Hyunjun Cho

Multiple selection filters ensure accurate tail-anchored membrane protein targeting

Accurate protein localization is crucial to generate and maintain organization in all cells. Achieving accuracy is challenging, as the molecular signals that dictate a protein’s cellular destination are often promiscuous. A salient example is the targeting of an essential class of tail-anchored (TA) proteins, whose sole defining feature is a transmembrane domain near their C-terminus. Here we show that the Guided Entry of Tail-anchored protein (GET) pathway selects TA proteins destined to the endoplasmic reticulum (ER) utilizing distinct molecular steps, including differential binding by the co-chaperone Sgt2 and kinetic proofreading after ATP hydrolysis by the targeting factor Get3. Further, the different steps select for distinct physicochemical features of the TA substrate. The use of multiple selection filters may be general to protein biogenesis pathways that must distinguish correct and incorrect substrates based on minor differences. DOI: http://dx.doi.org/10.7554/eLife.21301.001

Mechanisms of Tail-Anchored Membrane Protein Targeting and Insertion

Proper localization of membrane proteins is essential for the function of biological membranes and for the establishment of organelle identity within a cell. Molecular machineries that mediate membrane protein biogenesis need to not only achieve a high degree of efficiency and accuracy, but also prevent off-pathway aggregation events that can be detrimental to cells. The posttranslational targeting of tail-anchored proteins (TAs) provides tractable model systems to probe these fundamental issues. Recent advances in understanding TA-targeting pathways reveal sophisticated molecular machineries that drive and regulate these processes. These findings also suggest how an interconnected network of targeting factors, cochaperones, and quality control machineries together ensures robust membrane protein biogenesis.

Surface-Enhanced Raman Spectroscopy-Based Label-Free Insulin Detection at Physiological Concentrations for Analysis of Islet Performance

Label-free optical detection of insulin would allow in vitro assessment of pancreatic cell functions in their natural state and expedite diabetes-related clinical research and treatment; however, no existing method has met these criteria at physiological concentrations. Using spatially uniform 3D gold-nanoparticle sensors, we have demonstrated surface-enhanced Raman sensing of insulin in the secretions from human pancreatic islets under low and high glucose environments without the use of labels such as antibodies or aptamers. Label-free measurements of the islet secretions showed excellent correlation among the ambient glucose levels, secreted insulin concentrations, and measured Raman-emission intensities. When excited at 785 nm, plasmonic hotspots of the densely arranged 3D gold-nanoparticle pillars as well as strong interaction between sulfide linkages of the insulin molecules and the gold nanoparticles produced highly sensitive and reliable insulin measurements down to 100 pM. The sensors exhibited a dynamic range of 100 pM to 50 nM with an estimated detection limit of 35 pM, which covers the reported concentration range of insulin observed in pancreatic cell secretions. The sensitivity of this approach is approximately 4 orders of magnitude greater than previously reported results using label-free optical approaches, and it is much more cost-effective than immunoassay-based insulin detection widely used in clinics and laboratories. These promising results may open up new opportunities for insulin sensing in research and clinical applications.

Simple, Large-Scale Fabrication of Uniform Raman-Enhancing Substrate with Enhancement Saturation

It is well-known that gold nanoparticle (AuNP) clusters generate strong surface-enhanced Raman scattering (SERS). In order to produce spatially uniform Raman-enhancing substrates at a large scale, we synthesized vertically perforated three-dimensional (3D) AuNP stacks. The 3D stacks were fabricated by first hydrothermally synthesizing ZnO nanowires perpendicular to silicon wafers followed by repetitively performing liquid-phase deposition of AuNPs on the tops and side surfaces of the nanowires. During the deposition process, the nanowires were shown to gradually dissolve away, leaving hollow vestiges or perforations surrounded by stacks of AuNPs. Simulation studies and experimental measurements reveal these nanoscale perforations serve as light paths that allow the excitation light to excite deeper regions of the 3D stacks for stronger overall Raman emission. Combined with properly sized nanoparticles, this feature maximizes and saturates the Raman enhancement at 1-pM sensitivity across the entire wafer-scale substrate, and the saturation improves the wafer-scale uniformity by a factor of 6 when compared to nanoparticle layers deposited directly on a silicon wafer substrate. Using the 3D-stacked substrates, quantitative sensing of adenine molecules yielded concentrations measurements within 10% of the known value. Understanding the enhancing mechanisms and engineering the 3D stacks have opened a new method of harnessing the intense SERS observed in nanoparticle clusters and realize practical SERS substrates with significantly improved uniformity suitable for quantitative chemical sensing.

Substrate relay in an Hsp70‐cochaperone cascade safeguards tail‐anchored membrane protein targeting

Membrane proteins are aggregation‐prone in aqueous environments, and their biogenesis poses acute challenges to cellular protein homeostasis. How the chaperone network effectively protects integral membrane proteins during their post‐translational targeting is not well understood. Here, biochemical reconstitutions showed that the yeast cytosolic Hsp70 is responsible for capturing newly synthesized tail‐anchored membrane proteins (TAs) in the soluble form. Moreover, direct interaction of Hsp70 with the cochaperone Sgt2 initiates a sequential series of TA relays to the dedicated TA targeting factor Get3. In contrast to direct loading of TAs to downstream chaperones, stepwise substrate loading via Hsp70 maintains the solubility and targeting competence of TAs, ensuring their efficient delivery to the endoplasmic reticulum (ER). Inactivation of cytosolic Hsp70 severely impairs TA translocation in vivo. Our results demonstrate a new role of cytosolic Hsp70 in directly assisting the targeting of an essential class of integral membrane proteins and provide a paradigm for how “substrate funneling” through a chaperone cascade preserves the conformational quality of nascent membrane proteins during their biogenesis.

Scanning confocal vibrometer microscope for vibration analysis of energy-harvesting MEMS in wearables

Abstract We present a scanning confocal laser-Doppler vibrometer microscope for sensitive, contactless measurement of microelectromechanical systems (MEMS). This systems enables the dynamic analysis up to 3.2 MHz with a lateral resolution of few micrometers. We show measurements on developed MEMS for vocal-energy harvesting in wearables and medical implants. For efficient harvesting a cantilever beam with a serpentine form was designed with a fundamental resonance at 200 Hz. We verified the simulated mode shapes with our vibration measurements. The observed deviations in resonance frequencies between simulation and measurement are due to modelling and manufacturing dissimilarities. Zusammenfassung Wir stellen ein scannendes konfokales Laser-Doppler-Vibrometermikroskop für die empfindliche und berührungslose Messung von Mikrosystemen vor. Unser System erlaubt die dynamische Analyse der Bauteile bis zu einer Frequenz von 3.2 MHz bei einer lateralen Auflösung von wenigen Mikrometern. Wir zeigen Messungen an entwickelten Mikrosystemen zum Energie-Harvesting aus Stimmanregung für Wearables und medizinische Implantate. Für das effiziente Harvesting wurde ein serpentinenförmiger Cantilever entwickelt, der eine Resonanz bei 200 Hz besitzt. Wir verifizierten die simulierten Schwingformen mit unseren Schwingungsmessungen. Abweichungen der gemessenen Resonanzfrequenzen zwischen der Simulation und den Messungen sind auf Unterschiede zwischen Modell und gefertigtem Bauteil zurückzuführen.

Glucose measurement using Surface Enhanced Raman Scattering

Summary form only given. Surface Enhanced Raman Scattering (SERS) has a great potential to serve as a monitoring technology for biomolecules, but sensing biomolecules for practical purposes have remained challenging for two reasons. One of the challenges is securing SERS substrates with uniform spatial enhancement that is crucial for quantitative measurements, and the other is finding proper linker molecules that will promote the surface enhancement. To address these challenges, we have been developing a new approach of using highly sensitive surface enhanced Raman scattering (SERS) platform for glucose sensing. In the presentation, I will discuss the fabrication of high performance 3D SERS substrate based on straightforward, two successive wet chemical processes, with experimentally proven strong enhancement and excellent spatial uniformity as well as the use of new linker molecules for making glucose-specific SERS substrates and their use in performing quantitative glucose measurements. Glucose sensing results from different development stages will be discussed.

ZnO-nanowire morphology optimization for glucose-SERS sensing

Current technology requires diabetics to undergo painful, inconvenient, and discontinuous measurement processes several times a day. Thus, more convenient ways of measuring glucose by utilizing surface enhanced Raman spectroscopy (SERS) technique can be highly desirable. To accomplish commercially viable SERS technologies for glucose detection, an optimal substrate must be designed with higher electromagnetic (EM) enhancement and better spatial uniformity, and many parameters must be taken into consideration, such as nanoparticle sizes, shapes, dielectric environment, which all affect the EM enhancement and resonance properties of the designed structures. Our straightforward two-step substrate fabrication process involves (1) the hydrothermal synthesis to create ZnO-NWs perpendicularly standing on the substrate and (2) the liquid phase deposition (LPD) to create Au NPs on ZnO-NWs. In the presentation, we will share our most updated results on NW synthesis outcomes and Raman measurements. We will discuss (1) our extensively modified synthesis process, which includes the types of chemical compounds used, time-based synthesis temperature adjustments, and acidity levels; (2) resulting NW morphologies with different NW growth rates, lengths, diameters, and branching/tapering geometries; and (3) Raman enhancement and spatial uniformity on a large scale.

Efficient power generation from vocal folds vibrations for medical electronic implants

The availability of practical, implantable, efficient power generators will proliferate the use of medical electronic implants that can be very useful for treating and managing various medical conditions. Using a vibration-driven power generator, we have successfully generated 0.3-mW/cm2 of electric power continuously from the acousto-mechanical vibrations that originate from the human vocal folds and propagate along the skeletal frame and air passage throughout the head and neck. Our energy harvesters are highly efficient because vocal vibrations excite them at their designed resonant frequencies at 100 and 200 Hz, which are the dominant vocal vibrations of men and women, respectively. In addition, we use laser micromachining to pattern single crystal lead-zirconate-titanate (PZT) sheets for better efficiency. Our harvesters are designed to fit into a square area (1×1 cm2 or smaller) so that they can form a flexible large array to generate more power.