Chaeyeon Song

Simple super-resolution live-cell imaging based on diffusion-assisted Förster resonance energy transfer

Despite the recent development of several super-resolution fluorescence microscopic techniques, there are still few techniques that can be readily employed in conventional imaging systems. We present a very simple, rapid, general and cost-efficient super-resolution imaging method, which can be directly employed in a simple fluorescent imaging system with general fluorophores. Based on diffusion-assisted Förster resonance energy transfer (FRET), fluorescent donor molecules that label specific target structures can be stochastically quenched by diffusing acceptor molecules, thereby temporally separating otherwise spatially overlapped fluorescence signals and allowing super-resolution imaging. The proposed method provides two- to three-fold-enhancement in spatial resolution, a significant optical sectioning property, and favorable temporal resolution in live-cell imaging. We demonstrate super-resolution live-cell dynamic imaging using general fluorophores in a standard epi-fluorescence microscope with light-emitting diode (LED) illumination. Due to the simplicity of this approach, we expect that the proposed method will prove an attractive option for super-resolution imaging.

Breathing, crawling, budding, and splitting of a liquid droplet under laser heating

The manipulation of droplets with sizes on the millimetre scale and below has attracted considerable attention over the past few decades for applications in microfluidics, biology, and chemistry. In this paper, we report the response of an oil droplet floating in an aqueous solution to local laser heating. Depending on the laser power, distinct dynamic transitions of the shape and motion of the droplet are observed, namely, breathing, crawling, budding, and splitting. We found that the selection of the dynamic modes is determined by dynamic instabilities due to the interplay between the convection flows and capillary effects. Our findings can be useful for constructing microfluidic devices to control the motion and shape of a small droplet by simply altering the laser power, and for understanding thermal convective systems with fully soft boundaries.

Paclitaxel suppresses Tau-mediated microtubule bundling in a concentration-dependent manner.

BACKGROUND Microtubules (MTs) are protein nanotubes comprised of straight protofilaments (PFs), head to tail assemblies of αβ-tubulin heterodimers. Previously, it was shown that Tau, a microtubule-associated protein (MAP) localized to neuronal axons, regulates the average number of PFs in microtubules with increasing inner radius observed for increasing Tau/tubulin-dimer molar ratio ΦTau at paclitaxel/tubulin-dimer molar ratio ΛPtxl=1/1. METHODS We report a synchrotron SAXS and TEM study of the phase behavior of microtubules as a function of varying concentrations of paclitaxel (1/32≤ΛPtxl≤1/4) and Tau (human isoform 3RS, 0≤Φ3RS≤1/2) at room temperature. RESULTS Tau and paclitaxel have opposing regulatory effects on microtubule bundling architectures and microtubule diameter. Surprisingly and in contrast to previous results at ΛPtxl=1/1 where microtubule bundles are absent, in the lower paclitaxel concentration regime (ΛPtxl≤1/4), we observe both microtubule doublets and triplets with increasing Tau. Furthermore, increasing paclitaxel concentration (up to ΛPtxl=1/1) slightly decreased the average microtubule diameter (by ~1 PF) while increasing Tau concentration (up to Φ3RS=1/2) significantly increased the diameter (by ~2-3 PFs). CONCLUSIONS The suppression of Tau-mediated microtubule bundling with increasing paclitaxel is consistent with paclitaxel seeding more, but shorter, microtubules by rapidly exhausting tubulin available for polymerization. Microtubule bundles require the aggregate Tau-Tau attractions along the microtubule length to overcome individual microtubule thermal energies disrupting bundles. GENERAL SIGNIFICANCE Investigating MAP-mediated interactions between microtubules (as it relates to in vivo behavior) requires the elimination or minimization of paclitaxel.

The effect of multivalent cations and Tau on paclitaxel-stabilized microtubule assembly, disassembly, and structure

In this review we describe recent studies directed at understanding the formation of novel nanoscale assemblies in biological materials systems. In particular, we focus on the effects of multivalent cations, and separately, of microtubule-associated protein (MAP) Tau, on microtubule (MT) ordering (bundling), MT disassembly, and MT structure. Counter-ion directed bundling of paclitaxel-stabilized MTs is a model electrostatic system, which parallels efforts to understand MT bundling by intrinsically disordered proteins (typically biological polyampholytes) expressed in neurons. We describe studies, which reveal an unexpected transition from tightly spaced MT bundles to loose bundles consisting of strings of MTs as the valence of the cationic counter-ion decreases from Z=3 to Z=2. This transition is not predicted by any current theories of polyelectrolytes. Notably, studies of a larger series of divalent counter-ions reveal strong ion specific effects. Divalent counter-ions may either bundle or depolymerize paclitaxel-stabilized MTs. The ion concentration required for depolymerization decreases with increasing atomic number. In a more biologically related system we review synchrotron small angle x-ray scattering (SAXS) studies on the effect of the Tau on the structure of paclitaxel-stabilized MTs. The electrostatic binding of MAP Tau isoforms leads to an increase in the average radius of microtubules with increasing Tau coverage (i.e. a re-distribution of protofilament numbers in MTs). Finally, inspired by MTs as model nanotubes, we briefly describe other more robust lipid-based cylindrical nanostructures, which may have technological applications, for example, in drug encapsulation and delivery.

High-throughput nanoscale lipid vesicle synthesis in a semicircular contraction-expansion array microchannel

Towards potential applications in the field of nanomedicine, a new high-throughput synthesis method of lipid vesicles with tunable size as well as enhanced monodispersity is demonstrated using a semicircular contraction-expansion array (CEA) microchannel. Lipid vesicles are generated in the CEA microchannel by injecting lipids in isopropyl alcohol as a sample flow and phosphate buffered saline as a buffer flow, leading to spontaneous formation of lipid vesicles. In the CEA microchannel, Dean vortices cause three-dimensional (3D) lamination by continuously splitting and redirecting fluid streams, resulting in enhancement of fluid mixing. When considered only 3D laminating effect, it showed the best mixing efficiency in the range of flow rates of 12–15 mL/h. However, shear force effect also gives a strong influence on the formation of lipid vesicles, leading to the smallest size and uniform size distribution of lipid vesicles at a total flow rate of 18 mL/h. Consequently, from the interplay between high shear stress and 3D laminating effect, the lipid vesicles were generated with monodispersity and high throughput. The formation of lipid vesicles can be controlled with a total flow rate and a flow rate ratio between the sample and buffer fluids. The throughput of the lipid generation in the CEA microchannel was 10 times higher than previous works. In addition, the generated lipid vesicle populations were confirmed using a cryogenic transmission electron microscopy (cryo-TEM) technique.

Synchrotron small-angle X-ray scattering and electron microscopy characterization of structures and forces in microtubule/Tau mixtures

Tau, a neuronal protein known to bind to microtubules and thereby regulate microtubule dynamic instability, has been shown recently to not only undergo conformational transitions on the microtubule surface as a function of increasing microtubule coverage density (i.e., with increasing molar ratio of Tau to tubulin dimers) but also to mediate higher-order microtubule architectures, mimicking fascicles of microtubules found in the axon initial segment. These discoveries would not have been possible without fine structure characterization of microtubules, with and without applied osmotic pressure through the use of depletants. Herein, we discuss the two primary techniques used to elucidate the structure, phase behavior, and interactions in microtubule/Tau mixtures: transmission electron microscopy and synchrotron small-angle X-ray scattering. While the former is able to provide striking qualitative images of bundle morphologies and vacancies, the latter provides angstrom-level resolution of bundle structures and allows measurements in the presence of in situ probes, such as osmotic depletants. The presented structural characterization methods have been applied both to equilibrium mixtures, where paclitaxel is used to stabilize microtubules, and also to dissipative nonequilibrium mixtures at 37°C in the presence of GTP and lacking paclitaxel.

The Effect of Multivalent Cations on Microtubule-Protein Tau Ordering

In previous studies, it was found that paclitaxel-stabilized microtubules (MTs), hollow protein nanotubes comprised of assembled αβ-tubulin heterodimers, spontaneously assemble into bundles above a critical concentration (∼1.5 mM Spermine) of tetravalent spermine (PNAS 2004, 101, 16099). Further, at concentrations of spermine several-fold higher (∼10 mM Spermine), paclitaxel-stabilized MT bundles (BMT) quickly become unstable and undergo a shape transformation to bundles of inverted tubulin tubules (BITT), the outside surface of which corresponds to the inner surface of the BMT tubules (Nature Materials 2014, 13, 195). Here we will report on our current study of protein Tau-microtubule ordering (in an active system) in the absence of paclitaxel and, in particular, the effect of biological cations on microtubule self-assembly using transmission electron microscopy (TEM) and synchrotron small-angle X-ray scattering (SAXS).