Nela Durisic

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate.

Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.

Stoichiometry of the Human Glycine Receptor Revealed by Direct Subunit counting

The subunit stoichiometry of heteromeric glycine-gated channels determines fundamental properties of these key inhibitory neurotransmitter receptors; however, the ratio of α1- to β-subunits per receptor remains controversial. We used single-molecule imaging and stepwise photobleaching in Xenopus oocytes to directly determine the subunit stoichiometry of a glycine receptor to be 3α1:2β. This approach allowed us to determine the receptor stoichiometry in mixed populations consisting of both heteromeric and homomeric channels, additionally revealing the quantitative proportions for the two populations.

Probing the “Dark” Fraction of Core–Shell Quantum Dots by Ensemble and Single Particle pH-Dependent Spectroscopy

The optical properties of core-shell CdSe-ZnS quantum dots (QDs) are characterized by complex photophysics leading to difficulties in interpreting quantitative measurements based on QD emission. By comparing the pH dependence of fluorescence of single QDs to that of an ensemble, we have been able to propose a molecular scale model of how QD surface chemical and physical processes are affected by protons and oxygen. We show that the connection between the ensemble fluorescence intensity and the single QD fluorescence properties such as dark fraction, blinking, particle brightness, and a multiexponential fluorescence lifetime decay is not trivial. The ensemble fluorescence intensity is more weakly dependent on pH than the single particle fluorescence which, together with fluorescence lifetime analysis, provided evidence that the dark fraction of QDs emits photons with low quantum efficiency and long lifetime. We uncovered two surface-dependent mechanisms that affected the fluorescence emission: an immediate physical effect of charges surrounding the QD and an irreversible chemical effect from reaction of the H(+) and O(2) with the QD shell surface. These results will have important implications for those using QD-based fluorescence lifetime imaging as well as for proper implementation of these probes for quantitative cellular imaging applications.

Atomic Force Microscopy Reveals Important Differences in Axonal Resistance to Injury

Axonal degeneration after traumatic brain injury and nerve compression is considered a common underlying cause of temporary as well as permanent disability. Because a proper functioning of neural network requires phase coherence of all components, even subtle changes in circuitry may lead to network failure. However, it is still not possible to determine which axons will recover or degenerate after injury. Several groups have studied the pressure threshold for axonal injury within a nerve, but difficulty accessing the injured region; insufficient imaging methods and the extremely small dimensions involved have prevented the evaluation of the response of individual axons to injury. We combined microfluidics with atomic force microscopy and in vivo imaging to estimate the threshold force required to 1), uncouple axonal transport without impairing axonal survival, and 2), compromise axonal survival in both individual and bundled axons. We found that rat hippocampal axons completely recover axonal transport with no detectable axonal loss when compressed with pressures up to 65 ± 30 Pa for 10 min, while dorsal root ganglia axons can resist to pressures up to 540 ± 220 Pa. We investigated the reasons for the differential susceptibility of hippocampal and DRG axons to mechanical injury and estimated the elasticity of live axons. We found that dorsal root ganglia axons have a 20% lower elastic modulus than hippocampal axons. Our results emphasize the importance of the integrity of the axonal cytoskeleton in deciding the axonal fate after damage and open up new avenues to improve injury diagnosis and to identify ways to protect axons.

Quantitative super-resolution microscopy: pitfalls and strategies for image analysis

Super-resolution microscopy is an enabling technology that allows biologists to visualize cellular structures at nanometer length scales using far-field optics. To break the diffraction barrier, it is necessary to leverage the distinct molecular states of fluorescent probes. At the same time, the existence of these different molecular states and the photophysical properties of the fluorescent probes can complicate data quantification and interpretation. Here, we review the pitfalls in super-resolution data analysis that must be avoided for proper interpretation of images.

Characterization of blinking dynamics in quantum dot ensembles using image correlation spectroscopy

Quantum dots (QDs) are being increasingly applied as luminescent labels in optical studies for biophysical and cell biological applications due to their unique spectroscopic properties. However, their fluorescence “blinking” characteristics that follow power law statistics make it difficult to use QDs in some quantitative biophysical applications. We present image correlation spectroscopy (ICS) in combination with total internal reflection fluorescence microscopy as a tool to characterize blinking dynamics in QDs. We show that the rate of decay of the ICS measured ensemble correlation function reflects variation in blinking dynamics and can be used to distinguish different blinking distribution regimes. To test and confirm our hypothesis, we also analyze image time series simulations of ensembles of point emitters with set blinking statistics. We show that optimization of the temporal sampling and the number of QDs sampled is essential for detecting changes in blinking dynamics with ICS. We propose that t...

DNA–Protein Noncovalent Cross-Linking: Ruthenium Dipyridophenazine Biotin Complex for the Assembly of Proteins and Gold Nanoparticles on DNA Templates

We report the first example of a small molecule that can noncovalently cross‐link DNA with streptavidin and streptavidin‐labeled materials. Molecule 1 possesses a ruthenium dipyridophenazine DNA‐intercalating moiety and a biotin unit; these two units are adequately separated to ensure efficient cross‐linking of DNA with protein. Complex 1 is essentially nonemissive in aqueous solution and when bound to streptavidin, however, its luminescence is turned “on” when it binds to DNA. We have used these properties to establish that this complex can simultaneously bind to DNA and streptavidin, and can thus bring these two biomolecules together. We also synthesized a related molecule, 3, in which the biotin and DNA‐intercalating moieties are covalently bound. While complex 3 can intercalate into DNA through a threading mechanism, luminescence experiments show that it cannot simultaneously bind DNA and streptavidin, most likely due to the proximity of its two molecular‐recognition units. The cross‐linking ability of molecule 1 was used to template the assembly of streptavidin molecules on circular plasmid DNA, as visualized by atomic force microscopy. In addition, using 1, we show the organization of discrete groups of gold nanoparticles labeled with streptavidin on a linear DNA template of finite size, with transmission electron microscopy. In these experiments the DNA template acted as a “molecular ruler” that dictated the number of particles in the assembly.

Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles

Joensuu et al. describe a tool for subdiffractional tracking of internalized molecules. They reveal that synaptic vesicles exhibit stochastic switching between heterogeneous diffusive and transport states in live hippocampal nerve terminals.

Exosomes taken up by neurons hijack the endosomal pathway to spread to interconnected neurons

In Alzheimer disease and related disorders, the microtubule-associated protein tau aggregates and forms cytoplasmic lesions that impair neuronal physiology at many levels. In addition to affecting the host neuron, tau aggregates also spread to neighboring, recipient cells where the misfolded tau aggregates, in a manner similar to prions, actively corrupt the proper folding of soluble tau, and thereby impair cellular functions. One vehicle for the transmission of tau aggregates are secretory nanovesicles known as exosomes. Here, we established a simple model of a neuronal circuit using a microfluidics culture system in which hippocampal neurons A and B were seeded into chambers 1 and 2, respectively, extending axons via microgrooves in both directions and thereby interconnecting. This system served to establish two models to track exosome spreading. In the first model, we labeled the exosomal membrane by coupling tetraspanin CD9 with either a green or red fluorescent tag. This allowed us to reveal that interconnected neurons exchange exosomes only when their axons extend in close proximity. In the second model, we added exosomes isolated from the brains of tau transgenic rTg4510 mice (i.e. exogenous, neuron A-derived) to neurons in chamber 1 (neuron B) interconnected with neuron C in chamber 2. This allowed us to demonstrate that a substantial fraction of the exogenous exosomes were internalized by neuron B and passed then on to neuron C. This transportation from neuron B to C was achieved by a mechanism that is consistent with the hijacking of secretory endosomes by the exogenous exosomes, as revealed by confocal, super-resolution and electron microscopy. Together, these findings suggest that fusion events involving the endogenous endosomal secretory machinery increase the pathogenic potential and the radius of action of pathogenic cargoes carried by exogenous exosomes.

γ1-Containing GABA-A Receptors Cluster at Synapses Where they Mediate Slower Synaptic Currents than γ2-Containing GABA-A Receptors

GABA-A receptors (GABAARs) are pentameric ligand-gated ion channels that are assembled mainly from α (α1–6), β (β1–3) and γ (γ1–3) subunits. Although GABAARs containing γ2L subunits mediate most of the inhibitory neurotransmission in the brain, significant expression of γ1 subunits is seen in the amygdala, pallidum and substantia nigra. However, the location and function of γ1-containing GABAARs in these regions remains unclear. In “artificial” synapses, where the subunit composition of postsynaptic receptors is specifically controlled, γ1 incorporation slows the synaptic current decay rate without affecting channel deactivation, suggesting that γ1-containing receptors are not clustered and therefore activated by diffuse neurotransmitter. However, we show that γ1-containing receptors are localized at neuronal synapses and form clusters in both synaptic and extrasynaptic regions. In addition, they exhibit rapid membrane diffusion and a higher frequency of exchange between synaptic and perisynaptic populations compared to γ2L-containing GABAARs. A point mutation in the large intracellular domain and a pharmacological analysis reveal that when a single non-conserved γ2L residue is mutated to its γ1 counterpart (T349L), the synaptic current decay is slowed from γ2L- to γ1-like without changing the clustering or diffusion properties of the receptors. In addition, previous fast perfusion and single channel kinetic experiments revealed no difference in the intrinsic closing rates of γ2L- and γ1-containing receptors when expressed in HEK293 cells. These observations together with Monte Carlo simulations of synaptic function confirm that decreased clustering does not control γ1-containing GABAAR kinetics. Rather, they suggest that γ1- and γ2L-containing receptors exhibit differential synaptic current decay rates due to differential gating dynamics when localized at the synapse.