CONVERGENT AND CORRELATED EVOLUTION OF MAJOR LIFE‐HISTORY TRAITS IN THE ANGIOSPERM GENUS LEUCADENDRON (PROTEACEAE)

Natural selection is expected to cause convergence of life histories among taxa as well as correlated evolution of different life‐history traits. Here, we quantify the extent of convergence of five key life‐history traits (adult fire survival, seed storage, degree of sexual dimorphism, pollination mode, and seed‐dispersal mode) and test hypotheses about their correlated evolution in the genus Leucadendron (Proteaceae) from the fire‐prone South African fynbos. We reconstructed a new molecular phylogeny of this highly diverse genus that involves more taxa and molecular markers than previously. This reconstruction identifies new clades that were not detected by previous molecular study and morphological classifications. Using this new phylogeny and robust methods that account for phylogenetic uncertainty, we show that the five life‐history traits studied were labile during the evolutionary history of the genus. This diversity allowed us to tackle major questions about the correlated evolution of life‐history strategies. We found that species with longer seed‐dispersal distances tended to evolve lower pollen‐dispersal distance, that insect‐pollinated species evolved decreased sexual dimorphism, and that species with a persistent soil seed‐bank evolved toward reduced fire‐survival ability of adults.     

Gigantism among Late Jurassic limulids: New ichnological evidence from the Causses Basin (Lozère,France) and comments on body-size evolution among horseshoe crabs

An abundant ichnological material composed of xiphosuran trackways and isolated traces was discovered in Upper Jurassic limestones from the Causses Basin (Causse Méjean, Lozère, France). The morphology of the imprints supports their identification as Kouphichnium isp. In contrast to the most frequent case, the trackways are composed of omnipresent pusher imprints sometime associated with leg traces, but with no telson mark. We argue that this pattern reflects actual surface traces rather than an incomplete set of undertracks. The size distribution of the sampled ichnites is broadly bimodal. This is best explained by sexual dimorphism, a phenomenon frequently observed in modern xiphosurans. Analysis of the trace fossils further suggests that several growth stages are recorded and that the horseshoe crabs were walking in a protected and flat environment like a lagoon. This area, certainly close to a mating ground, was occasionally affected by a continental influence. The biometric study of the tracks suggests a gigantic size for the trackmakers whose body may have reached 84 cm in length. This discovery complements the few reports on other gigantic horseshoe crabs in the Jurassic of Western Europe, thus casting doubt on the postulated increase in body size from the Palaeozoic to the Recent. Furthermore, a literature review shows that there are still major gaps in the record of limulid body-fossils and tracks. Thus, neither of these archives can be taken at face value for quantifying the body-size evolution of horseshoe crabs.     

Fast Label-Free Cytoskeletal Network Imaging in Living Mammalian Cells

We present a full-field technique that allows label-free cytoskeletal network imaging inside living cells. This noninvasive technique allows monitoring of the cytoskeleton dynamics as well as interactions between the latter and organelles on any timescale. It is based on high-resolution quantitative phase imaging (modified Quadriwave lateral shearing interferometry) and can be directly implemented using any optical microscope without modification. We demonstrate the capability of our setup on fixed and living Chinese hamster ovary cells, showing the cytoskeleton dynamics in lamellipodia during protrusion and mitochondria displacement along the cytoskeletal network. In addition, using the quantitative function of the technique, along with simulation tools, we determined the refractive index of a single tubulin microtubule to be n_tubu = 2.36 ± 0.6 at λ = 527 nm.The cytoskeleton is mainly composed of an actin and tubulin microtubule network, and it has many important roles at the cellular scale (1). It allows the cell to modify its shape, is implied in cell migration and adhesion processes, and is used as a support for organelle displacement inside cells.Optical microscopy is useful for dynamic studies in which the cytoskeletal network is reorganizing quickly. However, due to the poor native interaction between light and this network fluorescence labeling is commonly used to image the cytoskeleton (2). Anisotropic approaches are also used on this kind of structure, as the cytoskeletal filaments may present refractive index anisotropy. Based on this property, polarized microscopy has been used to reveal the cytoskeleton (3). However, this technique is relatively slow compared to the cytoskeleton dynamics and requires perfectly stressless optics and a nondepolarizing sample. Differential interference contrast (DIC) approaches enhance the contrast in unstained cytoskeletal fibers (4) but also require precise light polarization control of both samples and optical components (for example, no plastic elements can be used for standard DIC). Moreover, the image has a gradient shape that induces loss of resolution and makes the images hard to interpret, especially in complex biological environments. Some DIC-based developments have been proposed that would make it possible to retrieve quantitative information from the sample and/or minimize the effects of depolarizing elements (5–8). Nonlinear interactions in second-harmonic generation (SHG) (9) that are sensitive to orientation and anisotropic refractive index are also applied to cytoskeleton imaging. Label-free imaging is thus obtained, but it requires a powerful laser and a scanning approach that may be too slow when fast dynamics need to be studied.Although light interaction with a nonlabeled cytoskeleton is weak, with barely any absorption, there is a signature on the beam that travels through the structure even with nonpolarized illumination/detection. Indeed, as tubulin microtubules and actin filaments are denser than the cytoplasm, their respective refractive indices are also higher (10). This means that the light is slightly delayed by the cytoskeleton, leading to a possible contrast when looking at the phase component of light. In this article, we consider quantitative phase microscopy (QPM) based on quadriwave lateral shearing interferometry (QWLSI) (11). QWLSI makes it possible to image nonlabeled cells with a conventional transillumination microscope equipped with a halogen lamp. We propose a modified version of the QWLSI presented in our previous publication (11) that allows the fast, sensitive, and highly resolved imaging required to reveal cytoskeletal network dynamics in living cells. After discussing the signal/noise ratio (SNR) of our approach, we compare QPM with immunostaining of actin and tubulin microtubules on Chinese hamster ovary (CHO) cells, demonstrating the capability of QPM to visualize the cytoskeleton. Living wild-type (wt) CHO cells are then imaged at a high frame rate (2.5 Hz) to illustrate the spatiotemporal resolution of the technique for cytoskeleton imaging.     

Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

The septal association of Mycobacterium tuberculosis MtrB, the kinase partner of the MtrAB two-component signal transduction system, is necessary for the optimal expression of the MtrA regulon targets, including ripA, fbpB, and ftsI, which are involved in cell division and cell wall synthesis. Here, we show that MtrB, irrespective of its phosphorylation status, interacts with Wag31, whereas only phosphorylation-competent MtrB interacts with FtsI. We provide evidence that FtsI depletion compromises the MtrB septal assembly and MtrA regulon expression; likewise, the absence of MtrB compromises FtsI localization and, possibly, FtsI activity. We conclude from these results that FtsI and MtrB are codependent for their activities and that FtsI functions as a positive modulator of MtrB activation and MtrA regulon expression. In contrast to FtsI, Wag31 depletion does not affect MtrB septal assembly and MtrA regulon expression, whereas the loss of MtrB increased Wag31 localization and the levels of PknA/PknB (PknA/B) serine-threonine protein kinase-mediated Wag31 phosphorylation. Interestingly, we found that FtsI decreased levels of phosphorylated Wag31 (Wag31∼P) and that MtrB interacted with PknA/B. Overall, our results indicate that MtrB interactions with FtsI, Wag31, and PknA/B are required for its optimal localization, MtrA regulon expression, and phosphorylation of Wag31. Our results emphasize a new role for MtrB in cell division and cell wall synthesis distinct from that regulating the MtrA phosphorylation activities.     

Exenatide does not evoke pancreatitis and attenuates chemically induced pancreatitis in normal and diabetic rodents

The risk of developing pancreatitis is elevated in type 2 diabetes and obesity. Cases of pancreatitis have been reported in type 2 diabetes patients treated with GLP-1 (GLP-1R) receptor agonists. To examine whether the GLP-1R agonist exenatide potentially induces or modulates pancreatitis, the effect of exenatide was evaluated in normal or diabetic rodents. Normal and diabetic rats received a single exenatide dose (0.072, 0.24, and 0.72 nmol/kg) or vehicle. Diabetic ob/ob or HF-STZ mice were infused with exenatide (1.2 and 7.2 nmol·kg(-1)·day(-1)) or vehicle for 4 wk. Post-exenatide treatment, pancreatitis was induced with caerulein (CRN) or sodium taurocholate (ST), and changes in plasma amylase and lipase were measured. In ob/ob mice, plasma cytokines (IL-1β, IL-2, IL-6, MCP-1, IFNγ, and TNFα) and pancreatitis-associated genes were assessed. Pancreata were weighed and examined histologically. Exenatide treatment alone did not modify plasma amylase or lipase in any models tested. Exenatide attenuated CRN-induced release of amylase and lipase in normal rats and ob/ob mice but did not modify the response to ST infusion. Plasma cytokines and pancreatic weight were unaffected by exenatide. Exenatide upregulated Reg3b but not Il6, Ccl2, Nfkb1, or Vamp8 expression. Histological analysis revealed that the highest doses of exenatide decreased CRN- or ST-induced acute inflammation, vacuolation, and acinar single cell necrosis in mice and rats, respectively. Ductal cell proliferation rates were low and similar across all groups of ob/ob mice. In conclusion, exenatide did not modify plasma amylase and lipase concentrations in rodents without pancreatitis and improved chemically induced pancreatitis in normal and diabetic rodents.     

Control of Cell Survival and Proliferation by Mammalian Eukaryotic Initiation Factor 4B

Translation initiation plays an important role in cell growth, proliferation, and survival. The translation initiation factor eIF4B (eukaryotic initiation factor 4B) stimulates the RNA helicase activity of eIF4A in unwinding secondary structures in the 5′ untranslated region (5′UTR) of the mRNA in vitro. Here, we studied the effects of eIF4B depletion in cells using RNA interference (RNAi). In agreement with the role of eIF4B in translation initiation, its depletion resulted in inhibition of this step. Selective reduction of translation was observed for mRNAs harboring strong to moderate secondary structures in their 5′UTRs. These mRNAs encode proteins, which function in cell proliferation (Cdc25C, c-myc, and ODC [ornithine decarboxylase]) and survival (Bcl-2 and XIAP [X-linked inhibitor of apoptosis]). Furthermore, eIF4B silencing led to decreased proliferation rates, promoted caspase-dependent apoptosis, and further sensitized cells to camptothecin-induced cell death. These results demonstrate that eIF4B is required for cell proliferation and survival by regulating the translation of proliferative and prosurvival mRNAs.Targeting the translation initiation pathway is emerging as a potential therapy for inhibiting cancer cell growth (35, 38). Ribosome recruitment to the 5′ ends of eukaryotic mRNAs proceeds via translation initiation mechanisms that are dependent either on the 5′ cap structure (m⁷GpppN, where N is any nucleotide) or an internal ribosome entry site (IRES). The majority of translation initiation events in eukaryotes are mediated through cap-dependent translation whereby the 40S ribosomal subunit is recruited to the vicinity of the mRNA 5′ cap structure by the eukaryotic initiation factor 4F (eIF4F) complex. eIF4F is comprised of eIF4E (the cap-binding subunit), eIF4A (an RNA helicase), and eIF4G (a large scaffolding protein for eIF4E, eIF4A, and other initiation factors). Once assembled at the 5′ cap, the 40S ribosomal subunit in association with several initiation factors scans the 5′ untranslated region (5′UTR) of the mRNA until it encounters a start codon in a favorable context, followed by polypeptide synthesis (37).Early in vitro studies have shown that the initiation factor eIF4B acts to potentiate ribosome recruitment to the mRNA (3, 45). eIF4B stimulates translation of both capped and uncapped mRNAs in vitro (1, 36). This function is exerted through stimulation of the helicase activity of eIF4A (43), possibly through direct interactions with eIF4A (44) or with mRNA, the ribosome-associated eIF3, and 18S rRNA (28, 29, 44). Thus, eIF4B is thought to form auxiliary bridges between the mRNA and the 40S ribosomal subunit. Toeprinting studies using mammalian eIF4B underscored its importance in the assembly of the 48S initiation complex, especially on mRNAs harboring secondary structures in the 5′UTRs (11).In vivo studies of eIF4B are limited. Ectopic expression of eIF4B in cultured Drosophila melanogaster cells and in developing eye imaginal discs stimulated cell proliferation (16). Enhanced cell proliferation is most likely mediated by increased translation of a subset of mRNAs, since knockdown of Drosophila eIF4B by RNA interference (RNAi) caused a modest reduction in global translation but compromised the survival of insect cells grown under low serum conditions (16). Studies of eIF4B in mammalian cells yielded contradictory results. Transient overexpression of eIF4B stimulated translation initiation in a phosphorylation-dependent manner in some cells (18, 49) while inhibiting translation in others (30, 31, 41). These differences might be attributed to disparate levels of eIF4B overexpression.To address the physiological role of eIF4B in mRNA translation in the cell, RNAi knockdown of eIF4B was used here. We demonstrate that eIF4B is required for optimal translation. Importantly, the translation of mRNAs bearing structured 5′UTRs, such as the cell cycle regulators Cdc25C, c-myc, and ODC (ornithine decarboxylase), and the antiapoptotic factors Bcl-2 and XIAP (X-linked inhibitor of apoptosis), was reduced as a result of eIF4B silencing by RNAi. Furthermore, eIF4B silencing promoted caspase-dependent apoptosis. Thus, we show that mammalian eIF4B is required for cell proliferation and survival, whereby it acts by regulating the translation of a functionally related subset of mRNAs.     

Genome-wide association analyses of common wheat (Triticum aestivum L.) germplasm identifies multiple loci for aluminium resistance

Aluminium (Al3+) toxicity restricts productivity and profitability of wheat (Triticum aestivum L.) crops grown on acid soils worldwide. Continued gains will be obtained by identifying superior alleles and novel Al3+ resistance loci that can be incorporated into breeding programs. We used association mapping to identify genomic regions associated with Al3+ resistance using 1055 accessions of common wheat from different geographic regions of the world and 178 polymorphic diversity arrays technology (DArT) markers. Bayesian analyses based on genetic distance matrices classified these accessions into 12 subgroups. Genome-wide association analyses detected markers that were significantly associated with Al3+ resistance on chromosomes 1A, 1B, 2A, 2B, 2D, 3A, 3B, 4A, 4B, 4D, 5B, 6A, 6B, 7A, and 7B. Some of these genomic regions correspond to previously identified loci for Al3+ resistance, whereas others appear to be novel. Among the markers targeting TaALMT1 (the major Al3+-resistance gene located on chromosome 4D), those that detected alleles in the promoter explained most of the phenotypic variance for Al3+ resistance, which is consistent with this region controlling the level of TaALMT1 expression. These results demonstrate that genome-wide association mapping cannot only confirm known Al3+-resistance loci, such as those on chromosomes 4D and 4B, but they also highlight the utility of this technique in identifying novel resistance loci.     

Evidence that yeast frataxin is not an iron storage protein in vivo

Yeast cells deficient in the yeast frataxin homolog (Yfh1p) accumulate iron in their mitochondria. Whether this iron is toxic, however, remains unclear. We showed that large excesses of iron in the growth medium did not inhibit growth and did not decrease cell viability. Increasing the ratio of mitochondrial iron-to-Yfh1p by decreasing the steady-state level of Yfh1p to less than 100 molecules per cell had very few deleterious effects on cell physiology, even though the mitochondrial iron concentration greatly exceeded the iron-binding capacity of Yfh1p in these conditions. Mössbauer spectroscopy and FPLC analyses of whole mitochondria or of isolated mitochondrial matrices showed that the chemical and biochemical forms of the accumulated iron in mitochondria of mutant yeast strains (Δyfh1, Δggc1 and Δssq1) displayed a nearly identical distribution. This was also the case for Δggc1 cells, in which Yfh1p was overproduced. In these mitochondria, most of the iron was insoluble, and the ratio of soluble-to-insoluble iron did not change when the amount of Yfh1p was increased up to 4500 molecules per cell. Our results do not privilege the hypothesis of Yfh1p being an iron storage protein in vivo.     

Mitochondria in Huntington's disease

Huntington's disease (HD) is an inherited progressive neurodegenerative disorder associated with involuntary abnormal movements (chorea), cognitive deficits and psychiatric disturbances. The disease is caused by an abnormal expansion of a CAG repeat located in exon 1 of the gene encoding the huntingtin protein (Htt) that confers a toxic function to the protein. The most striking neuropathological change in HD is the preferential loss of medium spiny GABAergic neurons in the striatum. The mechanisms underlying striatal vulnerability in HD are unknown, but compelling evidence suggests that mitochondrial defects may play a central role. Here we review recent findings supporting this hypothesis. Studies investigating the toxic effects of mutant Htt in cell culture or animal models reveal mitochondrial changes including reduction of Ca²⁺ buffering capacity, loss of membrane potential, and decreased expression of oxidative phosphorylation (OXPHOS) enzymes. Striatal neurons may be particularly vulnerable to these defects. One hypothesis is that neurotransmission systems such as dopamine and glutamate exacerbate mitochondrial defects in the striatum. In particular, mitochondrial dysfunction facilitates impaired Ca²⁺ homeostasis linked to the glutamate receptor-mediated excitotoxicity. Also dopamine receptors modulate mutant Htt toxicity, at least in part through regulation of the expression of mitochondrial complex II. All these observations support the hypothesis that mitochondria, acting as “sensors” of the neurochemical environment, play a central role in striatal degeneration in HD.     

Endoplasmic reticulum is a major target of cadmium toxicity in yeast

Cadmium (Cd²⁺) is a very toxic metal that causes DNA damage, oxidative stress and apoptosis. Despite many studies, the cellular and molecular mechanisms underlying its high toxicity are not clearly understood. We show here that very low doses of Cd²⁺ cause ER stress in Saccharomyces cerevisiae as evidenced by the induction of the unfolded protein response (UPR) and the splicing of HAC1 mRNA. Furthermore, mutant strains (Δire1 and Δhac1) unable to induce the UPR are hypersensitive to Cd²⁺, but not to arsenite and mercury. The full functionality of the pathways involved in ER stress response is required for Cd²⁺ tolerance. The data also suggest that Cd²⁺‐induced ER stress and Cd²⁺ toxicity are a direct consequence of Cd²⁺ accumulation in the ER. Cd²⁺ does not inhibit disulfide bond formation but perturbs calcium metabolism. In particular, Cd²⁺ activates the calcium channel Cch1/Mid1, which also contributes to Cd²⁺ entry into the cell. The results reinforce the interest of using yeast as a cellular model to study toxicity mechanisms in eukaryotic cells.