Neuronal paxillin and drebrin mediate BDNF-induced force transduction and growth cone turning in a soft-tissue-like environment.

Soft tissue environments govern neuronal morphogenesis. However, the precise molecular mechanisms underlying chemotropism-directed axonal growth cone movement in extremely soft environments remain unclear. Here, we show that drebrin, a growth cone T-zone protein, modulates growth cone turning in response to brain-derived neurotrophic factor (BDNF) coated on a soft substrate. Structurally, axonal growth cones of rodent hippocampal neurons grown on 0.1 kPa hydrogels possess an expanded T zone in which drebrin is highly integrated with both F-actin and microtubules. Biochemically, we identify paxillin as interacting with drebrin in cells grown on 0.1 kPa hydrogels but not on glass coverslips. When grown on 0.1 kPa substrates, growth cones asymmetrically exposed to BDNF-bound stripes exhibit enhanced paxillin-drebrin interaction on the side facing the stripes, an activity that is PKA and AAK1 dependent but independent of Src kinase. Functionally, we show that BDNF-induced growth cone turning and force generation on soft substrates require drebrin phosphorylation and paxillin-drebrin association.

Rapid high resolution 3D imaging of expanded biological specimens with lattice light sheet microscopy.

Expansion microscopy was invented to surpass the optical diffraction limit by physically expanding biological specimens with swellable polymers. Due to the large sizes of expanded specimens, 3D imaging techniques that are capable to acquire large volumetric data rapidly at high spatial resolution are therefore required for expansion microscopy. Lattice light sheet microscopy (LLSM) was developed to image biological specimens rapidly at high 3D spatial resolution by using a thin lattice light sheet for sample illumination. However, due to the current limitations of LLSM mechanism and the optical design of LLS microscopes, it is challenging to image large expanded specimens at isotropic high spatial resolution using LLSM. To address the problem, we first optimized the sample preparation and expansion procedure for LLSM. Then, we implement a tiling lattice light sheet method to minimize sample translation during imaging and achieve much faster 3D imaging speed at high spatial resolution with more isotropic performance. Taken together, we report a general and improved 3D super-resolution imaging method for expanded samples.

Rapid single-wavelength lightsheet localization microscopy for clarified tissue.

Optical super-resolution microscopy allows nanoscale imaging of protein molecules in intact biological tissues. However, it is still challenging to perform large volume super-resolution imaging for entire animal organs. Here we develop a single-wavelength Bessel lightsheet method, optimized for refractive-index matching with clarified specimens to overcome the aberrations encountered in imaging thick tissues. Using spontaneous blinking fluorophores to label proteins of interest, we resolve the morphology of most, if not all, dopaminergic neurons in the whole adult brain (3.64 × 107 µm3) of Drosophila melanogaster at the nanometer scale with high imaging speed (436 µm3 per second) for localization. Quantitative single-molecule localization reveals the subcellular distribution of a monoamine transporter protein in the axons of a single, identified serotonergic Dorsal Paired Medial (DPM) neuron. Large datasets are obtained from imaging one brain per day to provide a robust statistical analysis of these imaging data.

The Applications of Lattice Light-sheet Microscopy for Functional Volumetric Imaging of Hippocampal Neurons in a Three-Dimensional Culture System.

The characterization of individual cells in three-dimensions (3D) with very high spatiotemporal resolution is crucial for the development of organs-on-chips, in which 3D cell cultures are integrated with microfluidic systems. In this study, we report the applications of lattice light-sheet microscopy (LLSM) for monitoring neuronal activity in three-dimensional cell culture. We first established a 3D environment for culturing primary hippocampal neurons by applying a scaffold-based 3D tissue engineering technique. Fully differentiated and mature hippocampal neurons were observed in our system. With LLSM, we were able to monitor the behavior of individual cells in a 3D cell culture, which was very difficult under a conventional microscope due to strong light scattering from thick samples. We demonstrated that our system could study the membrane voltage and intracellular calcium dynamics at subcellular resolution in 3D under both chemical and electrical stimulation. From the volumetric images, it was found that the voltage indicators mainly resided in the cytosol instead of the membrane, which cannot be distinguished using conventional microscopy. Neuronal volumetric images were sheet scanned along the axial direction and recorded at a laser exposure of 6 ms, which covered an area up to 4800 µm2, with an image pixel size of 0.102 µm. When we analyzed the time-lapse volumetric images, we could quantify the voltage responses in different neurites in 3D extensions.

Lightsheet localization microscopy enables fast, large-scale, and three-dimensional super-resolution imaging.

Recent advances in super-resolution microscopy allow the localization of single molecules within individual cells but not within multiple whole cells due to weak signals from single molecules and slow acquisition process for point accumulation to reconstruct super-resolution images. Here, we report a fast, large-scale, and three-dimensional super-resolution fluorescence microscope based on single-wavelength Bessel lightsheet to selectively illuminate spontaneous blinking fluorophores tagged to the proteins of interest in space. Critical parameters such as labeling density, excitation power, and exposure time were systematically optimized resulting in a maximum imaging speed of 2.7 × 104 µm3 s-1. Fourier ring correlation analysis revealed a reconstructed image with a lateral resolution of ~75 nm through the accumulation of 250 image volumes on immobilized samples within 15 min. Hence, the designed system could open new insights into the discovery of complex biological structures and live 3D localization imaging.

Lattice light sheet microscopy using tiling lattice light sheets.

We present a novel method used to implement tiling lattice light sheets (LLS) in lattice light sheet microscopy (LLSM) on regular LLS microscopes without changing the LLS microscope hardware. A LLS is tiled by applying binary phase maps acquired from off-center cross-sections of the corresponding optical lattice to the binary SLM used in LLS microscopes, by which a thin LLS can be tiled to image large specimens while maintaining the 3D imaging ability in the entire field of view. We investigate the method via numerical simulations and experiments, and demonstrate the method by imaging fluorescent particles embedded in agarose gel and expanded cells in the dithered mode of LLSM.

5D imaging via light sheet microscopy reveals cell dynamics during the eye-antenna disc primordium formation in Drosophila.

5D images of engrailed (en) and eye gone (eyg) gene expressions during the course of the eye-antenna disc primordium (EADP) formation of Drosophila embryos from embryonic stages 13 through 16 were recorded via light sheet microscopy and analyzed to reveal the cell dynamics involved in the development of the EADP. Detailed analysis of the time-lapsed images revealed the process of EADP formation and its invagination trajectory, which involved an inversion of the EADP anterior-posterior axis relative to the body. Furthermore, analysis of the en-expression pattern in the EADP provided strong evidence that the EADP is derived from one of the en-expressing head segments.

Carbapenem resistant Enterobacteriaceae carrying New Delhi metallo-ß-lactamase gene (NDM-1) in Taiwan.

The spread of New Delhi metallo-ß-lactamase gene (NDM-1)-producing bacteria has become a growing concern to the medical community worldwide. In this study, we reported 4 NDM-1-positive Enterobacteriaceae isolates recovered from two Taiwanese patients having travel history. The ß-lactamase genetic background, antimicrobial susceptibility, clonal relationships, and plasmid sizes of the NDM-1-producing Enterobacteriaceae were investigated. This report highlights the alarming introduction of NDM-1 gene among Enterobacteriaceae in Taiwan.

The interaction of Glu294 at the subunit interface is important for the activity and stability of goose delta-crystallin.

PURPOSE: delta-Crystallin is a soluble structural protein in found in avian eye lenses; it shares high amino acid sequence identity with argininosuccinate lyase. E294 is the only residue located at the double dimer interface and it performs hydrogen bonding with the active site residues of H160 and K323 in the neighboring and diagonal subunits, respectively. H160 is reported to play an important role in catalysis due to its H-bond interaction with the fumarate moiety of the substrate. In order to clarify the function of E294 in either stabilization of the quaternary structure or in catalysis, we carried out site-directed mutagenesis and functional analysis. METHODS: The structure of both wild-type and mutant proteins were analyzed by circular dichroism (CD) spectroscopy, fluorescence spectra, and analytical ultracentrifugation. Structural stability was measured by CD and tryptophan fluorescence. A modeled structure of the E294L mutant was built and optimized with energy minimization. RESULTS: No gross structural changes were observed when E294 was substituted with leucine, as judged by circular dichroism, tryptophan fluorescence, ANS fluorescence, and sedimentation velocity analyses. However, this mutant enzyme had only about 10% of the activity of a wild-type enzyme and its secondary structure was more easily denatured by increased temperature than that of a wild-type enzyme. The mutant protein also underwent its first unfolding transition at a lower concentration of guanidinium-hydrochloride than the wild-type protein. CONCLUSIONS: These results indicate that the interactions offered by E294 in the dimer-dimer interface of delta-crystallin are required to maintain the hydrogen bonding network in the active site for catalysis. Disruption of the interaction had no significant effect on the conformation and quaternary structure of delta-crystallin but it did lead to instability in the double dimer structure.