner-alpine area (NW Italy), where the inhibited pedogenesis should enhance parent material influences on vegetation.

Methods

Site conditions, topsoil properties, plant associations and species on and off serpentinite were statistically associated (51 sites).

Results

Serpentine soils had higher Mg and Ni concentrations, but did not differ from non-serpentine ones in nutrient contents. The 15 vegetation clusters often showed substrate specificity. Two components of the Canonical Analysis of Principal Coordinates, respectively related to Mg and to Ni and heat load, identified serpentine vegetation. Random Forests showed that several species were positively correlated with Ni and/or Ca/Mg or Mg, some were negatively associated with high Ni, Mg excess affected only few species. Considering only serpentine sites, nutrients and microclimate were most important.

Conclusions

Ni excess most often precludes the presence of plant species on serpentinite, while an exclusion due to Mg is rarer. Endemic species are mostly adapted to both factors. Nutrient scarcity was not specific of serpentine soils in the considered environment. Considering only serpentine sites, nutrient and microclimatic gradients drove vegetation variability.     

Water deficit induces chlorophyll degradation via the ‘PAO/phyllobilin’ pathway in leaves of homoio‐ (Craterostigma pumilum) and poikilochlorophyllous (Xerophyta viscosa) resurrection plants

Angiosperm resurrection plants exhibit poikilo‐ or homoiochlorophylly as a response to water deficit. Both strategies are generally considered as effective mechanisms to reduce oxidative stress associated with photosynthetic activity under water deficiency. The mechanism of water deficit‐induced chlorophyll (Chl) degradation in resurrection plants is unknown but has previously been suggested to occur as a result of non‐enzymatic photooxidation. We investigated Chl degradation during dehydration in both poikilochlorophyllous (Xerophyta viscosa) and homoiochlorophyllous (Craterostigma pumilum) species. We demonstrate an increase in the abundance of PHEOPHORBIDE a OXYGENASE (PAO), a key enzyme of Chl breakdown, together with an accumulation of phyllobilins, that is, products of PAO‐dependent Chl breakdown, in both species. Phyllobilins and PAO levels diminished again in leaves from rehydrated plants. We conclude that water deficit‐induced poikilochlorophylly occurs via the well‐characterized PAO/phyllobilin pathway of Chl breakdown and that this mechanism also appears conserved in a resurrection species displaying homoiochlorophylly. The roles of the PAO/phyllobilin pathway during different plant developmental processes that involve Chl breakdown, such as leaf senescence and desiccation, fruit ripening and seed maturation, are discussed.     

The effect of short-term flooding on the sap flow, gas exchange and hydraulic conductivity of young apricot trees

The effect of short-term flooding was examined in 2-year-old apricot trees (Prunus armeniaca cv. Búlida). Six apricot trees of similar appearance were submitted to two treatments: three were irrigated daily, while the others were flooded for a period of 50 h by submerging the pots in plastic water tanks. The trees were removed from the water, drained and then placed in the same conditions as the control plants. A decrease in transpiration in the flooded trees with respect to the control plants was evident. The daily pattern of soil O₂ concentration and plant hydraulic resistance followed a similar trend during the flooding. However, this relationship was not maintained throughout the experiment, since the O₂ values increased rapidly when the waterlogging ceased, while plant hydraulic resistance only recovered at the end of the experiment when the original root system, damaged by flooded conditions, was replaced with new roots. In flooded trees, the midday leaf water potential decreased progressively from the beginning of flooding, but gradually recovered when the waterlogging ceased. Leaf conductance values of treated plants were slow to recover, reaching values of the control plants 8 days after the leaf water potential had recovered. The close relationship observed during most of the experiment between the leaf water parameters, leaf conductance and plant hydraulic conductance indicate that hydraulic messages are likely to play a dominant role in co-ordinating the observed responses of the shoot.     

Conformational transitions of the Spindly adaptor underlie its interaction with Dynein and Dynactin

Cytoplasmic Dynein 1, or Dynein, is a microtubule minus end–directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein–Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein–Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein–Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. Thus, our work illustrates how specific Dynein activation at a defined cellular locale may require multiple factors.     

Can network metrics predict vulnerability and species roles in bird‐dispersed plant communities? Not without behaviour

Network metrics are widely used to infer the roles of mutualistic animals in plant communities and to predict the effect of species' loss. However, their empirical validation is scarce. Here we parameterized a joint species model of frugivory and seed dispersal with bird movement and foraging data from tropical and temperate communities. With this model, we investigate the effect of frugivore loss on seed rain, and compare our predictions to those of standard coextinction models and network metrics. Topological coextinction models underestimated species loss after the removal of highly linked frugivores with unique foraging behaviours. Network metrics informed about changes in seed rain quantity after frugivore loss. However, changes in seed rain composition were only predicted by partner diversity. Nestedness, closeness, and d’ specialisation could not anticipate the effects of rearrangements in plant–frugivore communities following species loss. Accounting for behavioural differences among mutualists is critical to improve predictions from network models.     

Volatile components of grape pomaces from different cultivars of Sicilian Vitis vinifera L

The volatile components of grape pomace coming from the processing of some of the most important varieties of grape (Vitis vinifera L.) cultivated in Sicily, namely Nero d'Avola, Nerello Mascalese, Frappato and Cabernet Sauvignon, have been determined by gas-chromatography (GC) and gas-chromatography-mass spectrometry (GC-MS). According to the winemaking procedure that entails the removal of stalks before fermentation, two kinds of grape pomace are obtained. The first consists of skins, pulp residues and seeds, the proper grape pomace, which is partially used for grappa, a typical Italian spirit, and alcohol production, the second consists almost exclusively of stalks. On the whole, 38 components have been characterized in the samples of grape pomaces, with Frappato cv. showing the richest composition; instead, 88 components have been detected in the stalks of Frappato, Nero d'Avola, Nerello Mascalese and Cabernet Sauvignon varieties. In order to make a comparison between the grape varieties easier, the volatile components detected in the two sets of samples (grape pomaces and stalks) have been grouped in different classes. Significant differences among varieties have been detected and statistical treatment of data is also reported. This study is part of a wider project aimed at the possible exploitation of the main agro-industrial by-products. At the same time it is one of the first reports on the volatile components of this waste material.     

Kinetics of Na(+) transport in necturus proximal tubule

The dependence of proximal tubular sodium and fluid readsorption on the Na(+) concentration of the luminal and peritubular fluid was studied in the perfused necturus kidney. Fluid droplets, separated by oil from the tubular contents and identical in composition to the vascular perfusate, were introduced into proximal tubules, reaspirated, and analyzed for Na(+) and [(14)C]mannitol. In addition, fluid transport was measured in short-circuited fluid samples by observing the rate of change in length of the split droplets in the tubular lumen. Both reabsorptive fluid and calculated Na fluxes were simple, storable functions of the perfusate Na(+) concentration (K(m) = 35-39 mM/liter, V(max) = 1.37 control value). Intracellular Na(+), determined by tissue analysis, and open-circuit transepithelial electrical potential differences were also saturable functions of extracellular Na(+). In contrast, net reabsorptive fluid and Na(+) fluxes were linearly dependent on intracellular Na(+) and showed no saturation, even at sharply elevated cellular sodium concentrations. These concentrations were achieved by addition of amphotericin B to the luminal perfusate, a maneuver which increased the rate of Na(+) entry into the tubule cells and caused a proportionate rise in net Na(+) flux. It is concluded that active peritubular sodium transport in proximal tubule cells of necturus is normally unsaturated and remains so even after amphotericin-induced enhancement of luminal Na(+) entry. Transepithelial movement of NaCl may be described by a model with a saturable luminal entry step of Na(+) or NaCl into the cell and a second, unsaturated active transport step of Na(+) across the peritubular cell boundary.