Fabien Pinaud

Probing cellular events, one quantum dot at a time.

Monitoring the behavior of single molecules in living cells is a powerful approach to investigate the details of cellular processes. Owing to their optical, chemical and biofunctional properties, semiconductor quantum dot (QD) probes promise to be tools of choice in this endeavor. Here we review recent advances that allow ever more controlled experiments at the single-nanoparticle level in live cells. Several examples, related to membrane dynamics, cell signaling or intracellular transport, illustrate how single QD tracking can be readily used to decipher complex biological processes and address key concepts that underlie cellular organization and dynamics.

Time-gated biological imaging by use of colloidal quantum dots

The long (but not too long) fluorescence lifetime of CdSe semiconductor quantum dots was exploited to enhance fluorescence biological imaging contrast and sensitivity by time-gated detection. Significant and selective reduction of the autofluorescence contribution to the overall image was achieved, and enhancement of the signal-to-background ratio by more than an order of magnitude was demonstrated.

Dynamic partitioning of a glycosyl-phosphatidylinositol-anchored protein in glycosphingolipid-rich microdomains imaged by single-quantum dot tracking.

Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft‐associated glycosyl‐phosphatidyl‐inositol‐anchored avidin test probe (Av‐GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed high‐resolution and long‐term tracking of individual Av‐GPI and the classification of their various diffusive behaviors. Using dual‐color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av‐GPI and the location of glycosphingolipid GM1‐rich microdomains and caveolae. We show that Av‐GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1‐rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av‐GPI dynamically partition in and out of these microdomains in a cholesterol‐dependent manner. Our results provide direct evidence that cholesterol‐/sphingolipid‐rich microdomains can compartmentalize the diffusion of GPI‐anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft‐associated proteins across the plasma membrane.

Tracking bio-molecules in live cells using quantum dots

Single particle tracking (SPT) techniques were developed to explore bio-molecules dynamics in live cells at single molecule sensitivity and nanometer spatial resolution. Recent developments in quantum dots (Qdots) surface coating and bio-conjugation schemes have made them most suitable probes for live cell applications. Here we review recent advancements in using quantum dots as SPT probes for live cell experiments.

High-Affinity Labeling and Tracking of Individual Histidine-Tagged Proteins in Live Cells Using Ni2+ Tris-nitrilotriacetic Acid Quantum Dot Conjugates

Investigation of many cellular processes using fluorescent quantum dots (QDs) is hindered by the nontrivial requirements for QD surface functionalization and targeting. To address these challenges, we designed, characterized and applied QD-trisNTA, which integrates tris-nitrilotriacetic acid, a small and high-affinity recognition unit for the ubiquitous polyhistidine protein tag. Using QD-trisNTA, we demonstrate two-color QD tracking of the type-1 interferon receptor subunits in live cells, potentially enabling direct visualization of protein-protein interactions at the single molecule level.

Covalent Monofunctionalization of Peptide-Coated Quantum Dots for Single-Molecule Assays

Fluorescent probes for biological imaging of single molecules (SM) have many stringent design requirements. In the case of quantum dot (QD) probes, it remains a challenge to control their functional properties with high precision. Here, we describe the simple preparation of QDs with reduced size and monovalency. Our approach combines a peptide surface coating, stable covalent conjugation of targeting units and purification by gel electrophoresis. We precisely characterize these probes by ensemble and SM techniques and apply them to tracking individual proteins in living cells.

Self-Controlled Monofunctionalization of Quantum Dots for Multiplexed Protein Tracking in Live Cells†

Tracking the motion of individual proteins on the surface of live cells has contributed considerably towards unveiling the functional organization of proteins in the plasma membrane. Individual proteins labeled with quantum dots (QDs) can be imaged over long time periods with ultrahigh spatial and temporal resolution, yielding powerful information on the spatiotemporal dynamics of proteins at the plasma membrane in live cells. A key challenge for the application of QDs is to site-specifically attach proteins to the surface of these nanoparticles in a stoichiometric manner without affecting protein function. Several procedures for rendering surfaces of QDs biocompatible have been described, thus reducing non-specific binding and protein denaturation on the QD surface. However, functionalized biocompatible QDs used to target cell surface proteins generally result in multipoint attachment to the target proteins because the number of functional groups on the QDs is very difficult to control. Such multiply functionalized QDs induce clustering of target proteins on the cell surface, biasing not only lateral diffusion but also the functional properties of these proteins. As a consequence, increased endocytosis has been observed upon binding of QDs functionalized with multiple epidermal grow factor (EGF) molecules to cell-surface EGF receptors. Stochastic functionalization of multiple reactive sites on the QD offers the choice of obtaining only a minor fraction of the QDs with a single functional group, or a significant fraction of QDs with multiple functional groups. Preparation of homogeneous, monofunctional QDs currently relies on electrophoretic purification, which has been achieved only for very small QDs. These compact QDs are designed with very thin surface coatings, which have the disadvantage of showing relatively strong non-specific interactions. Most approaches for targeting proteins using QDs in live cells are based on biotin–streptavidin interactions, which form quasi-irreversible complexes. For multiplexed, generic labeling of proteins on the cell surface, further targeting strategies are required. We have recently described tris(hydroxymethyl)methylamine–nitrilotriacetic acid (Tris-NTA) moieties for highly specific and stable attachment of fluorophores and other functional units to histidine-tagged proteins in vitro and on the surface of live cells. The lifetime of Tris-NTA complexes with His-tagged proteins is in the order of several hours, which is well-suited to medium-term single molecule tracking applications. Herein, we have attempted to control the functionalization degree of QDs with Tris-NTA by means of electrostatic repulsion. We devised a bottom-up coupling chemistry based on a novel Tris-NTA derivative (1; Figure 1a), which comprises a thiol-terminated hepta(ethylene glycol) linker. This compound was generated in situ by reduction of the disulfide-linked dimer (1a ; see the Supporting Information, Scheme S1) and coupled to commercially available polymer-coated and amine-functionalized QDs by means of a hetero-bifunctional cross-linker (Figure 1b). Covalently attachment of 1 to surfaces modified with maleimide-functionalized polyethylene glycol (PEG) polymer brush and specific immobilization of His-tagged proteins was confirmed by label-free detection (Supporting Information, Figure S1). To control the degree of functionalization with Tris-NTA on the QD surface, the reaction of 1 with surface maleimide groups was performed at low ionic strength. Under these conditions, all QDs were reacted with Tris-NTA, as confirmed by an increase in negative charges detected by anion exchange chromatography and agarose gel electrophoreses (Figure 1c,d). These assays indicated relatively monodisperse electrostatic properties after coupling of 1, despite the fact that it was reacted at a large excess (660 mm of compound 1 to 1 mm QD). Coupling of 1 at higher ionic strength yielded QDs with a substantially higher degree of functionalization, as confirmed by a further shift of the signals both in anion exchange chromatography and agarose gel electrophoresis (Figure 1c,d). To characterize the functional properties of Tris-NTAcoupled QDs, binding to immobilized hexahistidine (H6) [*] Dr. C. You, S. Wilmes, O. Beutel, S. L chte, Y. Podoplelowa, F. Roder, C. Richter, T. Seine, D. Schaible, Prof. Dr. J. Piehler Division of Biophysics, Universit t Osnabr ck Barbarstrasse 11, 49076 Osnabr ck (Germany) Fax: (+49)541-969-2262 E-mail: piehler@uos.de Homepage: http://www.biologie.uni-osnabrueck.de/Biophysik/ Piehler/

Characterization and Mechanism of Stress-induced Translocation of 78-Kilodalton Glucose-regulated Protein (GRP78) to the Cell Surface

Background: ER stress induces cell surface translocation of GRP78/BiP. Results: GRP78 translocation to the cell surface requires its substrate binding domain and exists majorly as a peripheral protein. Conclusion: GRP78 anchors on the cell surface via interaction with other proteins, and the translocation mechanism is cell context-dependent. Significance: Learning how GRP78 exists on the cell surface is crucial for understanding its signaling regulatory functions. Glucose-regulated protein (GRP78)/BiP, a major chaperone in the endoplasmic reticulum, is recently discovered to be preferably expressed on the surface of stressed cancer cells, where it regulates critical oncogenic signaling pathways and is emerging as a target for anti-cancer therapy while sparing normal organs. However, because GRP78 does not contain classical transmembrane domains, its mechanism of transport and its anchoring at the cell surface are poorly understood. Using a combination of biochemical, mutational, FACS, and single molecule super-resolution imaging approaches, we discovered that GRP78 majorly exists as a peripheral protein on plasma membrane via interaction with other cell surface proteins including glycosylphosphatidylinositol-anchored proteins. Moreover, cell surface GRP78 expression requires its substrate binding activity but is independent of ATP binding or a membrane insertion motif conserved with HSP70. Unexpectedly, different cancer cell lines rely on different mechanisms for GRP78 cell surface translocation, implying that the process is cell context-dependent.

Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins

Single-molecule (SM) microscopy allows outstanding insight into biomolecular mechanisms in cells. However, selective detection of single biomolecules in their native environment remains particularly challenging. Here, we introduce an easy methodology that combines specific targeting and nanometer accuracy imaging of individual biomolecules in living cells. In this method, named complementation-activated light microscopy (CALM), proteins are fused to dark split-fluorescent proteins (split-FPs), which are activated into bright FPs by complementation with synthetic peptides. Using CALM, the diffusion dynamics of a controlled subset of extracellular and intracellular proteins are imaged with nanometer precision, and SM tracking can additionally be performed with fluorophores and quantum dots. In cells, site-specific labeling of these probes is verified by coincidence SM detection with the complemented split-FP fusion proteins or intramolecular single-pair Förster resonance energy transfer. CALM is simple and combines advantages from genetically encoded and synthetic fluorescent probes to allow high-accuracy imaging of single biomolecules in living cells, independently of their expression level and at very high probe concentrations.

Aromatic Aldehyde and Hydrazine Activated Peptide Coated Quantum Dots for Easy Bioconjugation and Live Cell Imaging

We present a robust scheme for preparation of semiconductor quantum dots (QDs) and cognate partners in a conjugation ready format. Our approach is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HyNic) activated peptide coated quantum dots. We demonstrate controlled preparation of antibody--QD bioconjugates for specific targeting of endogenous epidermal growth factor receptors in breast cancer cells and for single QD tracking of transmembrane proteins via an extracellular epitope. The same approach was also used for optical mapping of RNA polymerases bound to combed genomic DNA in vitro.