Hotheaded: TRPV1 as mediator of hippocampal synaptic plasticity

TRPV1 is a sensory transduction channel that mediates thermal nociception and some aspects of pathological pain. In this issue of Neuron, Gibson et al. report that TRPV1 also plays important roles in hippocampal synaptic plasticity, presenting a potential challenge for TRPV1-targeted therapeutics for the treatment of pain.     

Colonisation and mass rearing: learning from others

Mosquitoes, just as other insects produced for the sterile insect technique (SIT), are subjected to several unnatural processes including laboratory colonisation and large-scale factory production. After these processes, sterile male mosquitoes must perform the natural task of locating and mating with wild females. Therefore, the colonisation and production processes must preserve characters necessary for these functions. Fortunately, in contrast to natural selection which favours a suite of characteristics that improve overall fitness, colonisation and production practices for SIT strive to maximize only the few qualities that are necessary to effectively control populations.However, there is considerable uncertainty about some of the appropriate characteristics due to the lack of data. Development of biological products for other applications suggest that it is possible to identify and modify competitiveness characteristics in order to produce competitive mass produced sterile mosquitoes. This goal has been pursued - and sometimes achieved - by mosquito colonisation, production, and studies that have linked these characteristics to field performance. Parallels are drawn to studies in other insect SIT programmes and aquaculture which serve as vital technical reference points for mass-production of mosquitoes, most of whose development occurs - and characteristics of which are determined - in an aquatic environment. Poorly understood areas that require further study are numerous: diet, mass handling and genetic and physiological factors that influence mating competitiveness. Compromises in such traits due to demands to increase numbers or reduce costs, should be carefully considered in light of the desired field performance.     

Comparing the in Vivo Function of α-Carboxysomes and β-Carboxysomes in Two Model Cyanobacteria

The carbon dioxide (CO₂)-concentrating mechanism of cyanobacteria is characterized by the occurrence of Rubisco-containing microcompartments called carboxysomes within cells. The encapsulation of Rubisco allows for high-CO₂ concentrations at the site of fixation, providing an advantage in low-CO₂ environments. Cyanobacteria with Form-IA Rubisco contain α-carboxysomes, and cyanobacteria with Form-IB Rubisco contain β-carboxysomes. The two carboxysome types have arisen through convergent evolution, and α-cyanobacteria and β-cyanobacteria occupy different ecological niches. Here, we present, to our knowledge, the first direct comparison of the carboxysome function from α-cyanobacteria (Cyanobium spp. PCC7001) and β-cyanobacteria (Synechococcus spp. PCC7942) with similar inorganic carbon (C_i; as CO₂ and HCO₃⁻) transporter systems. Despite evolutionary and structural differences between α-carboxysomes and β-carboxysomes, we found that the two strains are remarkably similar in many physiological parameters, particularly the response of photosynthesis to light and external C_i and their modulation of internal ribulose-1,5-bisphosphate, phosphoglycerate, and C_i pools when grown under comparable conditions. In addition, the different Rubisco forms present in each carboxysome had almost identical kinetic parameters. The conclusions indicate that the possession of different carboxysome types does not significantly influence the physiological function of these species and that similar carboxysome function may be possessed by each carboxysome type. Interestingly, both carboxysome types showed a response to cytosolic C_i, which is of higher affinity than predicted by current models, being saturated by 5 to 15 mm C_i. This finding has bearing on the viability of transplanting functional carboxysomes into the C₃ chloroplast.Cyanobacteria inhabit a diverse range of ecological habitats, including both freshwater and marine ecosystems. The flexibility to occupy these different habitats is thought to come in part from the carbon-concentrating mechanism (CCM) present in all species (Badger et al., 2006). The CCM comprises inorganic carbon (C_i; as carbon dioxide [CO₂] and HCO₃⁻) transporters for C_i uptake and protein microbodies called carboxysomes for CO₂ concentration and fixation by Rubisco (Badger and Price, 2003). The CCM is believed to have evolved in response to changes in the absolute and relative levels of CO₂ and oxygen (O₂) in the atmosphere during the evolution of oxygenic photosynthesis in cyanobacteria (Price et al., 2008).There are two main phylogenetic groups within the cyanobacteria based on Rubisco and carboxysome phylogenies; α-cyanobacteria have α-carboxysomes with Form-IA Rubisco, whereas β-cyanobacteria have β-carboxysomes with Form-IB Rubisco (Tabita, 1999; Badger et al., 2002). Rubisco large subunit protein sequences from these two groups are closely related but nevertheless, distinguishable (Supplemental Fig. S1). In general, α-cyanobacteria and β-cyanobacteria occupy a quite different range of ecological habitats. The α-cyanobacteria are mostly marine organisms, with the majority of species living in the open ocean (Badger et al., 2006). Marine α-cyanobacteria live in very stable environments with high pH (pH 8.2) and dissolved carbon levels but low nutrients. They are characterized by small cells, very small genomes (1.6–2.8 Mb), and a few constitutively expressed carbon uptake transporters (Rae et al., 2011; Beck et al., 2012). They have been described as low flux, low energy cyanobacteria with a minimal CCM (Badger et al., 2006). Although these species are slow growing, oceanic cyanobacteria contribute as much as one-half of oceanic primary productivity (Liu et al., 1997, 1999; Field et al., 1998), suggesting that they may contribute up to 25% to net global productivity every year.In comparison, β-cyanobacteria occupy a much more diverse range of habitats, including freshwater, estuarine, and hot springs and never reach the same levels of global abundance (Badger et al., 2006). They are characterized by larger cells, larger genomes (2.2–3.6 Mb), and an array of carbon uptake transporters, including those transporters induced under low C_i (Rae et al., 2011, 2013). In addition to these broadly defined α-groups and β-groups, there are small numbers of α-cyanobacteria that have been termed transitional strains (Price, 2011; Rae et al., 2011). These species (e.g. Cyanobium spp. PCC7001, Synechococcus spp. WH5701, and Cyanobium spp. PCC6307; Supplemental Fig. S1) live in marginal marine and freshwater environments and have a number of characteristics similar to β-cyanobacteria. For example, they have a more diverse range of C_i uptake systems and a significantly larger genome than closely related α-cyanobacteria, and it has been suggested that the additional genes encoding transport systems were acquired by horizontal gene transfer (HGT) from β-cyanobacteria (Rae et al., 2011).Although the carboxysomes from α-cyanobacteria and β-cyanobacteria are very similar in overall structure, in that they share an outer protein shell of common phylogenetic origin (Kerfeld et al., 2005), they are distinguished from each other largely by differences in the proteins, which seem to make up or interact with the interior of the carboxysome compartment (Supplemental Table S1). This finding suggests that their different structures today have arisen through periods of common and convergent evolution. Certain carboxysome shell proteins from α-carboxysomes and β-carboxysomes show regions of significant sequence homology. These proteins are denoted as CsoS1 to CsoS4 (in α-cyanobacteria) and CcmKLO (in β-cyanobacteria), and the homologous regions have been termed bacterial microcompartment domains (Kerfeld et al., 2010; Rae et al., 2013). Proteins with these domains are also found in bacterial microcompartments in proteobacteria. However, other identified carboxysome proteins do not show any sequence homology between α-carboxysomes and β-carboxysomes but may perform similar functional roles. For example, carbonic anhydrase activity is essential for carboxysome function, but its activity seems to be provided by a range of different proteins (β-CcaA, β-CcmM, and α-CsoSCA; Kupriyanova et al., 2013). Similarly, β-CcmM and α-CsoS2 could play similar roles in organizing the interface between the shell and Rubisco within the carboxysomes (Gonzales et al., 2005; Long et al., 2007).The functioning of a carboxysome relies on a number of biochemical properties associated with the protein microbody structure. These properties include the biochemical/kinetic properties of Rubisco contained within carboxysomes, the conductance of the carboxysome shell to the influx of substrate ribulose-1,5-bisphosphate (RuBP) and the efflux of the carboxylation product phosphoglycerate (PGA), the conductance of the shell to the influx of bicarbonate and the efflux of CO₂, and lastly, the manner in which bicarbonate is converted to CO₂ within the carboxysomes. α-Carboxysomes and β-carboxysomes have the potential to differ in each of these properties. The flux of phosphorylated sugars across the shell has been postulated to be mediated by the pores in the hexameric shell proteins (Yeates et al., 2010; Kinney et al., 2011), which although similar, do differ between the two carboxysomes types. Bicarbonate and CO₂ uptake processes are less well-defined but probably involve aspects of the way in which unique shell interface proteins interact with Rubisco, which also differs in that CsoS2 and CsoSCA are probably the interacting proteins involved in α-carboxysomes (Espie and Kimber, 2011), whereas CcmM and β-carboxysomal CA are variably involved in β-carboxysomes (Long et al., 2010). Finally, the Form-IA and Form-IB Rubisco proteins at the heart of carboxylation, although similar, have the potential to show different kinetic properties. Although Form-IB Rubiscos from β-cyanobacteria are well-characterized, the Form-IA counterparts have received very little attention. In addition, the CCM of very few strains of cyanobacteria have been studied at the level of biochemistry and physiology, and they have been almost exclusively β-cyanobacteria. As a result, there are significant gaps in our knowledge about the similarities and differences in functional traits between α-cyanobacterial and β-cyanobacterial strains. One important question that remains to be answered is whether α-carboxysomes and β-carboxysomes have intrinsic differences in their biochemical properties that influence the nature of the CCM, which is established within each broad cell type.Because of the difficulties in isolating and assaying intact carboxysomes in vitro, the characterization of biochemical properties of carboxysomes is not easily addressed. One way forward is to study the properties of the CCM in detail in a model representative strain from each group and compare their characteristics to contrast the intracellular function of α-cell types and β-cell types. In the past, it has been restricted because of the difficulties in growing many of the open ocean α-cyanobacteria and their very different natures in relation to inorganic transporter composition. However, the availability of α-cyanobacteria transition strains, which grow well in the laboratory, has provided an opportunity to address this question. The α-cyanobacteria Cyanobium spp. PCC7001 (hereafter Cyanobium spp.), in particular, grows in standard freshwater media (BG11) and has growth and photosynthetic performance properties that closely match the model β-cyanobacteria, Synechococcus spp. PCC7942 (hereafter Synechococcus spp.); for this reason, Cyanobium spp. is ideal for a balanced comparison of the in vivo physiological properties of α-carboxysomes and β-carboxysomes in two species with relatively similar C_i-uptake properties.Genome analysis of both strains indicates that Cyanobium spp. have many of the same carbon uptake systems present in Synechococcus spp. (Rae et al., 2011). In using two strains with such similar transport capacities, we aimed to shed light on aspects of the functional properties of carboxysomes in each strain and how these properties affect the operation of the CCM in α-cyanobacteria and β-cyanobacteria. Using both membrane inlet mass spectrometry (MIMS) and silicon oil centrifugation, we investigated C_i pool sizes and CO₂ uptake rates in both species for cells grown at high and low CO₂. Comparative Rubisco properties and photosynthetic rates of each species were determined, and intracellular pools of RuBP and PGA were measured. In addition, we characterized a number of cellular properties to determine differences in the biochemical environments in which each carboxysome type exists. Together, the results provide a unique functional comparison of two distinct carboxysome types from phylogenetically disparate cyanobacteria.     

A novel interaction of CLN3 with nonmuscle myosin-IIB and defects in cell motility of Cln3 cells

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a pediatric lysosomal storage disorder characterized by accumulation of autofluorescent storage material and neurodegeneration, which result from mutations in CLN3. The function of CLN3, a lysosomal membrane protein, is currently unknown. We report that CLN3 interacts with cytoskeleton-associated nonmuscle myosin-IIB. Both CLN3 and myosin-IIB are ubiquitously expressed, yet mutations in either produce dramatic consequences in the CNS such as neurodegeneration in JNCL patients and Cln3^−/− mouse models, or developmental deficiencies in Myh10^−/− mice, respectively. A scratch assay revealed a migration defect associated with Cln3^−/− cells. Inhibition of nonmuscle myosin-II with blebbistatin in WT cells resulted in a phenotype that mimics the Cln3^−/− migration defect. Moreover, inhibiting lysosome function by treating cells with chloroquine exacerbated the migration defect in Cln3^−/−. Cln3^−/− cells traversing a transwell filter under gradient trophic factor conditions displayed altered migration, further linking lysosomal function and cell migration. The myosin-IIB distribution in Cln3^−/− cells is elongated, indicating a cytoskeleton defect caused by the loss of CLN3. In summary, cells lacking CLN3 have defects that suggest altered myosin-IIB activity, supporting a functional and physical interaction between CLN3 and myosin-IIB. We propose that the migration defect in Cln3^−/− results, in part, from the loss of the CLN3–myosin-IIB interaction.     

Over-expression of the ��-carboxysomal CcmM protein in Synechococcus PCC7942 reveals a tight co-regulation of carboxysomal carbonic anhydrase (CcaA) and M58 content

Carboxysomes, containing the cell's complement of RuBisCO surrounded by a specialized protein shell, are a central component of the cyanobacterial CO(2)-concentrating mechanism. The ratio of two forms of the β-carboxysomal protein CcmM (M58 and M35) may affect the carboxysomal carbonic anhydrase (CcaA) content. We have over-expressed both M35 and M58 in the β-cyanobacterium Synechococcus PCC7942. Over-expression of M58 resulted in a marked increase in the amount of this protein in carboxysomes at the expense of M35, with a concomitant increase in the observed CcaA content of carboxysomes. Conversely, M35 over-expression diminished M58 content of carboxysomes and led to a decrease in CcaA content. Carboxysomes of air-grown wild-type cells contained slightly elevated CcaA and M58 content and slightly lower M35 content compared to their 2% CO(2)-grown counterparts. Over a range of CcmM expression levels, there was a strong correlation between M58 and CcaA content, indicating a constant carboxysomal M58:CcaA stoichiometry. These results also confirm a role for M58 in the recruitment of CcaA into the carboxysome and suggest a tight regulation of M35 and M58 translation is required to produce carboxysomes with an appropriate CA content. Analysis of carboxysomal protein ratios, resulting from the afore-mentioned over-expression studies, revealed that β-carboxysomal protein stoichiometries are relatively flexible. Determination of absolute protein quantities supports the hypothesis that M35 is distributed throughout the β-carboxysome. A modified β-carboxysome packing model is presented.     

Viruses and the TNF-related cytokines,an evolving battle

Tumor necrosis factor (TNF)-related cytokines are critical effector molecules in the immune response to viral pathogens. Engagement of the TNF receptors by their cognate ligands activates apoptotic and non-apoptotic signaling pathways, both of which can mediate antiviral activity. In response, viruses have evolved mechanisms to inhibit signaling by some cytokines of the TNF superfamily. These strategies are largely unique to each class of virus, but are similar in that they all target key regulatory checkpoints of the TNF pathway. In recent years, studies directed towards dissecting the mechanisms of TNF signaling and the viral retort have led to several significant discoveries, and form the basis for this review.     

Moving in an Environment of Induced Sensorimotor Incongruence Does Not Influence Pain Sensitivity in Healthy Volunteers: A Randomised Within-Subject Experiment

Objectives

It has been proposed that in the same way that conflict between vestibular and visual inputs leads to motion sickness, conflict between motor commands and sensory information associated with these commands may contribute to some chronic pain states. Attempts to test this hypothesis by artificially inducing a state of sensorimotor incongruence and assessing self-reported pain have yielded equivocal results. To help clarify the effect sensorimotor incongruence has on pain we investigated the effect of moving in an environment of induced incongruence on pressure pain thresholds (PPT) and the pain experienced immediately on completion of PPT testing.

Methods

Thirty-five healthy subjects performed synchronous and asynchronous upper-limb movements with and without mirror visual feedback in random order. We measured PPT over the elbow and the pain evoked by testing. Generalised linear mixed-models were performed for each outcome. Condition (four levels) and baseline values for each outcome were within-subject factors.

Results

There was no effect of condition on PPT (p = 0.887) or pressure-evoked pain (p = 0.771). A sensitivity analysis using only the first PPT measure after each condition confirmed the result (p = 0.867).

Discussion

Inducing a state of movement related sensorimotor incongruence in the upper-limb of healthy volunteers does not influence PPT, nor the pain evoked by testing. We found no evidence that sensorimotor incongruence upregulates the nociceptive system in healthy volunteers.