A Highlights from MBoC Selection: Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell''s height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule–mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.     

Thermal performance of the Chagas disease vector,Triatoma infestans,under thermal variability

Vector-borne diseases (VBD) are particularly susceptible to climate change because most of the diseases’ vectors are ectotherms, which themselves are susceptible to thermal changes. The Chagas disease is one neglected tropical disease caused by the protozoan parasite, Trypanosoma cruzi. One of the main vectors of the Chagas disease in South America is Triatoma infestans, a species traditionally considered to be restricted to domestic or peridomestic habitats, but sylvatic foci have also been described along its distribution. The infestation of wild individuals, together with the projections of environmental changes due to global warming, urge the need to understand the relationship between temperature and the vector’s performance. Here, we evaluated the impact of temperature variability on the thermal response of T. infestans. We acclimated individuals to six thermal treatments for five weeks to then estimate their thermal performance curves (TPCs) by measuring the walking speed of the individuals. We found that the TPCs varied with thermal acclimation and body mass. Individuals acclimated to a low and variable ambient temperature (18°C ± 5°C) exhibited lower performances than those individuals acclimated to an optimal temperature (27°C ± 0°C); while those individuals acclimated to a low but constant temperature (18°C ± 0°C) did not differ in their maximal performance from those at an optimal temperature. Additionally, thermal variability (i.e., ± 5°C) at a high temperature (30°C) increased performance. These results evidenced the plastic response of T. infestans to thermal acclimation. This plastic response and the non-linear effect of thermal variability on the performance of T. infestans posit challenges when predicting changes in the vector’s distribution range under climate change.     

Protein disulfide isomerase in redox cell signaling and homeostasis

Thiol proteins may potentially act as redox signaling adaptor proteins, adjusting reactive oxygen species intermediates to specific signals and redox signals to cell homeostasis. In this review, we discuss redox effects of protein disulfide isomerase (PDI), a thioredoxin superfamily oxidoreductase from the endoplasmic reticulum (ER). Abundantly expressed PDI displays ubiquity, interactions with redox and nonredox proteins, versatile effects, and several posttranslational modifications. The PDI family contains >20 members with at least some apparent complementary actions. PDI has oxidoreductase, isomerase, and chaperone effects, the last not directly dependent on its thiols. PDI is a converging hub for pathways of disulfide bond introduction into ER-processed proteins, via hydrogen peroxide-generating mechanisms involving the oxidase Ero1α, as well as hydrogen peroxide-consuming reactions involving peroxiredoxin IV and the novel peroxidases Gpx7/8. PDI is a candidate pathway for coupling ER stress to oxidant generation. Emerging information suggests a convergence between PDI and Nox family NADPH oxidases. PDI silencing prevents Nox responses to angiotensin II and inhibits Akt phosphorylation in vascular cells and parasite phagocytosis in macrophages. PDI overexpression spontaneously enhances Nox activation and expression. In neutrophils, PDI redox-dependently associates with p47phox and supports the respiratory burst. At the cell surface, PDI exerts transnitrosation, thiol reductase, and apparent isomerase activities toward targets including adhesion and matrix proteins and proteases. Such effects mediate redox-dependent adhesion, coagulation/thrombosis, immune functions, and virus internalization. The route of PDI externalization remains elusive. Such multiple redox effects of PDI may contribute to its conspicuous expression and functional role in disease, rendering PDI family members putative redox cell signaling adaptors.     

Physiological and molecular mechanisms underlying the integration of light and temperature cues in Arabidopsis thaliana seeds

The relief of dormancy and the promotion of seed germination are of extreme importance for a successful seedling establishment. Although alternating temperatures and light are signals promoting the relief of seed dormancy, the underlying mechanisms of their interaction in seeds are scarcely known. By exposing imbibed Arabidopsis thaliana dormant seeds to two‐day temperature cycles previous of a red light pulse, we demonstrate that the germination mediated by phytochrome B requires the presence of functional PSEUDO‐RESPONSE REGULATOR 7 (PRR7) and TIMING OF CAB EXPRESSION 1 (TOC1) alleles. In addition, daily cycles of alternating temperatures in darkness reduce the protein levels of DELAY OF GERMINATION 1 (DOG1), allowing the expression of TOC1 to induce seed germination. Our results suggest a functional role for some components of the circadian clock related with the action of DOG1 for the integration of alternating temperatures and light signals in the relief of seed dormancy. The synchronization of germination by the synergic action of light and temperature through the activity of circadian clock might have ecological and adaptive consequences.     

Exploring the Structure-Function Loop Adaptability of a (β/α)(8)-Barrel Enzyme through Loop Swapping and Hinge Variability

Cellulosomes are large extracellular multi-enzyme complexes that exhibit elevated activity on plant cell-wall polysaccharides. In the present study, the relationships between the conformational flexibility and efficacy of cellulosomes, and the inter-modules linkers of their scaffold protein were investigated. For this purpose, the length of the intrinsically disordered Ser/Thr-rich 50-residue linker connecting a Clostridium thermocellum and a Clostridium cellulolyticum cohesin in a hybrid scaffoldin (Scaf4) was changed by sequences ranging from 4 to 128 residues. The composition was also modified and new linkers composed of series of N, S or repeats of the EPPV motif were generated. Two model cellulases (Cel48F and Cel9G) appended with appropriate dockerins were subsequently bound to the engineered scaffoldins. All the resulting minicomplexes displayed the same activity on crystalline cellulose as the complex based on the initial Scaf4, and were found to be 2-fold more active than Cel48F and Cel9G bound to separate cohesins. Small-angle X-ray scattering assays of the engineered scaffoldins confirmed, however, that the size and the conformational flexibility of some of the new inter-cohesins linkers differed significantly from that of the initial 50 residue linker displayed by the parental Scaf4. Our data suggest that the synergy induced by proximity does not require a specific inter-cohesins sequence or distance. The present study reveals that complexation onto the hybrid scaffoldins modifies the type of soluble sugars released from crystalline cellulose by the selected cellulases, compared to the free enzyme system.     

Reproductive biology and mating system estimates of two Andean melocacti, Melocactus schatzlii and M. andinus (Cactaceae)

BACKGROUND AND AIMS: The genus Melocactus comprises 36 species of globose cacti with the most derived traits in the Cereeae tribe. It is the proper study system to examine what are the most derived reproductive strategies within that tribe. This study aims to characterize the reproductive biology and to estimate the mating system parameters of two Andean melocacti, Melocactus schatzlii and M. andinus. METHODS: The reproductive attributes of the two species were described, including floral morphology, anthesis patterns, floral rewards, floral visitors and visitation patterns. Levels of self-compatibility and autonomous self-pollination were estimated by hand-pollination experiments. Mating system estimates were obtained by conducting progeny array analyses using isozymes. KEY RESULTS: The flowers of the two species present the typical hummingbird-pollination syndrome. Despite their morphological resemblance, the two species differ in flower size, pollen and ovule production and anthesis pattern. Their main pollinator agents are hummingbirds, four species in M. schatzlii and one species in M. andinus. Both cacti are self-compatible and capable of self-pollination without the aid of pollen vectors. Population-level outcrossing rate was higher for M. schatzlii (t(m)=0.9) than for M. andinus (t(m)=0.4). At the family level, outcrossing rates for most mothers of M. schatzlii were higher (t(m)>0.8) than for M. andinus (t(m)<0.5). CONCLUSIONS: Although the two cacti are capable of selfing, M. schatzlii is a predominantly outcrossing species, while M. andinus behaves as a mixed-mating cactus. Hummingbirds are the only pollinators responsible for outcrossing and gene flow events in these species. In their absence, both melocacti set seeds by selfing. Based on its low population size, restricted distribution in Venezuela, low rates of floral visits, and high levels of inbreeding, M. andinus is considered to be an endangered species deserving further study to define its conservation status.     

Hypothyroidism provides resistance to kidney mitochondria against the injury induced by renal ischemia-reperfusion

Massive Ca(2+) accumulation in mitochondria, plus the stimulating effect of an inducing agent, i.e., oxidative stress, induces the so-called permeability transition, which is characterized by the opening of a nonspecific pore. This work was aimed at studying the influence of thyroid hormone on the opening of such a nonspecific pore in kidney mitochondria, as induced by an oxidative stress. To meet this objective, membrane permeability transition was examined in mitochondria isolated from kidney of euthyroid and hypothyroid rats, after a period of ischemia/reperfusion. It was found that mitochondria from hypothyroid rats were able to retain accumulated Ca(2+) to sustain a transmembrane potential after Ca(2+) addition, as well as to maintain matrix NAD(+) and membrane cytochrome c content. The protective effect of hypothyroidism was clearly opposed to that occurring in ischemic reperfused mitochondria from euthyroid rats. Our findings demonstrate that these mitochondria were unable to preserve selective membrane permeability, except when cyclosporin A was added. It is proposed that the protection is conferred by the low content of cardiolipin found in the inner membrane. This phospholipid is required to switch adenine nucleotide translocase from specific carrier to a non-specific pore.     

Synthesis and potent antitumor activity of new arylamino derivatives of nor-beta-lapachone and nor-alpha-lapachone

Several arylamino derivatives of nor-beta-lapachone were synthesized in moderate to high yields and found to show very potent cytotoxicity against six neoplastic cancer cells: SF-295 (central nervous system), HCT-8 (colon), MDAMB-435 (breast), HL-60 (leukaemia), PC-3 (prostate), and B-16 (murine melanoma), with IC(50) below 1 microg/mL. Their cytotoxicities were compared to doxorubicin and with their synthetic precursors, beta-lapachone and nor-beta-lapachone. The activity against a normal murine fibroblast L-929 showed that some of the compounds were selective against cancer cells. The absence of hemolytic activity (EC(50)>200 microg/mL), performed with erythrocyte suspensions, suggests that the cytotoxicity of the compounds was not related to membrane damage of mouse erythrocytes. For comparison purposes, one isomeric compound based on nor-alpha-lapachone was also synthesized and showed lower activity than the related ortho-derivative. The modified arylamino quinones appear as interesting new lead compounds in anti-cancer drug development.