Hsien-Shun Liao

Tumor-Specific Formation of Enzyme-Instructed Supramolecular Self-Assemblies as Cancer Theranostics

Despite the effort of developing various nanodelivery systems, most of them suffer from undesired high uptakes by the reticuloendothelial system, such as liver and spleen. Herein we develop an endogenous phosphatase-triggered coassembly strategy to form tumor-specific indocyanine green (ICG)-doped nanofibers (5) for cancer theranostics. Based on coordinated intermolecular interactions, 5 significantly altered near-infrared absorbance of ICG, which improves the critical photoacoustic and photothermal properties. The phosphatase-instructed coassembly process, as well as its theranostic capability, was successfully conducted at different levels ranging from in vitro, living cell, tissue mimic, to in vivo. Specifically, the tumor uptake of ICG was markedly increased to 15.05 ± 3.78%ID/g, which was 25-fold higher than that of free ICG (0.59 ± 0.24%ID/g) at 4 h after intravenous injection. The resulting ultrahigh T/N ratios (>15) clearly differentiated tumors from the surrounding normal tissue. Complete tumor elimination with high therapeutic accuracy has been successfully achieved upon laser irradiation (0.8 W/cm(2), 5 min) within 24-48 h postinjection. As the first example, in vivo formation of tumor-specific ICG-doped nanofiber for PTT theranostics owns the immense potential for clinical translation of personalized nanomedicine with targeted drug delivery as well as for cancer theranostics.

Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane

Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30–300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging.

Clathrin-coat disassembly illuminates the mechanisms of Hsp70 force generation

Hsp70s use ATP hydrolysis to disrupt protein-protein associations and to move macromolecules. One example is the Hsc70- mediated disassembly of the clathrin coats that form on vesicles during endocytosis. Here, we exploited the exceptional features of these coats to test three models—Brownian ratchet, power-stroke and entropic pulling—proposed to explain how Hsp70s transform their substrates. Our data rule out the ratchet and power-stroke models and instead support a collision-pressure mechanism whereby collisions between clathrin-coat walls and Hsc70s drive coats apart. Collision pressure is the complement to the pulling force described in the entropic pulling model. We also found that self-association augments collision pressure, thereby allowing disassembly of clathrin lattices that have been predicted to be resistant to disassembly. These results illuminate how Hsp70s generate the forces that transform their substrates.

Polymeric Nanovehicle Regulated Spatiotemporal Real-Time Imaging of the Differentiation Dynamics of Transplanted Neural Stem Cells after Traumatic Brain Injury

Recent advances in neural stem cell (NSC) transplantation have led to an inspiring progress in alleviating central nervous system (CNS) damages and restoring brain functions from diseases or injuries. One challenge of NSC transplantation is directed differentiation of transplanted NSCs into desired neuronal subtypes, such as neurons, to compensate the adverse impact of brain injury; another challenge lies in the lack of tools to noninvasively monitor the dynamics of NSC differentiation after transplantation in vivo. In this study, we developed a polymer nanovehicle for morphogen sustained release to overcome the drawbacks of conventional methods to realize the long-term directed NSC differentiation in vivo. Moreover, we constructed a bicistronic vector with a unique neuron specific gene tubb3 promoter to drive reporter gene expression for real-time imaging of NSC differentiation and migration. The developed uniform nanovehicle showed efficient NSC uptake and achieved a controlled release of morphogen in cytosol to consistently stimulate NSC differentiation into neurons at a sustainably effective concentration. The spatiotemporal imaging results showed a multiplexed migration, proliferation, differentiation, and apoptosis orchestra of transplanted NSCs regulated by nanovehicles in TBI mice. The imaging results also uncovered the peak time of NSC differentiation in vivo. Although we observed only a handful of NSCs ultimately migrated to the TBI area and differentiated into neurons, those neurons were functional, ameliorating the detrimental impact of TBI. The imaging findings enabled by the nanovehicle and the neuron specific bicistronic vector provide additional understanding of the in vivo behaviors of transplanted NSCs in neuronal regenerative medicine.

Zinc-Induced Polymerization of Killer-Cell Ig-like Receptor into Filaments Promotes Its Inhibitory Function at Cytotoxic Immunological Synapses

The inhibitory function of killer cell immunoglobulin-like receptors (KIR) that bind HLA-C and block activation of human natural killer (NK) cells is dependent on zinc. We report that zinc induced the assembly of soluble KIR into filamentous polymers, as detected by electron microscopy, which depolymerized after zinc chelation. Similar KIR filaments were isolated from lysates of cells treated with zinc, and membrane protrusions enriched in zinc were detected on whole cells by scanning electron microscopy and imaging mass spectrometry. Two independent mutations in the extracellular domain of KIR, away from the HLA-C binding site, impaired zinc-driven polymerization and inhibitory function. KIR filaments formed spontaneously, without the addition of zinc, at functional inhibitory immunological synapses of NK cells with HLA-C(+) cells. Adding to the recent paradigm of signal transduction through higher order molecular assemblies, zinc-induced polymerization of inhibitory KIR represents an unusual mode of signaling by a receptor at the cell surface.

Operation of astigmatic-detection atomic force microscopy in liquid environments

The astigmatic detection system (ADS) based on commercial optical pickup head was demonstrated to achieve a sub-nanometer sensitivity in detecting the vertical movement of an object surface in air. The detection laser spot of the ADS was sub-μm and the detection bandwidth was over 80 MHz. These advantages allow detection of high-frequency mechanical resonance of very small objects, which would have many important applications in nanotechnology. In this work, we optimized the operation conditions of ADS to achieve good sensitivity in aqueous solutions. We demonstrated good contrast and good spatial resolution of cancer cells in water with the optical profilometry mode. We also built an ADS-AFM (atomic force microscopy) for imaging in water. A novel cantilever holder was designed, and the spurious peaks were suppressed down to 26.0% of the real resonance peak. Most importantly, we demonstrated that the ADS-AFM could resolve single atomic steps on a graphite substrate and image soft DNA molecules on mica in water.

Imaging soft matters in water with torsional mode atomic force microscopy

We have developed a high-sensitivity atomic force microscopy (AFM) mode operated in aqueous environment based on the torsional resonance of the cantilever. It is found that the torsional mode can achieve a good spatial resolution even with a relatively large tip. We have used this mode to image different soft materials in water, including DNA molecules and purple membrane. High-resolution images of purple membrane can be obtained at a relatively low ion concentration under a long-range electrostatic force. Thus the torsional mode allows investigators to probe surface structures and their properties under a wide range of solution conditions.

Cantilever-based mass sensor using high order resonances for liquid environment

For precisely measuring the mass variation, different modes of cantilever-based mass sensor were developed. The dynamic mode applied for mass sensor has advantages of less thermal drift and high mass sensitivity. However, mass sensor is usually operated in liquid environment for bio-chemical examination, but the liquid viscosity significantly dampens the performance of the dynamic mode. In this paper, the high order resonances are proposed for increasing the mass sensitivity in liquid. For comprehending the influence on the mass sensitivity, we analytically and experimentally studied the performances of the unmodified commercial cantilever the modified cantilever, which is made of commercial cantilever and processed to derive different shapes with mass variation. And in their thermal fluctuation spectra, the first five order resonances are detected for comparison. The mass sensitivity −5.397 Hz/pg of the fifth order resonance is 94 times that of the first order resonance of −0.057 Hz/pg.

High-speed atomic force microscope based on an astigmatic detection system

High-speed atomic force microscopy (HS-AFM) enables visualizing dynamic behaviors of biological molecules under physiological conditions at a temporal resolution of 1s or shorter. A small cantilever with a high resonance frequency is crucial in increasing the scan speed. However, detecting mechanical resonances of small cantilevers is technically challenging. In this study, we constructed an atomic force microscope using a digital versatile disc (DVD) pickup head to detect cantilever deflections. In addition, a flexure-guided scanner and a sinusoidal scan method were implemented. In this work, we imaged a grating sample in air by using a regular cantilever and a small cantilever with a resonance frequency of 5.5 MHz. Poor tracking was seen at the scan rate of 50 line/s when a cantilever for regular AFM imaging was used. Using a small cantilever at the scan rate of 100 line/s revealed no significant degradation in the topographic images. The results indicate that a smaller cantilever can achieve a higher scan rate and superior force sensitivity. This work shows the potential for using a DVD pickup head in future HS-AFM technology.

Torsional resonance mode atomic force microscopy in liquid with Lorentz force actuation

In this work, we present a design based on Lorentz force induction to excite pure torsional resonances of different types of cantilevers in air as well as in water. To demonstrate the atomic force microscopy imaging capability, the phase-modulation torsional resonance mode is employed to resolve fine features of purple membranes in a buffer solution. Most importantly, force-versus-distance curves using a relatively stiff cantilever can clearly detect the characteristic oscillatory profiles of hydration layers at a water-mica interface, indicating the high force sensitivity of the torsional mode. The high resonance frequencies and high quality-factors for the torsional mode may be of great potential for high-speed and high-sensitivity imaging in aqueous environment.