Several aquaporin (AQP) water channels are short-term regulated by the messenger cyclic adenosine monophosphate (cAMP), including AQP3. Bulk measurements show that cAMP can change diffusive properties of AQP3; however, it remains unknown how elevated cAMP affects AQP3 organization at the nanoscale. Here we analyzed AQP3 nano-organization following cAMP stimulation using photoactivated localization microscopy (PALM) of fixed cells combined with pair correlation analysis. Moreover, in live cells, we combined PALM acquisitions of single fluorophores with single-particle tracking (spt-PALM). These analyses revealed that AQP3 tends to cluster and that the diffusive mobility is confined to nanodomains with radii of ∼150 nm. This domain size increases by ∼30% upon elevation of cAMP, which, however, is not accompanied by a significant increase in the confined diffusion coefficient. This regulation of AQP3 organization at the nanoscale may be important for understanding the mechanisms of water AQP3-mediated water transport across plasma membranes.

RNA granules have been likened to liquid droplets whose dynamics depend on the controlled dissolution and condensation of internal components. The molecules and reactions that drive these dynamics in vivo are not well understood. In this study, we present evidence that a group of intrinsically disordered, serine-rich proteins regulate the dynamics of P granules in C. elegans embryos. The MEG (maternal-effect germline defective) proteins are germ plasm components that are required redundantly for fertility. We demonstrate that MEG-1 and MEG-3 are substrates of the kinase MBK-2/DYRK and the phosphatase PP2A(PPTR-½). Phosphorylation of the MEGs promotes granule disassembly and dephosphorylation promotes granule assembly. Using lattice light sheet microscopy on live embryos, we show that GFP-tagged MEG-3 localizes to a dynamic domain that surrounds and penetrates each granule. We conclude that, despite their liquid-like behavior, P granules are non-homogeneous structures whose assembly in embryos is regulated by phosphorylation.

In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.

Transcription is a stepwise process that involves many specialized proteins and protein complexes, all of which must work together to express a given gene in a spatially and temporally regulated manner. An integral step in this regulatory process is carried out by large, multisubunit co-activator complexes, which have diverse roles in transcriptional control. Their diversity and large size allows for many potential regulatory inputs, but how is the versatility and specificity of these co-activator complexes determined?

Cases of convergent evolution that involve changes in the same developmental pathway, called parallelism, provide evidence that a limited number of developmental changes are available to evolve a particular phenotype. To our knowledge, in no case are the genetic changes underlying morphological convergence understood. However, morphological convergence is not generally assumed to imply developmental parallelism. Here we investigate a case of convergence of larval morphology in insects and show that the loss of particular trichomes, observed in one species of the Drosophila melanogaster species group, has independently evolved multiple times in the distantly related D. virilis species group. We present genetic and gene expression data showing that regulatory changes of the shavenbaby/ovo (svb/ovo) gene underlie all independent cases of this morphological convergence. Our results indicate that some developmental regulators might preferentially accumulate evolutionary changes and that morphological parallelism might therefore be more common than previously appreciated.

Painful events establish opponent memories: cues that precede pain are remembered negatively, whereas cues that follow pain, thus coinciding with relief are recalled positively. How do individual reinforcement-signaling neurons contribute to this "timing-dependent valence-reversal?" We addressed this question using an optogenetic approach in the fruit fly. Two types of fly dopaminergic neuron, each comprising just one paired cell, indeed established learned avoidance of odors that preceded their photostimulation during training, and learned approach to odors that followed the photostimulation. This is in striking parallel to punishment versus relief memories reinforced by a real noxious event. For only one of these neuron types, both effects were strong enough for further analyses. Notably, interfering with dopamine biosynthesis in these neurons partially impaired the punishing effect, but not the relieving after-effect of their photostimulation. We discuss how this finding constraints existing computational models of punishment versus relief memories and introduce a new model, which also incorporates findings from mammals. Furthermore, whether using dopaminergic neuron photostimulation or a real noxious event, more prolonged punishment led to stronger relief. This parametric feature of relief may also apply to other animals and may explain particular aspects of related behavioral dysfunction in humans.

The field of organic chemistry began with 19th century scientists identifying and then expanding upon synthetic dye molecules for textiles. In the 20th century, dye chemistry continued with the aim of developing photographic sensitizers and laser dyes. Now, in the 21st century, the rapid evolution of biological imaging techniques provides a new driving force for dye chemistry. Of the extant collection of synthetic fluorescent dyes for biological imaging, two classes reign supreme: rhodamines and cyanines. Here, we provide an overview of recent examples where modern chemistry is used to build these old-but-venerable classes of optically responsive molecules. These new synthetic methods access new fluorophores, which then enable sophisticated imaging experiments leading to new biological insights.

Although cellular mitochondrial DNA (mtDNA) copy number varies widely among cell lines and tissues, little is known about the mechanism of mtDNA copy number control. Most nascent replication strands from the leading, heavy-strand origin (O(H)) are prematurely terminated, defining the 3’ boundary of the displacement loop (D-loop). We have depleted mouse LA9 cell mtDNA to approximately 20% of normal levels by treating with 2’,3’-dideoxycytidine (ddC) and subsequently allowed recovery to normal levels of mtDNA. A quantitative ligation-mediated PCR assay was used to determine the levels of both terminated and extended nascent O(H) strands during mtDNA depletion and repopulation. Depleting mtDNA leads to a release of replication termination until mtDNA copy number approaches a normal level. Detectable total nascent strands per mtDNA genome remain below normal. Therefore, it is likely that the level of replication termination plays a significant role in copy number regulation in this system. However, termination of D-loop strand synthesis is persistent, indicating formation of the D-loop structure has a purpose that is required under conditions of rapid recovery of depleted mtDNA.

Hydrogen peroxide (H(2)O(2)) is the major reactive oxygen species (ROS) produced in sperm. High concentrations of H(2)O(2) in sperm induce nuclear DNA fragmentation and lipid peroxidation and result in cell death. The respiratory chain of the mitochondrion is one of the most productive ROS generating systems in sperm, and thus the destruction of ROS in mitochondria is critical for the cell. It was recently reported that H(2)O(2) generated by the respiratory chain of the mitochondrion can be efficiently destroyed by the cytochrome c-mediated electron-leak pathway where the electron of ferrocytochrome c migrates directly to H(2)O(2) instead of to cytochrome c oxidase. In our studies, we found that mouse testis-specific cytochrome c (T-Cc) can catalyze the reduction of H(2)O(2) three times faster than its counterpart in somatic cells (S-Cc) and that the T-Cc heme has the greater resistance to being degraded by H(2)O(2). Together, these findings strongly imply that T-Cc can protect sperm from the damages caused by H(2)O(2). Moreover, the apoptotic activity of T-Cc is three to five times greater than that of S-Cc in a well established apoptosis measurement system using Xenopus egg extract. The dramatically stronger apoptotic activity of T-Cc might be important for the suicide of male germ cells, considered a physiological mechanism that regulates the number of sperm produced and eliminates those with damaged DNA. Thus, it is very likely that T-Cc has evolved to guarantee the biological integrity of sperm produced in mammalian testis.