Rosette Nanotube Porins as Ion Selective Transporters and Single-Molecule Sensors.

Rosette nanotubes (RNTs) are a class of materials formed by molecular self-assembly of a fused guanine-cytosine base (G∧C base). An important feature of these self-assembled nanotubes is their precise atomic structure, intriguing for rational design and optimization as synthetic transmembrane porins. Here, we present experimental observations of ion transport across 1.1 nm inner diameter RNT porins (RNTPs) of various lengths in the range 5-200 nm. In a typical experiment, custom lipophilic RNTPs were first inserted into lipid vesicles; the vesicles then spontaneously fused with a planar lipid bilayer, which produced stepwise increases of ion current across the bilayer. Our measurements in 1 M KCl solution indicate ion transport rates of ∼50 ions s-1 V-1 m, which for short channels amounts to conductance values of ∼1 nS, commensurate with naturally occurring toxin channels such as α-hemolysin. Measurements of interaction times of α-cyclodextrin with RNTPs reveal two distinct unbinding time scales, which suggest that interactions of either face of α-cyclodextrin with the RNTP face are differentiable, backed with all-atom molecular dynamics simulations. Our results highlight the potential of RNTPs as self-assembled nonproteinaceous single-molecule sensors and selective nanofilters with tunable functionality through chemistry.

A method for freeze-fracture and scanning electron microscopy of isolated mitochondria.

Electron microscopy as a methodology for the study of mitochondria based on morphological features is a standard technique that has experienced little evolution over the course of several decades. This technology has identified heterogeneity of mitochondria populations across both whole tissues, as well between individual cells, using primarily ultrathin sections for transmission electron microscopy (TEM). However, this technique constrains the evaluation of a sample to a single two-dimensional plane. To overcome this limitation, scanning electron microscopy (SEM) has been successfully utilized to observe three-dimensional mitochondria structures within the complex microenvironment containing total cellular components. In response to these dual technical caveats of existing electron microscopy protocols, we developed a methodology to evaluate the three-dimensional ultrastructure of isolated mitochondria, utilizing a freeze-fracture step and rigorous preservation of sample morphology. This protocol allows for a more high-throughput analysis of mitochondria populations from a specimen of interest, as the sample has been previously purified, as well as a finer resolution of complex intra-mitochondrial structures, using the depth of field created by SEM. •Protocol designed for SEM of isolated mitochondria samples.•SEM visualizes mitochondria ultrastructure in 3-D.•Freeze-fracture creates cross-sectional plane for view of interior organelle structures.

New insights on mitochondrial heterogeneity observed in prepared mitochondrial samples following a method for freeze-fracture and scanning electron microscopy.

Mitochondria are dynamic intracellular organelles with diverse roles in tissue- and cell type-specific processes, extending beyond bioenergetics. In keeping with this array of functions, mitochondria are described as heterogeneous both between and within tissue types based on multiple parameters, including a broad spectrum of morphological features, and new research points toward a need for the evaluation of mitochondria as isolated organelles. Although transmission electron microscopy (TEM) is commonly used for the evaluation of mitochondria in tissues and renders mitochondrial structures in ultra-thin sections in two-dimensions, additional information regarding complex features within these organelles can be ascertained using scanning electron microscopy (SEM), which allows for analysis of phenotypic differences in three-dimensions. One challenge in producing mitochondrial images for evaluation of ultrastructure using SEM has been the ability to reliably visualize important intramitochondrial features, the inner membrane and cristae structures, on a large-scale (e.g. multiple mitochondria) within a sample preparation, as mitochondria are enclosed within a double membrane. This can be overcome using a 'freeze-fracture' technique in which mitochondrial preparations are snap-frozen followed by application of intense pressure to break open the organelles, revealing the intact components within. Previously published reports using freeze-fracture strategies for mitochondrial SEM have demonstrated feasibility in whole tissue specimens, but a detailed methodology for SEM analysis on isolated mitochondrial fractions has not been reported. By combining previously reported tissue freeze-fracture strategies, along with utilizing the depth of field created by SEM, herein we present a complete method reliant on the freeze-fracture of mitochondrial fractions prepared by differential centrifugation to produce a comprehensive and direct evaluation of three-dimensional mitochondrial ultrastructure by SEM. Image analysis of internal mitochondrial features demonstrates heterogeneity in mitochondrial ultrastructure from a single sample preparation.

Class Cariacotrichea, a novel ciliate taxon from the anoxic Cariaco Basin, Venezuela.

The majority of environmental micro-organisms identified with the rRNA approach have never been visualized. Thus, their reliable classification and taxonomic assignment is often difficult or even impossible. In our preliminary 18S rRNA gene sequencing work from the world's largest anoxic marine environment, the Cariaco Basin (Caribbean Sea, Venezuela), we detected a ciliate clade, designated previously as CAR_H [Stoeck, S., Taylor, G. T. & Epstein, S. S. (2003). Appl Environ Microbiol 63, 5656-5663]. Here, we combine the traditional rRNA detection method of fluorescent in situ hybridization (FISH) with scanning electron microscopy (SEM) and confirm the phylogenetic separation of the CAR_H sequences from all other ciliate classes by showing an outstanding morphological feature of this group: a unique, archway-shaped kinety surrounding the oral apparatus and extending to the posterior body end in CAR_H cells. Based on this specific feature and the molecular phylogenies, we propose a novel ciliate class, Cariacotrichea nov. cl.

Influence of micro-well biomimetic topography on intestinal epithelial Caco-2 cell phenotype.

A microfabrication approach was utilized to create topographic analogs of intestinal crypts on a polymer substrate. It was hypothesized that biomimetic crypt-like micro-architecture may induce changes in small intestinal cell (i.e. Caco-2 cell) phenotype. A test pattern of micro-well features with similar dimensions (50, 100, and 500 microm diameter, 50 microm spacing, 120 microm in depth) to the crypt structures found in native basal lamina was produced in the surface of a poly(dimethylsiloxane) (PDMS) substrate. PDMS surfaces were coated with fibronectin, seeded with intestinal-epithelial-cell-like Caco-2 cells, and cultured up to fourteen days. The cells were able to crawl along the steep side walls and migrated from the bottom to the top of the well structures, completely covering the surface by 4-5 days in culture. The topography of the PDMS substrates influenced cell spreading after seeding; cells spread faster and in a more uniform fashion on flat surfaces than on those with micro-well structures, where cell protrusions extending to micro-well side walls was evident. Substrate topography also affected cell metabolic activity and differentiation; cells had higher mitochondrial activity but lower alkaline phosphatase activity at early time points in culture (2-3 days post-seeding) when seeded on micro-well patterned PDMS substrates compared to flat substrates. These results emphasize the importance of topographical design properties of a scaffolds used for tissue engineered intestine.

Mitochondrial disruption in Drosophila apoptosis.

Mitochondrial disruption is a conserved aspect of apoptosis, seen in many species from mammals to nematodes. Despite significant conservation of other elements of the apoptotic pathway in Drosophila, a broad role for mitochondrial changes in apoptosis in flies remains unconfirmed. Here, we show that Drosophila mitochondria become permeable in response to the expression of Reaper and Hid, endogenous regulators of developmental apoptosis. Caspase activation in the absence of Reaper and Hid is not sufficient to permeabilize mitochondria, but caspases play a role in Reaper- and Hid-induced mitochondrial changes. Reaper and Hid rapidly localize to mitochondria, resulting in changes in mitochondrial ultrastructure. The dynamin-related protein, Drp1, is important for Reaper- and DNA-damage-induced mitochondrial disruption. Significantly, we show that inhibition of Reaper or Hid mitochondrial localization or inhibition of Drp1 significantly inhibits apoptosis, indicating a role for mitochondrial disruption in fly apoptosis.

Methodology of protistan discovery: from rRNA detection to quality scanning electron microscope images.

Each year, thousands of new protistan 18S rRNA sequences are detected in environmental samples. Many of these sequences are molecular signatures of new protistan species, classes, and/or kingdoms that have never been seen before. The main goal of this study was to enable visualization of these novel organisms and to conduct quality ultrastructural examination. We achieved this goal by modifying standard procedures for cell fixation, fluorescence in situ hybridization, and scanning electron microscopy (SEM) and by making these methodologies work in concert. As a result, the same individual cell can now be detected by 18S rRNA-targeted fluorochrome-labeled probes and then viewed by SEM to reveal its diagnostic morphological characteristics. The method was successfully tested on a wide range of protists (alveolates, stramenopiles, kinetoplastids, and cryptomonads). The new methodology thus opens a way for fine microscopy studies of many organisms previously known exclusively by their 18S rRNA sequences.