The antennae of male silk moths are extremely sensitive to the female sex pheromone such that a male moth can find a female up to 4.5 km away. This remarkable sensitivity is due to both the morphological and biochemical design of these antennae. Along the branches of the plumose antennae are the sensilla trichodea, each consisting of a hollow cuticular hair containing two unbranched dendrites bathed in a fluid, the receptor lymph ,3. The dendrites and receptor lymph are isolated from the haemolymph by a barrier of epidermal cells which secreted the cuticular hair. Pheromone molecules are thought to diffuse down 100 A-wide pore tubules through the cuticular wall and across the receptor lymph space to receptors located in the dendritic membrane. To prevent the accumulation of residual stimulant and hence sensory adaptation, the pheromone molecules are subsequently inactivated in an apparent two-step process of rapid ’early inactivation’ followed by much slower enzymatic degradation. The biochemistry involved in this sequence of events is largely unknown. We report here the identification of three proteins which interact with the pheromone of the wild silk moth Antheraea polyphemus: a pheromone-binding protein and a pheromone-degrading esterase, both uniquely located in the pheromone-sensitive sensilla; and a second esterase common to all cuticular tissues except the sensilla.

Proteomic studies have identified thousands of eukaryotic phosphorylation sites (phosphosites), but few are functionally characterized. Nishi et al., in this issue of Structure, characterize phosphosites at protein-protein interfaces and estimate the effect of their phosphorylation on interaction affinity, by combining proteomics data with protein structures.

{At distal dendritic locations, the threshold for action potential generation is higher and the amplitude of back-propagating spikes is decreased. To study whether these characteristics depend upon Na+ channels, their voltage-dependent properties at proximal and distal dendritic locations were compared in CA1 hippocampal neurons. Distal Na+ channels activated at more hyperpolarized voltages than proximal (half-activation voltages were -20.4 +/- 2.4 mV vs. -12.0 +/- 1.7 mV for distal and proximal patches, respectively

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

Key to understanding a protein’s biological function is the accurate determination of its spatial distribution inside a cell. Although fluorescent protein markers allow the targeting of specific proteins with molecular precision, much of this information is lost when the resultant fusion proteins are imaged with conventional, diffraction-limited optics. In response, several imaging modalities that are capable of resolution below the diffraction limit (approximately 200 nm) have emerged. Here, both single- and dual-color superresolution imaging of biological structures using photoactivated localization microscopy (PALM) are described. The examples discussed focus on adhesion complexes: dense, protein-filled assemblies that form at the interface between cells and their substrata. A particular emphasis is placed on the instrumentation and photoactivatable fluorescent protein (PA-FP) tags necessary to achieve PALM images at approximately 20 nm resolution in 5 to 30 min in fixed cells.

Commentary: A paper spearheaded by Hari which gives a thorough description of the methods and hardware needed to successfully practice PALM, including cover slip preparation, cell transfection and fixation, drift correction with fiducials, characterization of on/off contrast ratios for different photoactivted fluorescent proteins, identifying PALM-suitable cells, and mechanical and optical components of a PALM system.

Superresolution fluorescence microscopy permits the study of biological processes at scales small enough to visualize fine subcellular structures that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable fluorescent proteins. The fluorescence of these proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysical properties of photocontrollable fluorescent proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitchable fluorescent proteins, and we detail their particular usefulness in single-molecule localization-based and nonlinear ensemble-based superresolution techniques. Finally, we discuss recent applications of photocontrollable proteins in superresolution imaging, as well as how these applications help to clarify properties of intracellular structures and processes that are relevant to cell and developmental biology, neuroscience, cancer biology and biomedicine.

By providing quantitative, visual data of live cells, fluorescent protein-based microscopy techniques are furnishing novel insights into the complexities of membrane trafficking pathways and organelle dynamics. In this chapter, we describe experimental protocols employing fluorescent protein-based photohighlighting techniques to quantify protein movement into and out of the Golgi apparatus, an organelle that serves as the central sorting and processing station of the secretory pathway. The methods allow kinetic characteristics of Golgi-associated protein trafficking to be deciphered, which can help clarify how the Golgi maintains itself as a steady-state structure despite a continuous flux of secretory cargo passing into and out of this organelle. The guidelines presented in this chapter can also be applied to examine the dynamics of other intracellular organelle systems, elucidating mechanisms for how proteins are maintained in specific organelles and/or circulated to other destinations within the cell.

Optical approaches for tracking neural dynamics are of widespread interest, but a theoretical framework quantifying the physical limits of these techniques has been lacking. We formulate such a framework by using signal detection and estimation theory to obtain physical bounds on the detection of neural spikes and the estimation of their occurrence times as set by photon counting statistics (shot noise). These bounds are succinctly expressed via a discriminability index that depends on the kinetics of the optical indicator and the relative fluxes of signal and background photons. This approach facilitates quantitative evaluations of different indicators, detector technologies, and data analyses. Our treatment also provides optimal filtering techniques for optical detection of spikes. We compare various types of Ca(2+) indicators and show that background photons are a chief impediment to voltage sensing. Thus, voltage indicators that change color in response to membrane depolarization may offer a key advantage over those that change intensity. We also examine fluorescence resonance energy transfer indicators and identify the regimes in which the widely used ratiometric analysis of signals is substantially suboptimal. Overall, by showing how different optical factors interact to affect signal quality, our treatment offers a valuable guide to experimental design and provides measures of confidence to assess optically extracted traces of neural activity.

A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622 nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621 nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

Many diverse taxa have evolved independently the habit of living in plant galls. For all but some viral galls, it is unknown whether plants produce galls as a specialized plant reaction to certain types of herbivory, or whether herbivores direct gall development. Here I present a phylogenetic analysis of gallforming cerataphidine aphids which demonstrates that gall morphology is extremely conservative with respect to aphid phylogeny, but variable with respect to plant taxonomy. In addition, the phylogeny reveals at least three host plant switches where the aphids produce galls most similar to the galls of their closest relatives, rather than galls similar to the galls of aphids already present on the host plant. These results suggest that aphids determine the details of gall morphology essentially extending their phenotype to include plant material. Based on this and other evidence, I suggest that the aphids and other galling insects manipulate latent plant developmental programmes to produce modified atavistic plant morphologies rather than create new forms de novo.