Live analysis of endodermal layer formation identifies random walk as a novel gastrulation movement.

During gastrulation, dramatic movements rearrange cells into three germ layers expanded over the entire embryo [1-3]. In fish, both endoderm and mesoderm are specified as a belt at the embryo margin. Mesodermal layer expansion is achieved through the combination of two directed migrations. The outer ring of precursors moves toward the vegetal pole and continuously seeds mesodermal cells inside the embryo, which then reverse their movement in the direction of the animal pole [3-6]. Unlike mesoderm, endodermal cells internalize at once and must therefore adopt a different strategy to expand over the embryo [7, 8]. With live imaging of YFP-expressing zebrafish endodermal cells, we demonstrate that in contrast to mesoderm, internalized endodermal cells display a nonoriented/noncoordinated movement fit by a random walk that rapidly disperses them over the yolk surface. Transplantation experiments reveal that this behaviour is largely cell autonomous, induced by TGF-beta/Nodal, and dependent on the downstream effector Casanova. At midgastrulation, endodermal cells switch to a convergence movement. We demonstrate that this switch is triggered by environmental cues. These results uncover random walk as a novel Nodal-induced gastrulation movement and as an efficient strategy to transform a localized cell group into a layer expanded over the embryo.

Velocity, processivity, and individual steps of single myosin V molecules in live cells.

We report the tracking of single myosin V molecules in their natural environment, the cell. Myosin V molecules, labeled with quantum dots, are introduced into the cytoplasm of living HeLa cells and their motion is recorded at the single molecule level with high spatial and temporal resolution. We perform an intracellular measurement of key parameters of this molecular transporter: velocity, processivity, step size, and dwell time. Our experiments bridge the gap between in vitro single molecule assays and the indirect measurements of the motor features deduced from the tracking of organelles in live cells.

Tracking individual proteins in living cells using single quantum dot imaging.

Single quantum dot imaging is a powerful approach to probe the complex dynamics of individual biomolecules in living systems. Due to their remarkable photophysical properties and relatively small size, quantum dots can be used as ultrasensitive detection probes. They make possible the study of biological processes, both in the membrane or in the cytoplasm, at a truly molecular scale and with high spatial and temporal resolutions. This chapter presents methods used for tracking single biomolecules coupled to quantum dots in living cells from labeling procedures to the analysis of the quantum dot motion.

Tracking individual membrane proteins using quantum dots.

Single-particle tracking of individual membrane molecules is now the method of choice to decipher the molecular organization of the plasma membrane. By labeling proteins or lipids with latex beads, 40-nm gold nanoparticles, or small organic fluorophores, it is possible to analyze the mechanisms controlling their lateral dynamics. Semiconductor quantum dots (QDs) provide several advantages for tracking membrane molecules: (1) Their size, which is intermediate between those of organic dyes (1-4 nm) and large beads (100 nm to 1 µm), remains close to the molecular scale; (2) their photostability allows observation over long durations; (3) parallel detection of multiple spots in a field of view is easy; and (4) multicolor imaging is facilitated by their absorption properties. In general, the labeling of membrane molecules is based on the targeting of an extracellular epitope by a tagged antibody or ligand. By progressively decreasing the concentration of markers, a regime is reached where isolated tags can be detected and tracked. We present here a protocol based on the successive use of biotinylated primary antibodies and streptavidin-coated QDs.

Purification of functionalized quantum dots.

Semiconductor quantum dots (QDs) are fluorescent nanoparticles that can be used for biological imaging. Because of their brightness and photostability, which is far superior to those of organic dyes and fluorescent proteins, they can be detected at the single-particle level over long periods of time using standard fluorescence microscopy techniques. QDs can be conjugated to biomolecules and then used to track the motion of these molecules. Commercial, soluble QDs are available either unconjugated or functionalized with specific biomolecules. In the latter case, biomolecules such as streptavidin, Protein A, or antibodies are attached to the QD surface. Free biomolecules are often present in the QD solution, and these can be detrimental for live-cell imaging or other fluorescence assays. It is thus desirable to purify the functionalized QDs from these contaminating free biomolecules using size-exclusion chromatography. This article describes a simple procedure for purifying functionalized QDs using MicroSpin SR-400 columns.

Ultrasensitive imaging in live cells using fluorescent quantum dots.

Semiconductor quantum dots (QDs) are fluorescent nanoparticles that can be used for biological imaging. Because of their brightness and photostability, which are far superior to those of organic dyes and fluorescent proteins, they can be detected at the single-particle level over long periods of time using standard fluorescence microscopy techniques. QDs can be conjugated to biomolecules and then used to track the motion of these molecules.

Tracking individual intracellular proteins using quantum dots.

Single-molecule detection of quantum dot (QD)-tagged proteins located in the cytoplasm or the nucleus presents a significant challenge in live-cell imaging. First, QDs must enter the cell cytoplasm and reach their molecular target but still preserve cell integrity. Second, the fluorescence of individual QDs must be detected in a noisy environment and distinguished from the autofluorescence of intracellular compartments and organelles. Finally, molecular motion in the cytosol is likely to be three-dimensional, compared to two-dimensional diffusion in the membrane. In this protocol, streptavidin-coated QDs (QD-SAVs) are coupled with biotinylated proteins (ideally in a 1:1 molar ratio) in hypertonic medium. The coupled reaction product (QD-P) is then added to live cells (e.g., mammalian HeLa cells) using a cell-loading technique based on the osmotic lysis of pinocytic vesicles. The osmotic lysis of pinocytic vesicles in hypotonic solution does not alter the viability of cultured cells and does not result in lysosomal enzyme release. By comparison with other internalization techniques, such as microinjection, this method is much simpler and more reproducible because all of the cells are simultaneously loaded under the same conditions. It can provide quantitative information on the movement of intracellular biomolecules, enhancing our understanding of complex biological processes such as signal transduction, cell division, or motility.

Mechanically induced helix-coil transition in biopolymer networks.

The quasi-equilibrium evolution of the helical fraction occurring in a biopolymer network (gelatin gel) under an applied stress has been investigated by observing modulation in its optical activity. Its variation with the imposed chain extension is distinctly nonmonotonic and corresponds to the transition of initially coiled strands to induced left-handed helices. The experimental results are in qualitative agreement with theoretical predictions of helices induced on chain extension. This new effect of mechanically stimulated helix-coil transition has been studied further as a function of the elastic properties of the polymer network: crosslink density and network aging.

Tracking individual kinesin motors in living cells using single quantum-dot imaging.

We report a simple method using semiconductor quantum dots (QDs) to track the motion of intracellular proteins with a high sensitivity. We characterized the in vivo motion of individual QD-tagged kinesin motors in living HeLa cells. Single-molecule measurements provided important parameters of the motor, such as its velocity and processivity, as well as an estimate of the force necessary to carry a QD. Our measurements demonstrate the importance of single-molecule experiments in the investigation of intracellular transport as well as the potential of single quantum-dot imaging for the study of important processes such as cellular trafficking, cell polarization, and division.