The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins.

Imaging and localizing single molecules with high accuracy in a 3D volume is a challenging task. Here we combine multifocal microscopy, a recently developed volumetric imaging technique, with point spread function engineering to achieve an increased depth for single molecule imaging. Applications in 3D single molecule localization-based super-resolution imaging is shown over an axial depth of 4 µm as well as for the tracking of diffusing beads in a fluid environment over 8 µm.

Holometabolous insects pass through a sedentary pupal stage and often choose a location for pupation that is different from the site of larval feeding. We have characterized a difference in pupariation site choice within and between sibling species of Drosophila. We found that, in nature, Drosophila sechellia pupariate within their host fruit, Morinda citrifolia, and that they perform this behavior in laboratory assays. In contrast, in the laboratory, geographically diverse strains of Drosophila simulans vary in their pupariation site preference; D. simulans lines from the ancestral range in southeast Africa pupariate on fruit, or a fruit substitute, whereas populations from Europe or the New World select sites off of fruit. We explored the genetic basis for the evolved preference in puariation site preference by performing quantitative trait locus mapping within and between species. We found that the interspecific difference is controlled largely by loci on chromosomes X and II. In contrast, variation between two strains of D. simulans appears to be highly polygenic, with the majority of phenotypic effects due to loci on chromosome III. These data address the genetic basis of how new traits arise as species diverge and populations disperse.

The intrinsic aberrations of high-NA gradient refractive index (GRIN) lenses limit their image quality as well as field of view. Here we used a pupil-segmentation-based adaptive optical approach to correct the inherent aberrations in a two-photon fluorescence endoscope utilizing a 0.8 NA GRIN lens. By correcting the field-dependent aberrations, we recovered diffraction-limited performance across a large imaging field. The consequent improvements in imaging signal and resolution allowed us to detect fine structures that were otherwise invisible inside mouse brain slices.

Optical aberrations deteriorate the performance of microscopes. Adaptive optics can be used to improve imaging performance via wavefront shaping. Here, we demonstrate a pupil-segmentation based adaptive optical approach with full-pupil illumination. When implemented in a two-photon fluorescence microscope, it recovers diffraction-limited performance and improves imaging signal and resolution.

Inhomogeneous optical properties of biological samples make it difficult to obtain diffraction-limited resolution in depth. Correcting the sample-induced optical aberrations needs adaptive optics (AO). However, the direct wavefront-sensing approach commonly used in astronomy is not suitable for most biological samples due to their strong scattering of light. We developed an image-based AO approach that is insensitive to sample scattering. By comparing images of the sample taken with different segments of the pupil illuminated, local tilt in the wavefront is measured from image shift. The aberrated wavefront is then obtained either by measuring the local phase directly using interference or with phase reconstruction algorithms similar to those used in astronomical AO. We implemented this pupil-segmentation-based approach in a two-photon fluorescence microscope and demonstrated that diffraction-limited resolution can be recovered from nonbiological and biological samples.

Mitochondrial fractions isolated from tissue culture cells or tissue such as liver after differential centrifugation can be purified further by density gradient centrifugation. Here we describe the use of sucrose for this purpose because it is commonly used and inexpensive and the resulting mitochondria preparations are useful for many purposes.

Laboratory behavioural tasks are an essential research tool. As questions asked of behaviour and brain activity become more sophisticated, the ability to specify and run richly structured tasks becomes more important. An increasing focus on reproducibility also necessitates accurate communication of task logic to other researchers. To these ends we developed pyControl, a system of open source hardware and software for controlling behavioural experiments comprising; a simple yet flexible Python-based syntax for specifying tasks as extended state machines, hardware modules for building behavioural setups, and a graphical user interface designed for efficiently running high throughput experiments on many setups in parallel, all with extensive online documentation. These tools make it quicker, easier and cheaper to implement rich behavioural tasks at scale. As important, pyControl facilitates communication and reproducibility of behavioural experiments through a highly readable task definition syntax and self-documenting features.

Quality assessment of tree-like structures obtained from a neuron reconstruction algorithm is necessary for evaluating the performance of the algorithm. The lack of user-friendly software for calculating common metrics motivated us to develop a Python toolbox called PyNeval, which is the first open-source toolbox designed to evaluate reconstruction results conveniently as far as we know. The toolbox supports popular metrics in two major categories, geometrical metrics and topological metrics, with an easy way to configure custom parameters for each metric. We tested the toolbox on both synthetic data and real data to show its reliability and robustness. As a demonstration of the toolbox in real applications, we used the toolbox to improve the performance of a tracing algorithm successfully by integrating it into an optimization procedure.

Pyramidal neurons are characterized by their distinct apical and basal dendritic trees and the pyramidal shape of their soma. They are found in several regions of the CNS and, although the reasons for their abundance remain unclear, functional studies--especially of CA1 hippocampal and layer V neocortical pyramidal neurons--have offered insights into the functions of their unique cellular architecture. Pyramidal neurons are not all identical, but some shared functional principles can be identified. In particular, the existence of dendritic domains with distinct synaptic inputs, excitability, modulation and plasticity appears to be a common feature that allows synapses throughout the dendritic tree to contribute to action-potential generation. These properties support a variety of coincidence-detection mechanisms, which are likely to be crucial for synaptic integration and plasticity.