Left–right asymmetry: cilia stir up new surprises in the node

Cilia are microtubule-based hair-like organelles that project from the surface of most eukaryotic cells. They play critical roles in cellular motility, fluid transport and a variety of signal transduction pathways. While we have a good appreciation of the mechanisms of ciliary biogenesis and the details of their structure, many of their functions demand a more lucid understanding. One such function, which remains as intriguing as the time when it was first discovered, is how beating cilia in the node drive the establishment of left–right asymmetry in the vertebrate embryo. The bone of contention has been the two schools of thought that have been put forth to explain this phenomenon. While the ‘morphogen hypothesis’ believes that ciliary motility is responsible for the transport of a morphogen preferentially to the left side, the ‘two-cilia model’ posits that the motile cilia generate a leftward-directed fluid flow that is somehow sensed by the immotile sensory cilia on the periphery of the node. Recent studies with the mouse embryo argue in favour of the latter scenario. Yet this principle may not be generally conserved in other vertebrates that use nodal flow to specify their left–right axis. Work with the teleost fish medaka raises the tantalizing possibility that motility as well as sensory functions of the nodal cilia could be residing within the same organelle. In the end, how ciliary signalling is transmitted to institute asymmetric gene expression that ultimately induces asymmetric organogenesis remains unresolved.     

The molecular basis of differential subcellular localization of C2 domains of protein kinase C-alpha and group IVa cytosolic phospholipase A2

The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. C2 domains are unique among membrane targeting domains in that they show a wide range of lipid selectivity for the major components of cell membranes, including phosphatidylserine and phosphatidylcholine. To understand how C2 domains show diverse lipid selectivity and how this functional diversity affects their subcellular targeting behaviors, we measured the binding of the C2 domains of group IVa cytosolic phospholipase A(2) (cPLA(2)) and protein kinase C-alpha (PKC-alpha) to vesicles that model cell membranes they are targeted to, and we monitored their subcellular targeting in living cells. The surface plasmon resonance analysis indicates that the PKC-alpha C2 domain strongly prefers the cytoplasmic plasma membrane mimic to the nuclear membrane mimic due to high phosphatidylserine content in the former and that Asn(189) plays a key role in this specificity. In contrast, the cPLA(2) C2 domain has specificity for the nuclear membrane mimic over the cytoplasmic plasma membrane mimic due to high phosphatidylcholine content in the former and aromatic and hydrophobic residues in the calcium binding loops of the cPLA(2) C2 domain are important for its lipid specificity. The subcellular localization of enhanced green fluorescent protein-tagged C2 domains and mutants transfected into HEK293 cells showed that the subcellular localization of the C2 domains is consistent with their lipid specificity and could be tailored by altering their in vitro lipid specificity. The relative cell membrane translocation rate of selected C2 domains was also consistent with their relative affinity for model membranes. Together, these results suggest that biophysical principles that govern the in vitro membrane binding of C2 domains can account for most of their subcellular targeting properties.     

Perinuclear localization of cytosolic phospholipase A(2)alpha is important but not obligatory for coupling with cyclooxygenases

In response to Ca(2+) signaling, cytosolic phospholipase A(2)alpha (cPLA(2)alpha) translocates from the cytosol to the perinuclear membrane, where downstream eicosanoid-synthetic enzymes, such as cyclooxygenase (COX), are localized. Although the spatiotemporal perinuclear colocalization of cPLA(2)alpha and COXs has been proposed to be critical for their functional coupling leading to prostanoid production, definitive evidence for this paradigm has remained elusive. To circumstantiate this issue, we took advantage of a chimeric cPLA(2)alpha mutant harboring the C2 domain of protein kinase Calpha, which translocates to the plasma membrane following cell activation. Transfection analyses of the native or chimeric cPLA(2)alpha in combination with COX-1 or COX-2 revealed that, even though the arachidonate-releasing capacities of native and mutant cPLA(2)alpha were comparable, prostaglandin production by mutant cPLA(2)alpha was markedly impaired as compared with that by native cPLA(2)alpha. We thus conclude that the perinuclear localization of cPLA(2)alpha is preferential, even if not obligatory, for efficient coupling with COXs.     

Frailty modeling for spatially correlated survival data,with application to infant mortality in Minnesota

The use of survival models involving a random effect or 'frailty' term is becoming more common. Usually the random effects are assumed to represent different clusters, and clusters are assumed to be independent. In this paper, we consider random effects corresponding to clusters that are spatially arranged, such as clinical sites or geographical regions. That is, we might suspect that random effects corresponding to strata in closer proximity to each other might also be similar in magnitude. Such spatial arrangement of the strata can be modeled in several ways, but we group these ways into two general settings: geostatistical approaches, where we use the exact geographic locations (e.g. latitude and longitude) of the strata, and lattice approaches, where we use only the positions of the strata relative to each other (e.g. which counties neighbor which others). We compare our approaches in the context of a dataset on infant mortality in Minnesota counties between 1992 and 1996. Our main substantive goal here is to explain the pattern of infant mortality using important covariates (sex, race, birth weight, age of mother, etc.) while accounting for possible (spatially correlated) differences in hazard among the counties. We use the GIS ArcView to map resulting fitted hazard rates, to help search for possible lingering spatial correlation. The DIC criterion (Spiegelhalter et al., Journal of the Royal Statistical Society, Series B 2002, to appear) is used to choose among various competing models. We investigate the quality of fit of our chosen model, and compare its results when used to investigate neonatal versus post-neonatal mortality. We also compare use of our time-to-event outcome survival model with the simpler dichotomous outcome logistic model. Finally, we summarize our findings and suggest directions for future research.     

Effect of temperature and arsenic on Aeromonas hydrophila growth,a modelling approach

Aeromonas hydrophila is frequently reported from arsenic affected areas. Present study was aimed to determine the effect of arsenic and temperature on growth of A. hydrophila. The bacteria were isolated from naturally infected fish from a water body in Birbhum, West-Bengal, India, which is reported to be an arsenic-free area. Arsenic concentration in natural aquatic reservoirs (e.g., pond, lake or river) varies from 0–6 mg/L. No significant change in bacterial growth was observed within this range of arsenic exposure. However, variation in temperature impacted the growth of A. hydrophila. A single dimension model was constructed using simple logistic equation. Rate parameters of the model were derived from the experimental observations. Comparison of model results and laboratory observations gives a good conformity regarding the effect of variation of arsenic concentration and temperature change on growth of this bacterium. From the analysis of this model we further get the idea that the maximum growth of A. hydrophila is supposed to be at 31.4°C in absence of arsenic, whereas at 477 mg/L arsenic concentration, the growth of the bacteria totally stops at 30°C.     

Comparison of strong cation exchange and SDS-PAGE fractionation for analysis of multiprotein complexes

Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.     

Support Vector Machine-based method for predicting subcellular localization of mycobacterial proteins using evolutionary information and motifs

Background

Annotations that describe the function of sequences are enormously important to researchers during laboratory investigations and when making computational inferences. However, there has been little investigation into the data quality of sequence function annotations. Here we have developed a new method of estimating the error rate of curated sequence annotations, and applied this to the Gene Ontology (GO) sequence database (GOSeqLite). This method involved artificially adding errors to sequence annotations at known rates, and used regression to model the impact on the precision of annotations based on BLAST matched sequences.

Results

We estimated the error rate of curated GO sequence annotations in the GOSeqLite database (March 2006) at between 28% and 30%. Annotations made without use of sequence similarity based methods (non-ISS) had an estimated error rate of between 13% and 18%. Annotations made with the use of sequence similarity methodology (ISS) had an estimated error rate of 49%.

Conclusion

While the overall error rate is reasonably low, it would be prudent to treat all ISS annotations with caution. Electronic annotators that use ISS annotations as the basis of predictions are likely to have higher false prediction rates, and for this reason designers of these systems should consider avoiding ISS annotations where possible. Electronic annotators that use ISS annotations to make predictions should be viewed sceptically. We recommend that curators thoroughly review ISS annotations before accepting them as valid. Overall, users of curated sequence annotations from the GO database should feel assured that they are using a comparatively high quality source of information.     

Microsecond simulations of mdm2 and its complex with p53 yield insight into force field accuracy and conformational dynamics

The p53‐MDM2 complex is both a major target for cancer drug development and a valuable model system for computational predictions of protein‐ligand binding. To investigate the accuracy of molecular simulations of MDM2 and its complex with p53, we performed a number of long (200 ns to 1 µs) explicit‐solvent simulations using a range of force fields. We systematically compared nine popular force fields (AMBER ff03, ff12sb, ff14sb, ff99sb, ff99sb‐ildn, ff99sb‐ildn‐nmr, ff99sb‐ildn‐phi, CHARMM22*, and CHARMM36) against experimental chemical shift data, and found similarly accurate results, with microsecond simulations achieving better agreement compared to 200‐ns trajectories. Although the experimentally determined apo structure has a closed binding cleft, simulations in all force fields suggest the apo state of MDM2 is highly flexible, and able to sample holo‐like conformations, consistent with a conformational selection model. Initial structuring of the MDM2 lid region, known to competitively bind the binding cleft, is also observed in long simulations. Taken together, these results show molecular simulations can accurately sample conformations relevant for ligand binding. We expect this study to inform future computational work on folding and binding of MDM2 ligands. Proteins 2015; 83:1665–1676. © 2015 Wiley Periodicals, Inc.