Semiconductor quantum dots (QDs) are nanometer-sized fluorescent probes suitable for advanced biological imaging. We used QDs to track individual glycine receptors (GlyRs) and analyze their lateral dynamics in the neuronal membrane of living cells for periods ranging from milliseconds to minutes. We characterized multiple diffusion domains in relation to the synaptic, perisynaptic, or extrasynaptic GlyR localization. The entry of GlyRs into the synapse by diffusion was observed and further confirmed by electron microscopy imaging of QD-tagged receptors.
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.
In migrating neurons, the centrosome nucleates and anchors a polarized network of microtubules that directs organelle movements. We report here that the mother centriole of neurons migrating tangentially from the medial ganglionic eminence (MGE) assembles a short primary cilium and exposes this cilium to the cell surface by docking to the plasma membrane in the leading process. Primary cilia are built by intraflagellar transport (IFT), which is also required for Sonic hedgehog (Shh) signal transduction in vertebrates. We show that Shh pathway perturbations influenced the leading process morphology and dynamics of MGE cells. Whereas Shh favored the exit of MGE cells away from their tangential migratory paths in the developing cortex, cyclopamine or invalidation of IFT genes maintained MGE cells in the tangential paths. Our findings show that signals transmitted through the primary cilium promote the escape of future GABAergic interneurons from their tangential routes to colonize the cortical plate.
A fluorescence resonance energy transfer pair consisting of a colloidal quantum dot donor and multiple organic fluorophores as acceptors is reported and the photophysics of the system is characterized. Most nanoparticle-based biosensors reported so far use the detection of specific changes of the donor/acceptor distance under the influence of analyte binding. Our nanoparticle design on the other hand leads to sensors that detect spectral changes of the acceptor (under the influence of analyte binding) at fixed donor/acceptor distance by the introduction of the acceptor into the polymer coating. This approach allows for short acceptor-donor separation and thus for high-energy transfer efficiencies. Advantageously, the binding properties of the hydrophilic polymer coating further allows for addition of poly(ethylene glycol) shells for improved colloidal stability.
Semiconductor colloidal quantum dots (QDs) are promising fluorescent probes for biology. Initially synthesized in organic solvents, they can be dispersed in aqueous solution by noncovalent coating with amphiphilic macromolecules, which renders the particles hydrophilic and modifies their interactions with other biological compounds. Here, after coating QDs with an alkyl-modified polyacrilic acid, we investigated their colloidal properties in aqueous buffers by electrophoresis, electron microscopy, light scattering, and rate zonal centrifugation. Despite polymer dispersity and variation of the size of the inorganic nanoparticles, the polymer-dot complexes appeared relatively well-defined in terms of hydrodynamic radius and surface charge. Our data show that these complexes contain isolated QD surrounded by a polymer layer with thickness 8-10 nm. We then analyzed their interaction with giant unilamellar vesicles, either neutral or cationic, by optical microscopy. At neutral pH, we found the absence of binding of the coated particles to lipid membrane, irrespective of their lipid composition. An abrupt surface aggregation of the nanoparticles on the lipid membrane was observed in a narrow pH range (6.0-6.2), indicative of critical binding triggered by polymer properties. The overall features of QDs coated with amphiphilic polymers open the route to using these nanoparticles in vivo as optically stable probes with switchable properties.
In the developing brain, cortical GABAergic interneurons migrate long distances from the medial ganglionic eminence (MGE) in which they are generated, to the cortex in which they settle. MGE cells express the cell adhesion molecule N-cadherin, a homophilic cell-cell adhesion molecule that regulates numerous steps of brain development, from neuroepithelium morphogenesis to synapse formation. N-cadherin is also expressed in embryonic territories crossed by MGE cells during their migration. In this study, we demonstrate that N-cadherin is a key player in the long-distance migration of future cortical interneurons. Using N-cadherin-coated substrate, we show that N-cadherin-dependent adhesion promotes the migration of mouse MGE cells in vitro. Conversely, mouse MGE cells electroporated with a construct interfering with cadherin function show reduced cell motility, leading process instability, and impaired polarization associated with abnormal myosin IIB dynamics. In vivo, the capability of electroporated MGE cells to invade the developing cortical plate is altered. Using genetic ablation of N-cadherin in mouse embryos, we show that N-cadherin-depleted MGEs are severely disorganized. MGE cells hardly exit the disorganized proliferative area. N-cadherin ablation at the postmitotic stage, which does not affect MGE morphogenesis, alters MGE cell motility and directionality. The tangential migration to the cortex of N-cadherin ablated MGE cells is delayed, and their radial migration within the cortical plate is perturbed. Altogether, these results identify N-cadherin as a pivotal adhesion substrate that activates cell motility in future cortical interneurons and maintains cell polarity over their long-distance migration to the developing cortex.
Calcium Ruby m-Cl (X = H, Y = Cl) is a visible-light excited red-emitting calcium concentration ([Ca2+]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or click chemistry. Its large molar extinction and high quantum yield rank it among the brightest long-wavelength Ca2+ indicators. Calcium Ruby is a promising alternative to existing dyes for imaging [Ca2+] in multicolor fluorescence applications or in the presence of yellow-green cellular autofluorescence.
Colloidal nanocrystal (NC) donors wrapped with a polymer coating including multiple organic acceptor molecules are promising scaffolds for fluorescence resonance energy transfer (FRET)-based nanobiosensors. Over other self-assembling donor-acceptor configurations, our preloaded polymers have the virtue of producing compact assemblies with a fixed donor/acceptor distance. This property, together with the possibility of stoichiometric polymer loading, allowed us to directly address how the FRET efficiency depended on the donor/acceptor. At the population level, nanoprobes based on commercial as well as custom CdSe/ZnS donors displayed the expected dose-dependent rise in transfer efficiency, saturating from about five ATTO dyes/NC. However, for a given acceptor concentration, both the intensity and lifetime of single-pair FRET data revealed a large dispersion of transfer efficiencies, highlighting an important heterogeneity among nominally identical FRET-based nanoprobes. Rigorous quality check during synthesis and shell assembly as well as postsynthesis sorting and purification are required to make hybrid semiconductor-organic nanoprobes a robust and viable alternative to organic or genetically encoded nanobiosensors.
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