Androgen signaling via the androgen receptor (AR) transcription factor is crucial to normal prostate homeostasis and prostate tumorigenesis. Current models of AR function are predominantly based on studies of prostate-specific antigen regulation in androgen-responsive cell lines. To expand on these in vitro paradigms, we used the mouse prostate to elucidate the mechanisms through which AR regulates another direct target, FKBP5, in vivo. FKBP5 encodes an immunophilin that has been previously implicated in glucocorticoid and progestin signaling pathways and that likely influences prostate physiology in the presence of androgens. In this work, we show that androgens directly regulate FKBP5 via an interaction between the AR and a distal enhancer located 65 kb downstream of the transcription start site in the fifth intron of the FKBP5 gene. We have found that AR selectively recruits cAMP response element-binding protein to this enhancer. These interactions, in turn, result in chromatin remodeling that affects the enhancer proper but not the FKBP5 locus as a whole. Furthermore, in contrast to prostate-specific antigen-regulatory mechanisms, we show that transactivation of the FKBP5 gene does not rely on a single looping complex to mediate communication between the distal enhancer and proximal promoter. Rather, the distal enhancer complex and basal transcription apparatus communicate indirectly with one another, implicating a regulatory mechanism that has not been previously appreciated for AR target genes.

The ability of synapses throughout the dendritic tree to influence neuronal output is crucial for information processing in the brain. Synaptic potentials attenuate dramatically, however, as they propagate along dendrites toward the soma. To examine whether excitatory axospinous synapses on CA1 pyramidal neurons compensate for their distance from the soma to counteract such dendritic filtering, we evaluated axospinous synapse number and receptor expression in three progressively distal regions: proximal and distal stratum radiatum (SR), and stratum lacunosum-moleculare (SLM). We found that the proportion of perforated synapses increases as a function of distance from the soma and that their AMPAR, but not NMDAR, expression is highest in distal SR and lowest in SLM. Computational models of pyramidal neurons derived from these results suggest that they arise from the compartment-specific use of conductance scaling in SR and dendritic spikes in SLM to minimize the influence of distance on synaptic efficacy.

In most organisms, low ethanol doses induce increased activity, while high doses are sedating. To investigate the underlying mechanisms, we isolated Drosophila mutants with altered ethanol responsiveness. Mutations in white rabbit (whir), disrupting RhoGAP18B, are strongly resistant to the sedating effects of ethanol. This resistance can be suppressed by reducing the levels of Rho1 or Rac, implicating these GTPases in the behavioral response to ethanol. Indeed, expression of constitutively active forms of Rho1 or Rac1 in adult flies results in ethanol resistance similar to that observed in whir mutants. The whir locus produces several transcripts, RA-RD, which are predicted to encode three distinct RhoGAPs that share only the GAP domain. The RC transcript mediates the sedating effects of ethanol, while the RA transcript regulates its stimulant effects. Thus, distinct RhoGAPs, encoded by the same gene, regulate different manifestations of acute ethanol intoxication.

Accurate cell division in Escherichia coli requires the Min proteins MinC, MinD, and MinE as well as the presence of nucleoids. MinD and MinE exhibit spatial oscillations, moving from pole to pole of the bacterium, resulting in an average MinD concentration that is low at the center of the cell and high at the poles. This concentration minimum is thought to signal the site of cell division. Deterministic models of the Min oscillations reproduce many observed features of the system, including the concentration minimum of MinD. However, there are only a few thousand Min proteins in a bacterium, so stochastic effects are likely to play an important role. Here, we show that Monte Carlo simulations with a large number of proteins agree well with the results from a deterministic treatment of the equations. The location of minimum local MinD concentration is too variable to account for cell division accuracy in wild type but is consistent with the accuracy of cell division in cells without nucleoids. This finding confirms the need to include additional mechanisms, such as reciprocal interactions with the cell division ring or positioning of the nucleoids, to explain wild-type accuracy.

The early planarian embryo presents a complete ciliated epidermis and a pharynx and feeds on maternal yolk cells. In this paper, we report on all the elements involved in the formation of such an autonomous embryo, which we name cryptic larva. First, we provide a description of the spherical and fusiform yolk cells and their relationship with the blastomeres, from the laying of the egg capsule up to their final fate in mid embryonic stages. Then, we describe the early cleavage and the subsequent development of the tissues of the cryptic larva, namely, the primary epidermis, the embryonic pharynx, and a new cell type, the star cells. Finally, we discuss the possibility that the cryptic larva either constitutes a vestigial larva or, more likely, is the evolutionary result of the competition between multiple embryos for the limited and shared maternal resources in the egg capsule.

Biophysically accurate multicompartmental models of individual neurons have significantly advanced our understanding of the input-output function of single cells. These models depend on a large number of parameters that are difficult to estimate. In practice, they are often hand-tuned to match measured physiological behaviors, thus raising questions of identifiability and interpretability. We propose a statistical approach to the automatic estimation of various biologically relevant parameters, including 1) the distribution of channel densities, 2) the spatiotemporal pattern of synaptic input, and 3) axial resistances across extended dendrites. Recent experimental advances, notably in voltage-sensitive imaging, motivate us to assume access to: i) the spatiotemporal voltage signal in the dendrite and ii) an approximate description of the channel kinetics of interest. We show here that, given i and ii, parameters 1-3 can be inferred simultaneously by nonnegative linear regression; that this optimization problem possesses a unique solution and is guaranteed to converge despite the large number of parameters and their complex nonlinear interaction; and that standard optimization algorithms efficiently reach this optimum with modest computational and data requirements. We demonstrate that the method leads to accurate estimations on a wide variety of challenging model data sets that include up to about 10(4) parameters (roughly two orders of magnitude more than previously feasible) and describe how the method gives insights into the functional interaction of groups of channels.

Odors evoke complex responses in locust antennal lobe projection neurons (PNs)-the mitral cell analogs. These patterns evolve over hundreds of milliseconds and contain information about odor identity and concentration. In nature, animals often encounter many odorants in short temporal succession. We explored the effects of such conditions by presenting two different odors with variable intervening delays. PN ensemble representations tracked stimulus changes and, in some delay conditions, reached states that corresponded neither to the representation of either odor alone nor to the static mixture of the two. We then recorded from Kenyon cells (KCs), the PNs’ targets. Their responses were consistent with the PN population’s behavior: in some conditions, KCs were recruited that did not fire during single-odor or mixture stimuli. Thus, PN population dynamics are history dependent, and responses of individual KCs are consistent with piecewise temporal decoding of PN output over large sections of the PN population.

The enzymatic aldose ketose isomerisation of glucose and fructose sugars involves the transfer of a hydrogen between their C1 and C2 carbon atoms and, in principle, can proceed through either a direct hydride shift or via a cis-enediol intermediate. Pyrococcus furiosus phosphoglucose isomerase (PfPGI), an archaeal metalloenzyme, which catalyses the interconversion of glucose 6-phosphate and fructose 6-phosphate, has been suggested to operate via a hydride shift mechanism. In contrast, the structurally distinct PGIs of eukaryotic or bacterial origin are thought to catalyse isomerisation via a cis-enediol intermediate. We have shown by NMR that hydrogen exchange between substrate and solvent occurs during the reaction catalysed by PfPGI eliminating the possibility of a hydride-shift-based mechanism. In addition, kinetic measurements on this enzyme have shown that 5-phospho-d-arabinonohydroxamate, a stable analogue of the putative cis-enediol intermediate, is the most potent inhibitor of the enzyme yet discovered. Furthermore, determination and analysis of crystal structures of PfPGI with bound zinc and the substrate F6P, and with a number of competitive inhibitors, and EPR analysis of the coordination of the metal ion within PfPGI, have suggested that a cis-enediol intermediate-based mechanism is used by PfPGI with Glu97 acting as the catalytic base responsible for isomerisation.

Traditional small-molecule fluorophores are always fluorescent. This attribute can obscure valuable information in biological experiments. Here, we report on a versatile "latent" fluorophore that overcomes this limitation. At the core of the latent fluorophore is a derivative of rhodamine in which one nitrogen is modified as a urea. That modification enables rhodamine to retain half of its fluorescence while facilitating conjugation to a target molecule. The other nitrogen of rhodamine is modified with a "trimethyl lock", which enables fluorescence to be unmasked fully by a single user-designated chemical reaction. An esterase-reactive latent fluorophore was synthesized in high yield and attached covalently to a cationic protein. The resulting conjugate was not fluorescent in the absence of esterases. The enzymatic activity of esterases in endocytic vesicles and the cytosol educed fluorescence, enabling the time-lapse imaging of endocytosis into live human cells and thus providing unprecedented spatiotemporal resolution of this process. The modular design of this "fluorogenic label" enables the facile synthesis of an ensemble of small-molecule probes for the illumination of numerous biochemical and cell biological processes.

A subset of Drosophila neurons that expresses crustacean cardioactive peptide (CCAP) has been shown previously to make the hormone bursicon, which is required for cuticle tanning and wing expansion after eclosion. Here we present evidence that CCAP-expressing neurons (NCCAP) consist of two functionally distinct groups, one of which releases bursicon into the hemolymph and the other of which regulates its release. The first group, which we call NCCAP-c929, includes 14 bursicon-expressing neurons of the abdominal ganglion that lie within the expression pattern of the enhancer-trap line c929-Gal4. We show that suppression of activity within this group blocks bursicon release into the hemolymph together with tanning and wing expansion. The second group, which we call NCCAP-R, consists of NCCAP neurons outside the c929-Gal4 pattern. Because suppression of synaptic transmission and protein kinase A (PKA) activity throughout NCCAP, but not in NCCAP-c929, also blocks tanning and wing expansion, we conclude that neurotransmission and PKA are required in NCCAP-R to regulate bursicon secretion from NCCAP-c929. Enhancement of electrical activity in NCCAP-R by expression of the bacterial sodium channel NaChBac also blocks tanning and wing expansion and leads to depletion of bursicon from central processes. NaChBac expression in NCCAP-c929 is without effect, suggesting that the abdominal bursicon-secreting neurons are likely to be silent until stimulated to release the hormone. Our results suggest that NCCAP form an interacting neuronal network responsible for the regulation and release of bursicon and suggest a model in which PKA-mediated stimulation of inputs to normally quiescent bursicon-expressing neurons activates release of the hormone.