Inhibitor-3 ensures bipolar mitotic spindle attachment by limiting association of SDS22 with kinetochore-bound protein phosphatase-1

Faithful chromosome segregation during mitosis is tightly regulated by opposing activities of Aurora B kinase and protein phosphatase-1 (PP1). PP1 function at kinetochores has been linked to SDS22, but the exact localization of SDS22 and how it affects PP1 are controversial. Here, we confirm that SDS22 is required for PP1 activity, but show that SDS22 does not normally localize to kinetochores. Instead, SDS22 is kept in solution by formation of a ternary complex with PP1 and inhibitor-3 (I3). Depletion of I3 does not affect the amount of PP1 at kinetochores but causes quantitative association of SDS22 with PP1 on KNL1 at the kinetochore. Such accumulation of SDS22 at kinetochores interferes with PP1 activity and inhibits Aurora B threonine-232 dephosphorylation, which leads to increased Aurora B activity in metaphase and persistence in anaphase accompanied with segregation defects. We propose a model in which I3 regulates an SDS22-mediated PP1 activation step in solution that precedes SDS22 dissociation and transfer of PP1 to kinetochores, and which is required for PP1 to efficiently antagonize Aurora B.     

Differential Protein Phosphorylation Regulates Chloroplast Movement in Response to Strong Light and Darkness in Arabidopsis thaliana

Phototropin-dependent chloroplast movement is essential to the photosynthetic acclimation of mesophyll cells to incident light. Chloroplast movement involves many cellular actors, such as chloroplast-associated actin filaments and proteins that mediate signalling between phototropins and chloroplast motion. In the past few years, genetic approaches have identified several key proteins but the intrinsic mechanisms of the signalling cascade, such as phosphorylation events, remain undefined. Here, we took advantage of phosphoproteomics to examine the involvement of protein phosphorylation in chloroplast movement in darkness or under high light, at different CO₂ mole fractions (100, 380 or 1,000 ppm) to vary photosynthetic activity. Amongst the 100 relevant identified phosphopeptides, 19 (corresponding to 8 proteins) were differentially phosphorylated in darkness vs. high light. There was no significant CO₂ effect on the observed phosphorylation patterns. We further characterized the phosphorylation sites in THRUMIN1, which is believed to be crucial for the attachment of chloroplast-associated actin filaments to the plasma membrane and thus for chloroplast movements. The mutant thrumin1 was complemented with a mutated protein in which phospho-sites were substituted to a phosphomimetic (Asp) or a non-phosphorylatable (Ala) residue. While the phosphomimetic substitution altered the chloroplast response in the light only, both light and dark responses were altered with the non-phosphorylatable substitution. Our data suggest a key role of protein phosphorylation, including that of THRUMIN1, in the light/dark control of chloroplast movements.     

Stimulation-induced ectopicity and propagation windows in model damaged axons

Neural tissue injuries render voltage-gated Na⁺ channels (Nav) leaky, thereby altering excitability, disrupting propagation and causing neuropathic pain related ectopic activity. In both recombinant systems and native excitable membranes, membrane damage causes the kinetically-coupled activation and inactivation processes of Nav channels to undergo hyperpolarizing shifts. This damage-intensity dependent change, called coupled left-shift (CLS), yields a persistent or “subthreshold” Nav window conductance. Nodes of Ranvier simulations involving various degrees of mild CLS showed that, as the system’s channel/pump fluxes attempt to re-establish ion homeostasis, the CLS elicits hyperexcitability, subthreshold oscillations and neuropathic type action potential (AP) bursts. CLS-induced intermittent propagation failure was studied in simulations of stimulated axons, but pump contributions were ignored, leaving open an important question: does mild-injury (small CLS values, pumps functioning well) render propagation-competent but still quiescent axons vulnerable to further impairments as the system attempts to cope with its normal excitatory inputs? We probe this incipient diffuse axonal injury scenario using a 10-node myelinated axon model. Fully restabilized nodes with mild damage can, we show, become ectopic signal generators (“ectopic nodes”) because incoming APs stress Na⁺/K⁺ gradients, thereby altering spike thresholds. Comparable changes could contribute to acquired sodium channelopathies as diverse as epileptic phenomena and to the neuropathic amplification of normally benign sensory inputs. Input spike patterns, we found, propagate with good fidelity through an ectopically firing site only when their frequencies exceed the ectopic frequency. This “propagation window” is a robust phenomenon, occurring despite Gaussian noise, large jitter and the presence of several consecutive ectopic nodes.     

Interaction of Nck1 and PERK phosphorylated at Y561 negatively modulates PERK activity and PERK regulation of pancreatic β-cell proinsulin content

PERK, the PKR-like endoplasmic reticulum (ER) kinase, is an ER transmembrane serine/threonine protein kinase activated during ER stress. In this study, we provide evidence that the Src-homology domain–containing adaptor Nck1 negatively regulates PERK. We show that Nck directly binds to phosphorylated Y⁵⁶¹ in the PERK juxtamembrane domain through its SH2 domain. We demonstrate that mutation of Y⁵⁶¹ to a nonphosphorylatable residue (Y561F) promotes PERK activity, suggesting that PERK phosphorylation at Y⁵⁶¹ (pY⁵⁶¹PERK) negatively regulates PERK. In agreement, we show that pY⁵⁶¹PERK delays PERK activation and signaling during ER stress. Compatible with a role for PERK in pancreatic β-cells, we provide strong evidence that Nck1 contributes to PERK regulation of pancreatic β-cell proteostasis. In fact, we demonstrated that down-regulation of Nck1 in mouse insulinoma MIN6 cells results in faster dephosphorylation of pY⁵⁶¹PERK, which correlates with enhanced PERK activation, increased insulin biosynthesis, and PERK-dependent increase in proinsulin content. Furthermore, we report that pancreatic islets in whole-body Nck1-knockout mice contain more insulin than control littermates. Together our data strongly suggest that Nck1 negatively regulates PERK by interacting with PERK and protecting PERK from being dephosphorylated at its inhibitory site pY⁵⁶¹ and in this way affects pancreatic β-cell proinsulin biogenesis.     

Tumor microenvironment–associated modifications of alternative splicing

Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.     

Long-term changes in above ground biomass after disturbance in a neotropical dry forest,Hellshire Hills,Jamaica

We used data from experimental plots (control, partially cut and clear-cut) established in 1998, in a tropical dry forest (TDF) in Jamaica, to assess changes in above ground biomass (AGB) 10 years after disturbance. The treatments reduced AGB significantly in 1999 (partially cut: 37.6 %, clear-cut: 94.4 %) and after 10 years, AGB did not recover overall, nor did it recover in the clear-cut plots. Partially cut plots, however, recovered the lost AGB in 10 years via growth of uncut trees, which contributed significantly to biomass recovery, with only minor contributions from recruited trees and coppice shoots. For clear-cut plots, coppice shoots contributed significantly to the recovered AGB when compared with recruited biomass. Together, they recovered 26 % of AGB lost, despite recovering 78 % of the density and height of the control plots. The probability of survivorship decreased for trees with higher pre-treatment AGB values, but was higher for trees with multiple stems in 1998, regardless of treatment. The magnitude of biomass reduction varied among the species assessed and this had a differential effect on their ability to recover AGB. We estimate that it will take approximately 45.4 years for the clear-cut plots to recover pre-treatment AGB; this is significantly longer than AGB recovery time for some successional rainforests on abandoned pastures/farmland. Consequently, this TDF may not be as resilient as tropical rainforests.     

Life‐history syndromes: Integrating dispersal through space and time

Recent research has highlighted interdependencies between dispersal and other life‐history traits, i.e. dispersal syndromes, thereby revealing constraints on the evolution of dispersal and opportunities for improved ability to predict dispersal by considering suites of dispersal‐related traits. This review adds to the growing list of life‐history traits linked to spatial dispersal by emphasising the interdependence between dispersal through space and time, i.e. life‐history diversity that distributes individuals into separate reproductive events. We reviewed the literature that has simultaneously investigated spatial and temporal dispersal to examine the prediction that traits of these two dispersal strategies are negatively correlated. Our results suggest that negative covariation is widely anticipated from theory. Empirical studies often reported evidence of weak negative covariation, although more complicated patterns were also evident, including across levels of biological organisation. Existing literature has largely focused on plants with dormancy capability, one or two phases of the dispersal process (emigration and/or transfer) and a single level of biological organisation (theory: individual; empirical: species). We highlight patterns of covariation across levels of organisation and conclude with a discussion of the consequences of dispersal through space and time and future research areas that should improve our understanding of dispersal‐related life‐history syndromes.     

Omics technologies provide new insights into the molecular physiopathology of equine osteochondrosis

Background

Osteochondrosis (OC(D)) is a juvenile osteo-articular disorder affecting several mammalian species. In horses, OC(D) is considered as a multifactorial disease and has been described as a focal disruption of endochondral ossification leading to the development of osteoarticular lesions. Nevertheless, OC(D) physiopathology is poorly understood. Affected horses may present joint swelling, stiffness and lameness. Thus, OC(D) is a major concern for the equine industry. Our study was designed as an integrative approach using omics technologies for the identification of constitutive defects in epiphyseal cartilage and/or subchondral bone associated with the development of primary lesions to further understand OC(D) pathology. This study compared samples from non-affected joints (hence lesion-free) from OC(D)-affected foals (n = 5, considered predisposed samples) with samples from OC-free foals (n = 5) considered as control samples. Consequently, results are not confounded by changes associated with the evolution of the lesion, but focus on altered constitutive molecular mechanisms. Comparative proteomics and micro computed tomography analyses were performed on predisposed and OC-free bone and cartilage samples. Metabolomics was also performed on synovial fluid from OC-free, OC(D)-affected and predisposed joints.

Results

Two lesion subtypes were identified: OCD (lesion with fragment) and OC (osteochondral defects). Modulated proteins were identified using omics technologies (2-DE proteomics) in cartilage and bone from affected foals compare to OC-free foals. These were associated with cellular processes including cell cycle, energy production, cell signaling and adhesion as well as tissue-specific processes such as chondrocyte maturation, extracellular matrix and mineral metabolism. Of these, five had already been identified in synovial fluid of OC-affected foals: ACTG1 (actin, gamma 1), albumin, haptoglobin, FBG (fibrinogen beta chain) and C4BPA (complement component 4 binding protein, alpha).

Conclusion

This study suggests that OCD lesions may result from a cartilage defect whereas OC lesions may be triggered by both bone and cartilage defects, suggesting that different molecular mechanisms responsible for the equine osteochondrosis lesion subtypes and predisposition could be due to a defect in both bone and cartilage. This study will contribute to refining the definition of OC(D) lesions and may improve diagnosis and development of therapies for horses and other species, including humans.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-947) contains supplementary material, which is available to authorized users.     

Probing druggability and biological function of essential proteins in Leishmania combining facilitated null mutant and plasmid shuffle analyses

Leishmania parasites cause important human morbidity and mortality. Essential Leishmania genes escape genetic assessment by loss‐of‐function analyses due to lethal null mutant phenotypes, even though these genes and their products are biologically most significant and represent validated drug targets. Here we overcome this limitation using a facilitated null mutant approach applied for the functional genetic analysis of the MAP kinase LmaMPK4. This system relies on the episomal expression of the target gene from vector pXNG that expresses the Herpes simplex virus thymidine kinase gene thus rendering transgenic parasites susceptible for negative selection using the antiviral drug ganciclovir. Using this system we establish the genetic proof of LmaMPK4 as essential kinase in promastigotes. LmaMPK4 structure/function analysis by plasmid shuffle allowed us to identify regulatory kinase sequence elements relevant for chemotherapeutic intervention. A partial null mutant, expressing an MPK4 derivative with altered ATP‐binding properties, showed defects in metacyclogenesis, establishing a first link of MPK4 function to parasite differentiation. The approaches presented here are broadly applicable to any essential gene in Leishmania thus overcoming major bottlenecks for their functional genetic analysis and their exploitation for structure‐informed drug development.     

MiR-145 Expression Accelerates Esophageal Adenocarcinoma Progression by Enhancing Cell Invasion and Anoikis Resistance

Background

Carcinoma of the esophagus has a high case fatality ratio and is now the 6th most common cause of cancer deaths in the world. We previously conducted a study to profile the expression of miRNAs in esophageal adenocarcinoma (EAC) pre and post induction therapy. Of the miRNAs differentially expressed post induction chemoradiation, miR-145, a known tumor suppressor miRNA, was upregulated 8-fold following induction therapy, however, its expression was associated with shorter disease-free survival. This unexpected result was explored in this current study.

Methods

In order to study the role of miR-145 in EAC, miRNA-145 was overexpressed in 3 EAC cell lines (OE33, FLO-1, SK-GT-4) and one ESCC cell line (KYSE-410). After validation of the expression of miR-145, hallmarks of cancer such as cell proliferation, resistance to chemotherapy drugs or anoikis, and cell invasion were analyzed.

Results

There were no differences in cell proliferation and 5 FU resistance between miR145 cell lines and the control cell lines. miR-145 expression also had no effect on cisplatin resistance in two of three cell lines (OE33 and FLO-1), but miR-145 appeared to protect SK-GT-4 cells against cisplatin treatment. However, there was a significant difference in cell invasion, cell adhesion and resistance to anoikis. All three EAC miR-145 cell lines invaded more than their respective controls. Similarly, OE33 and SK-GT-4 miR-145 cell lines were able to survive longer in a suspension state.

Discussion

While expression of miR-145 in ESCC stopped proliferation and invasion, expression of miR-145 in EAC cells enhanced invasion and anoikis resistance. Although more work is required to understand how miR-145 conveys these effects, expression of miR-145 appears to promote EAC progression by enhancing invasion and protection against anoikis, which could in turn facilitate distant metastasis.