Tania Q. Vu

Ligand-bound quantum dot probes for studying the molecular scale dynamics of receptor endocytic trafficking in live cells.

Endocytic receptor trafficking is a complex, dynamic process underlying fundamental cell function. An integrated understanding of endocytosis at the level of single or small numbers of ligand bound-receptor complexes inside live cells is currently hampered by technical limitations. Here, we develop and test ligand nerve growth factor-bound quantum dot (NGF-QD) bioconjugates for imaging discrete receptor endocytic events inside live NGF-responsive PC12 cells. Using single particle tracking, QD hybrid gel coimmunoprecipitation, and immuno-colocalization, we illustrate and validate the use of QD-receptor complexes for imaging receptor trafficking at synchronized time points after QD-ligand-receptor binding and internalization (t = 15-150 min). The unique value of these probes is illustrated by new dynamic observations: (1) that endocytosis proceeds at strikingly regulated fashion, and (2) that diffusive and active forms of transport inside cells are rapid and efficient. QDs are powerful intracellular probes that can provide biologists with new capabilities and fresh insight for studying endocytic receptor signaling events, in real time, and at the resolution of single or small numbers of receptors in live cells.

Kinetics of G-protein–coupled receptor endosomal trafficking pathways revealed by single quantum dots

G-protein–coupled receptors (GPCRs) are the largest protein superfamily in the human genome; they comprise 30% of current drug targets and regulate diverse cellular signaling responses. The role of endosomal trafficking in GPCR signaling regulation is gaining substantial consideration. However, this process remains difficult to study due to the inability to distinguish among many individual receptors, simultaneously trafficking within multiple endosomal pathways. Here we show accurate measurement of the internalization and endosomal trafficking of single groups of serotonin (5-hydroxytryptamine, 5-HT) receptors using single quantum dot (QD) probes and quantitative colocalization. We demonstrate that the presence of a QD tag does not interfere with 5-HT receptor internalization or endosomal recycling. Direct measurements show simultaneous trafficking of the 5-HT1A receptor in two distinct endosomal recycling pathways. Single-molecule imaging of endosomal trafficking will significantly impact the understanding of cellular signaling and provide powerful tools to elucidate the actions of GPCR-targeted therapeutics.

Quantum dots for quantitative imaging: from single molecules to tissue

Since their introduction to biological imaging, quantum dots (QDs) have progressed from a little known, but attractive, technology to one that has gained broad application in many areas of biology. The versatile properties of these fluorescent nanoparticles have allowed investigators to conduct biological studies with extended spatiotemporal capabilities that were previously not possible. In this review, we focus on QD applications that provide enhanced quantitative information concerning protein dynamics and localization, including single particle tracking and immunohistochemistry, and finish by examining the prospects of upcoming applications, such as correlative light and electron microscopy and super-resolution. Advances in single molecule imaging, including multi-color and three-dimensional QD tracking, have provided new insights into the mechanisms of cell signaling and protein trafficking. New forms of QD tracking in vivo have allowed the observation of biological processes at molecular level resolution in the physiological context of the whole animal. Further methodological development of multiplexed QD-based immunohistochemistry assays should enable more quantitative analysis of key proteins in tissue samples. These advances highlight the unique quantitative data sets that QDs can provide to further our understanding of biological and disease processes.

Distinct extracellular matrix microenvironments of progenitor and carotid endothelial cells.

Endothelial cells (ECs) produce and maintain the local extracellular matrix (ECM), a critical function that contributes to EC and blood vessel health. This function is also crucial to vascular tissue engineering, where endothelialization of vascular constructs require a cell source that readily produces and maintains ECM. In this study, baboon endothelial progenitor cell (EPC) deposition of ECM (laminin, collagen IV, and fibronectin) was characterized and compared to mature carotid ECs, evaluated in both elongated and cobblestone morphologies typically found in vivo. Microfluidic micropatterning was used to create 15-microm wide adhesive lanes with 45-microm spacing to reproduce the elongated EC morphology without the influence of external forces. Both EPCs and ECs elongated on micropatterned lanes had aligned actin cytoskeleton and readily deposited ECM. EPCs deposited and remodeled the ECM to a greater extent than ECs. Since a readily produced ECM can improve graft patency, EPCs are an advantageous cell source for endothelializing vascular constructs. Furthermore, EC deposition of ECM was dependent on cell morphology, where elongated ECs deposited more collagen IV and less fibronectin compared to matched cobblestone controls. Thus micropatterned surfaces controlled EC shape and ECM deposition, which ultimately has implications for the design of tissue-engineered vascular constructs.

Microcontact printing of quantum dot bioconjugate arrays for localized capture and detection of biomolecules.

The nanometer size scale of quantum dots (QDs) along with their unique luminescent properties offers great potential as photostable, color-metrically addressable nanoparticle platforms for high-throughput detection and identification of proteins. Here we apply microcontact printing for assembling quantum dot nanoparticle arrays with retained biomolecular capture functionality onto glass surfaces. This method allows the creation of addressable QD arrays on macroscopic glass surfaces. Using fluorescence and AFM imaging, we find that microcontact-printed QDs self-assemble predominantly as monolayers with highly resolved definition. Microcontact-printed streptavidin-conjugated red QDs exhibit retained adsorption onto silane-treated glass and exhibit functionality as demonstrated by the capture of discrete groups of biotin-conjugated red QDs by printed streptavidin-green QD bioconjugates that is at the detection limit of a few discrete protein binding events. These results indicate that microcontact printing of QD bioconjugate arrays serves as a simple technique that allows localized spatial capture and sensitive detection of proteins. This technique may be useful for development of future fluorescent QD-based systems aimed at the parallel capture and detection of trace concentrations of protein.

Quantitative analysis of multivesicular bodies (MVBs) in the hypoglossal nerve: Evidence that neurotrophic factors do not use MVBs for retrograde axonal transport

Multivesicular bodies (MVBs) are defined by multiple internal vesicles enclosed within an outer, limiting membrane. MVBs have previously been quantified in neuronal cell bodies and in dendrites, but their frequencies and significance in axons are controversial. Despite lack of conclusive evidence, it is widely believed that MVBs are the primary organelle that carries neurotrophic factors in axons. Reliable information about axonal MVBs under physiological and pathological conditions is needed for a realistic assessment of their functional roles in neurons. We provide a quantitative ultrastructural analysis of MVBs in the normal postnatal rat hypoglossal nerve and under a variety of experimental conditions. MVBs were about 50 times less frequent in axons than in neuronal cell bodies or dendrites. Five distinct types of MVBs were distinguished in axons, based on MVB size, electron density, and size of internal vesicles. Although target manipulations did not significantly change MVBs in axons, dystrophic conditions such as delayed fixation substantially increased the number of axonal MVBs. Radiolabeled brain‐ and glial‐cell derived neurotrophic factors (BDNF and GDNF) injected into the tongue did not accumulate during retrograde axonal transport in MVBs, as determined by quantitative ultrastructural autoradiography, and confirmed by analysis of quantum dot‐labeled BDNF. We conclude that for axonal transport, neurotrophic factors utilize small vesicles or endosomes that can be inconspicuous at transmission electron microscopic resolution, rather than MVBs. Previous reports of axonal MVBs may be based, in part, on artificial generation of such organelles in axons due to dystrophic conditions. J. Comp. Neurol. 514:641–657, 2009.

Single particle quantum dot imaging achieves ultrasensitive detection capabilities for Western immunoblot analysis

Substantially improved detection methods are needed to detect fractionated protein samples present at trace concentrations in complex, heterogeneous tissue and biofluid samples. Here we describe a modification of traditional Western immunoblotting using a technique to count quantum-dot-tagged proteins on optically transparent PVDF membranes. Counts of quantum-dot-tagged proteins on immunoblots achieved optimal detection sensitivity of 0.2 pg and a sample size of 100 cells. This translates to a 10(3)-fold improvement in detection sensitivity and a 10(2)-fold reduction in required cell sample, compared to traditional Westerns processed using the same membrane immunoblots. Quantum dot fluorescent blinking analysis showed that detection of single QD-tagged proteins is possible and that detected points of fluorescence consist of one or a few (<9) QDs. The application of single nanoparticle detection capabilities to Western blotting technologies may provide a new solution to a broad range of applications currently limited by insufficient detection sensitivity and/or sample availability.

Heterogeneous Intracellular Trafficking Dynamics of Brain-Derived Neurotrophic Factor Complexes in the Neuronal Soma Revealed by Single Quantum Dot Tracking

Accumulating evidence underscores the importance of ligand-receptor dynamics in shaping cellular signaling. In the nervous system, growth factor-activated Trk receptor trafficking serves to convey biochemical signaling that underlies fundamental neural functions. Focus has been placed on axonal trafficking but little is known about growth factor-activated Trk dynamics in the neuronal soma, particularly at the molecular scale, due in large part to technical hurdles in observing individual growth factor-Trk complexes for long periods of time inside live cells. Quantum dots (QDs) are intensely fluorescent nanoparticles that have been used to study the dynamics of ligand-receptor complexes at the plasma membrane but the value of QDs for investigating ligand-receptor intracellular dynamics has not been well exploited. The current study establishes that QD conjugated brain-derived neurotrophic factor (QD-BDNF) binds to TrkB receptors with high specificity, activates TrkB downstream signaling, and allows single QD tracking capability for long recording durations deep within the soma of live neurons. QD-BDNF complexes undergo internalization, recycling, and intracellular trafficking in the neuronal soma. These trafficking events exhibit little time-synchrony and diverse heterogeneity in underlying dynamics that include phases of sustained rapid motor transport without pause as well as immobility of surprisingly long-lasting duration (several minutes). Moreover, the trajectories formed by dynamic individual BDNF complexes show no apparent end destination; BDNF complexes can be found meandering over long distances of several microns throughout the expanse of the neuronal soma in a circuitous fashion. The complex, heterogeneous nature of neuronal soma trafficking dynamics contrasts the reported linear nature of axonal transport data and calls for models that surpass our generally limited notions of nuclear-directed transport in the soma. QD-ligand probes are poised to provide understanding of how the molecular mechanisms underlying intracellular ligand-receptor trafficking shape cell signaling under conditions of both healthy and dysfunctional neurological disease models.

Fabrication of Submicron IrO2 Nanowire Array Biosensor Platform by Conventional Complementary Metal-Oxide-Semiconductor Process

To explore the most dynamic cross field of nanotechnology and life science, and to find a more practical method of fabricating nanodevices on a very large scale with advantages of low manufacturing cost, high reliability and reproducibility, we successfully fabricated a submicron IrO2 nanowire array biosensor platform by conventional complementary metal–oxide–semiconductor (CMOS) process. Single crystal IrO2 nanowire array was grown uniformly on a 6-in. wafer surface by chemical vapor deposition (CVD) method and patterned into submicron array clusters. The obtained clusters were positioned in a designed pattern similar to a multiple electrode array (MEA) format, and each was individually addressed. The fabrication method was found to be reliable, low cost and robust. The final chip showed excellent transparency, functionality and durability.

Spatial structure and diffusive dynamics from single-particle trajectories using spline analysis.

Single-particle tracking of biomolecular probes has provided a wealth of information about intracellular trafficking and the dynamics of proteins and lipids in the cell membrane. Conventional mean-square displacement (MSD) analysis of single-particle trajectories often assumes that probes are moving in a uniform environment. However, the observed two-dimensional motion of probe particles is influenced by the local three-dimensional geometry of the cell membrane and intracellular structures, which are rarely flat at the submicron scale. This complex geometry can lead to spatially confined trajectories that are difficult to analyze and interpret using conventional two-dimensional MSD analysis. Here we present two methods to analyze spatially confined trajectories: spline-curve dynamics analysis, which extends conventional MSD analysis to measure diffusive motion in confined trajectories; and spline-curve spatial analysis, which measures spatial structures smaller than the limits of optical resolution. We show, using simulated random walks and experimental trajectories of quantum dot probes, that differences in measured two-dimensional diffusion coefficients do not always reflect differences in underlying diffusive dynamics, but can instead be due to differences in confinement geometries of cellular structures.