Hung-Jen Wu

Ciliated micropillars for the microfluidic-based isolation of nanoscale lipid vesicles

We fabricated a microfluidic device consisting of ciliated micropillars, forming a porous silicon nanowire-on-micropillar structure. We demonstrated that the prototype device can preferentially trap exosome-like lipid vesicles, while simultaneously filtering out proteins and cell debris. Trapped lipid vesicles can be recovered intact by dissolving the porous nanowires in PBS buffer.

Membrane-protein binding measured with solution-phase plasmonic nanocube sensors

We describe a solution-phase sensor of lipid-protein binding based on localized surface plasmon resonance (LSPR) of silver nanocubes. When silica-coated nanocubes are mixed in a suspension of lipid vesicles, supported membranes spontaneously assemble on their surfaces. Using a standard laboratory spectrophotometer, we calibrated the LSPR peak shift due to protein binding to the membrane surface and then characterized the lipid-binding specificity of a pleckstrin homology domain protein.

Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics

Outliers dominate signaling at cell membrane SOS enzymes act at cell membranes to activate Ras, a regulatory protein often overactive in cancer cells. Iversen et al. devised a system where they could observe the activity of individual enzymes at work. The single SOS molecules occupied stable states that varied greatly in their catalytic activity. Regulation appeared to occur by altering the time spent in active states. The overall activity of SOS was determined by just a few molecules that achieved the highest catalytic activity. The methods described should allow further detailed kinetic analysis of this and other signaling events that occur at the cell membrane — properties that it is not possible to discern from bulk biochemical measurements. Science, this issue p. 50 Single-molecule measurements reveal insights into regulation of the small GTPase Ras. Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras–guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average.

Altered Actin Centripetal Retrograde Flow in Physically Restricted Immunological Synapses

Antigen recognition by T cells involves large scale spatial reorganization of numerous receptor, adhesion, and costimulatory proteins within the T cell-antigen presenting cell (APC) junction. The resulting patterns can be distinctive, and are collectively known as the immunological synapse. Dynamical assembly of cytoskeletal network is believed to play an important role in driving these assembly processes. In one experimental strategy, the APC is replaced with a synthetic supported membrane. An advantage of this configuration is that solid structures patterned onto the underlying substrate can guide immunological synapse assembly into altered patterns. Here, we use mobile anti-CD3ε on the spatial-partitioned supported bilayer to ligate and trigger T cell receptor (TCR) in live Jurkat T cells. Simultaneous tracking of both TCR clusters and GFP-actin speckles reveals their dynamic association and individual flow patterns. Actin retrograde flow directs the inward transport of TCR clusters. Flow-based particle tracking algorithms allow us to investigate the velocity distribution of actin flow field across the whole synapse, and centripetal velocity of actin flow decreases as it moves toward the center of synapse. Localized actin flow analysis reveals that, while there is no influence on actin motion from substrate patterns directly, velocity differences of actin are observed over physically trapped TCR clusters. Actin flow regains its velocity immediately after passing through confined TCR clusters. These observations are consistent with a dynamic and dissipative coupling between TCR clusters and viscoelastic actin network.

Silicon Micro- and Nanofabrication for Medicine

This manuscript constitutes a review of several innovative biomedical technologies fabricated using the precision and accuracy of silicon micro- and nanofabrication. The technologies to be reviewed are subcutaneous nanochannel drug delivery implants for the continuous tunable zero-order release of therapeutics, multi-stage logic embedded vectors for the targeted systemic distribution of both therapeutic and imaging contrast agents, silicon and porous silicon nanowires for investigating cellular interactions and processes as well as for molecular and drug delivery applications, porous silicon (pSi) as inclusions into biocomposites for tissue engineering, especially as it applies to bone repair and regrowth, and porous silica chips for proteomic profiling. In the case of the biocomposites, the specifically designed pSi inclusions not only add to the structural robustness, but can also promote tissue and bone regrowth, fight infection, and reduce pain by releasing stimulating factors and other therapeutic agents stored within their porous network. The common material thread throughout all of these constructs, silicon and its associated dielectrics (silicon dioxide, silicon nitride, etc.), can be precisely and accurately machined using the same scalable micro- and nanofabrication protocols that are ubiquitous within the semiconductor industry. These techniques lend themselves to the high throughput production of exquisitely defined and monodispersed nanoscale features that should eliminate architectural randomness as a source of experimental variation thereby potentially leading to more rapid clinical translation.

Myosin IIA Modulates T Cell Receptor Transport and CasL Phosphorylation during Early Immunological Synapse Formation

Activation of T cell receptor (TCR) by antigens occurs in concert with an elaborate multi-scale spatial reorganization of proteins at the immunological synapse, the junction between a T cell and an antigen-presenting cell (APC). The directed movement of molecules, which intrinsically requires physical forces, is known to modulate biochemical signaling. It remains unclear, however, if mechanical forces exert any direct influence on the signaling cascades. We use T cells from AND transgenic mice expressing TCRs specific to the moth cytochrome c 88–103 peptide, and replace the APC with a synthetic supported lipid membrane. Through a series of high spatiotemporal molecular tracking studies in live T cells, we demonstrate that the molecular motor, non-muscle myosin IIA, transiently drives TCR transport during the first one to two minutes of immunological synapse formation. Myosin inhibition reduces calcium influx and colocalization of active ZAP-70 (zeta-chain associated protein kinase 70) with TCR, revealing an influence on signaling activity. More tellingly, its inhibition also significantly reduces phosphorylation of the mechanosensing protein CasL (Crk-associated substrate the lymphocyte type), raising the possibility of a direct mechanical mechanism of signal modulation involving CasL.

Characterization of dynamic actin associations with T-cell receptor microclusters in primary T cells

T cell triggering through T-cell antigen receptors (TCRs) results in spatial assembly of the receptors on multiple length scales. This assembly is mediated by the T cell actin cytoskeleton, which reorganizes in response to TCR phosphorylation and then induces the coalescence of TCRs into microclusters, followed by their unification into a micrometer-scale structure. The exact outcomes of the association of TCRs with a dynamic and fluctuating actin network across these length scales are not well characterized, but it is clear that weak and transient interactions at the single-molecule level sum to yield significant receptor rearrangements at the plasma membrane. We used the hybrid live cell–nanopatterned supported lipid bilayer system to quantitatively probe the actin–TCR interaction in primary T cells. A specialized tracking algorithm revealed that actin slows as it passes over TCR clusters in a direction-dependent manner with respect to the resistance against TCR motion. We also observed transient actin enrichments at sites corresponding to putative TCR clusters that far exceeded pure stochastic fluctuations and described an image time-autocorrelation analysis method to quantify these accumulations.

Quantification of circulating Mycobacterium tuberculosis antigen peptides allows rapid diagnosis of active disease and treatment monitoring

Significance Active Mycobacterium tuberculosis (Mtb) infections represent a significant global health threat, but can be difficult to diagnose and manage owing to the nonquantitative nature and relatively poor performance of current frontline sputum-based diagnostic assays, which can be further degraded by certain Mtb manifestations. This study describes the development of a rapid and quantitative blood-based assay with high sensitivity and specificity for active Mtb infections that can be used to monitor responses to antimycobacterial therapy. Our method combines antibody-labeled, energy-focusing nanodisks with high-throughput mass spectrometry to enhance the detection of Mtb-specific peptides in digested serum samples, and should allow rapid clinical translation. This approach also should be applicable to other infectious diseases, particularly those for which conventional immunoassays exhibit suboptimal performance. Tuberculosis (TB) is a major global health threat, resulting in an urgent unmet need for a rapid, non–sputum-based quantitative test to detect active Mycobacterium tuberculosis (Mtb) infections in clinically diverse populations and quickly assess Mtb treatment responses for emerging drug-resistant strains. We have identified Mtb-specific peptide fragments and developed a method to rapidly quantify their serum concentrations, using antibody-labeled and energy-focusing porous discoidal silicon nanoparticles (nanodisks) and high-throughput mass spectrometry (MS) to enhance sensitivity and specificity. NanoDisk-MS diagnosed active Mtb cases with high sensitivity and specificity in a case-control study with cohorts reflecting the complexity of clinical practice. Similar robust sensitivities were obtained for cases of culture-positive pulmonary TB (PTB; 91.3%) and extrapulmonary TB (EPTB; 92.3%), and the sensitivities obtained for culture-negative PTB (82.4%) and EPTB (75.0%) in HIV-positive patients significantly outperformed those reported for other available assays. NanoDisk-MS also exhibited high specificity (87.1–100%) in both healthy and high-risk groups. Absolute quantification of serum Mtb antigen concentration was informative in assessing responses to antimycobacterial treatment. Thus, a NanoDisk-MS assay approach could significantly improve the diagnosis and management of active TB cases, and perhaps other infectious diseases as well.

Like-charge interactions between colloidal particles are asymmetric with respect to sign

Two-dimensional dispersions of colloidal particles with a range of surface chemistries and electrostatic potentials are characterized under a series of solution ionic strengths. A combination of optical imaging techniques are employed to monitor both the colloid structure and the electrostatic surface potential of individual particles in situ. We find that like-charge multiparticle interactions can be tuned from exclusively repulsive to long-range attractive by changing the particle surface composition. This behavior is strongly asymmetric with respect to the sign of the surface potential. Collective long-range attractive interactions are only observed among negatively charged particles.

Serum peptidomic biomarkers for pulmonary metastatic melanoma identified by means of a nanopore-based assay.

The significant mortality rate associated with metastatic melanoma, which exceeds the number of deaths attributed to the primary tumor, is primarily due to poor diagnosis and increased resistance to systemic therapy. Early detection and treatment of invasive melanoma are therefore crucial to increase survival rates. Low molecular weight proteins and peptides have garnered significant interest as biomarker candidates as they potentially represent a snap shot of pathological condition within the body and, by extension, the organism as a whole. We have developed a nanoporous silica-based platform to segregate the low molecular weight from the high molecular weight protein fraction to aid in the detection of peptides from serum samples using mass spectrometry. The combination of sample treatment with our platform, MALDI-TOF MS and following biostatistical analysis led to the discovery and identification of 27 peptides that are potential biomarkers associated with the development of pulmonary metastatic melanoma. We strongly believe our findings can assist to discover stage-specific peptide signatures and lead to more specific and personalized treatments for patients suffering from pulmonary metastatic melanoma.