Orai1 pore residues control CRAC channel inactivation independently of calmodulin

Ca²⁺ entry through CRAC channels causes fast Ca²⁺-dependent inactivation (CDI). Previous mutagenesis studies have implicated Orai1 residues W76 and Y80 in CDI through their role in binding calmodulin (CaM), in agreement with the crystal structure of Ca²⁺–CaM bound to an Orai1 N-terminal peptide. However, a subsequent Drosophila melanogaster Orai crystal structure raises concerns about this model, as the side chains of W76 and Y80 are predicted to face the pore lumen and create a steric clash between bound CaM and other Orai1 pore helices. We further tested the functional role of CaM using several dominant-negative CaM mutants, none of which affected CDI. Given this evidence against a role for pretethered CaM, we altered side-chain volume and charge at the Y80 and W76 positions to better understand their roles in CDI. Small side chain volume had different effects at the two positions: it accelerated CDI at position Y80 but reduced the extent of CDI at position W76. Positive charges at Y80 and W76 permitted partial CDI with accelerated kinetics, whereas introducing negative charge at any of five consecutive pore-lining residues (W76, Y80, R83, K87, or R91) completely eliminated CDI. Noise analysis of Orai1 Y80E and Y80K currents indicated that reductions in CDI for these mutations could not be accounted for by changes in unitary current or open probability. The sensitivity of CDI to negative charge introduced into the pore suggested a possible role for anion binding in the pore. However, although Cl⁻ modulated the kinetics and extent of CDI, we found no evidence that CDI requires any single diffusible cytosolic anion. Together, our results argue against a CDI mechanism involving CaM binding to W76 and Y80, and instead support a model in which Orai1 residues Y80 and W76 enable conformational changes within the pore, leading to CRAC channel inactivation.     

Inbreeding depression in monarch butterflies

Monarch butterflies and their unique system of multigenerational migration have long fascinated the public, and concerns for the fate of this charismatic insect have grown due to the consistent declines in overwintering colony size over the last 20 years. Risks to this migratory insect have been considered in terms of climate change, habitat and thus population fragmentation, and decreased host plant availability. However, another obvious danger, that of decreased heterozygosity resulting from decreasing population size, has yet to be explored. Here we report experimental evidence for immediate inbreeding depression in individuals from the migratory population. Inbred matings produced less viable eggs and inbred offspring had higher developmental mortality and shorter lifespans. We discuss these results in the context of monarch migration extinction risk and suggest that additional genetic monitoring should be undertaken to protect this iconic animal.     

Readily Accessible Multiplane Microscopy: 3D Tracking the HIV‐1 Genome in Living Cells

Human immunodeficiency virus (HIV)‐1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV‐1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV‐1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV‐1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high‐resolution 3D imaging with single‐macromolecule sensitivity. We show with high temporal and spatial resolution that HIV‐1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV‐1 RNA genome is transported to the sites of assembly of nascent virions.      

Role of maternal provisioning in controlling interpopulation variation in hatching size in the marine snail Nucella ostrina

This study examined the role of maternal provisioning in controlling interpopulation variation in hatching size in nine isolated populations of the intertidal gastropod Nucella ostrina, in which development to the early juvenile stage takes place within an egg capsule. Variation among populations was almost entirely due to the ratio of nurse eggs to embryo, which explained 65% of the variation in hatching size. Egg size was not a significant predictor of hatching size. Differences among seven of these populations in the nurse egg/embryo ratio were entirely due to the number of nurse eggs allocated per capsule; these populations allocated different numbers of nurse eggs per capsule but allocated the same number of embryos. Intriguingly, the two most wave-sheltered populations allocated significantly more nurse eggs and more embryos to each capsule than did the seven other populations, but they maintained nurse egg/embryo ratios consistent with patterns observed in the other populations. Inter- and intrapopulation variation in hatching size appears to be controlled largely by different mechanisms: within-population variation being controlled mainly by differences in allocation of embryos per capsule, whereas most among-population variation being due to differences in allocation of nurse eggs per capsule.