Nanoscale organization is changed in native, surface AMPARs by mouse brain region and tauopathy.

Synaptic AMPA receptors (AMPARs) on neuronal plasma membranes are correlated with learning and memory. Using a unique labeling and super-resolution imaging, we have visualized the nanoscale synaptic and extra-synaptic organization of native AMPARs for the first time in mouse brain slices as a function of brain region and tauopathy. We find that the fraction of surface AMPARs organized in synaptic clusters is two-times smaller in the hippocampus compared to the motor and somatosensory cortex.

Automatic dendritic spine segmentation in widefield fluorescence images reveal synaptic nanostructures distribution with super-resolution imaging.

Dendritic spines are the main sites for synaptic communication in neurons, and alterations in their density, size, and shapes occur in many brain disorders. Current spine segmentation methods perform poorly in conditions with low signal-to-noise and resolution, particularly in the widefield images of thick (10 μm) brain slices. Here, we combined two open-source machine-learning models to achieve automatic 3D spine segmentation in widefield diffraction-limited fluorescence images of neurons in thick brain slices.

Stepping dynamics of dynein characterized by MINFLUX.

Cytoplasmic dynein is a dimeric motor that drives minus-end directed transport on microtubules (MTs). To couple ATP hydrolysis to a mechanical step, a dynein monomer must be released from the MT before undergoing a conformational change that generates a bias towards the minus end. However, the dynamics of dynein stepping have been poorly characterized by tracking flexible regions of the motor with limited resolution.

Synapses without tension fail to fire in an in vitro network of hippocampal neurons.

Neurons in the brain communicate with each other at their synapses. It has long been understood that this communication occurs through biochemical processes. Here, we reveal that mechanical tension in neurons is essential for communication.

Structural Design of Multidentate Copolymers as Compact Quantum Dot Coatings for Live-Cell Single-Particle Imaging.

Quantum dots (QDs) are a class of semiconductor nanocrystal used broadly as fluorescent emitters for analytical studies in the life sciences. These nanomaterials are particularly valuable for single-particle imaging and tracking applications in cells and tissues. An ongoing technological goal is to reduce the hydrodynamic size of QDs to enhance access to sterically hindered biological targets.

Quantitative DNA-PAINT imaging of AMPA receptors in live neurons.

DNA-point accumulation for imaging at nanoscale topography (DNA-PAINT) can image fixed biological specimens with nanometer resolution and absolute stoichiometry. In living systems, however, the usage of DNA-PAINT has been limited due to high salt concentration in the buffer required for specific binding of the imager to the docker attached to the target. Here, we used multiple binding motifs of the docker, from 2 to 16, to accelerate the binding speed of the imager under physiological buffer conditions without compromising spatial resolution and maintaining the basal level homeostasis during the measurement.

Peptide-PAINT Using a Transfected-Docker Enables Live- and Fixed-Cell Super-Resolution Imaging.

Point accumulation for imaging in nanoscale topography (PAINT) is a single-molecule technique for super-resolution microscopy, which uses exchangeable single stranded DNA oligos or peptide-pairs to create blinking phenomenon and achieves ≈5-25 nanometer resolution. Here, it is shown that by transfecting the protein-of-interest with a docker-coil, rather than by adding the docker externally-as is the norm when using DNA tethers or antibodies as dockers-similar localization can be achieved, ≈10 nm. However, using a transfected docker has several experimental advances and simplifications.

High-throughput force measurement of individual kinesin-1 motors during multi-motor transport.

Molecular motors often work in teams to move a cellular cargo. Yet measuring the forces exerted by each motor is challenging. Using a sensor made with denatured ssDNA and multi-color fluorescence, we measured picoNewtons of forces and nanometer distances exerted by individual constrained kinesin-1 motors acting together while driving a common microtubule .

Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line.

Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O.

Dextran-Mimetic Quantum Dots for Multimodal Macrophage Imaging , and .

Macrophages are white blood cells with diverse functions contributing to a healthy immune response as well as the pathogenesis of cancer, osteoarthritis, atherosclerosis, and obesity. Due to their pleiotropic and dynamic nature, tools for imaging and tracking these cells at scales spanning the whole body down to microns could help to understand their role in disease states. Here we report fluorescent and radioisotopic quantum dots (QDs) for multimodal imaging of macrophage cells , , and .

TALEN outperforms Cas9 in editing heterochromatin target sites.

Genome editing critically relies on selective recognition of target sites. However, despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. Here, we use single-molecule imaging in live cells to directly study the behavior of CRISPR/Cas9 and TALEN.

Functional analysis of low-grade glioma genetic variants predicts key target genes and transcription factors.

Background: Large-scale genome-wide association studies (GWAS) have implicated thousands of germline genetic variants in modulating individuals' risk to various diseases, including cancer. At least 25 risk loci have been identified for low-grade gliomas (LGGs), but their molecular functions remain largely unknown.

Methods: We hypothesized that GWAS loci contain causal single nucleotide polymorphisms (SNPs) that reside in accessible open chromatin regions and modulate the expression of target genes by perturbing the binding affinity of transcription factors (TFs).

Optimizing Quantum Dot Probe Size for Single-Receptor Imaging.

Quantum dots (QDs) are nanocrystals with bright fluorescence and long-term photostability, attributes particularly beneficial for single-molecule imaging and molecular counting in the life sciences. The size of a QD nanocrystal determines its physicochemical and photophysical properties, both of which dictate the success of imaging applications. Larger nanocrystals typically have better optical properties, with higher brightness, red-shifted emission, reduced blinking, and greater stability.

Pin1 Binding to Phosphorylated PSD-95 Regulates the Number of Functional Excitatory Synapses.

The post-synaptic density protein 95 (PSD-95) plays a central role in excitatory synapse development and synaptic plasticity. Phosphorylation of the N-terminus of PSD-95 at threonine 19 (T19) and serine 25 (S25) decreases PSD-95 stability at synapses; however, a molecular mechanism linking PSD-95 phosphorylation to altered synaptic stability is lacking. Here, we show that phosphorylation of T19/S25 recruits the phosphorylation-dependent peptidyl-prolyl isomerase (Pin1) and reduces the palmitoylation of Cysteine 3 and Cysteine 5 in PSD-95.

Short-Wave Infrared Quantum Dots with Compact Sizes as Molecular Probes for Fluorescence Microscopy.

Materials with short-wave infrared (SWIR) emission are promising contrast agents for in vivo animal imaging, providing high-contrast and high-resolution images of blood vessels in deep tissues. However, SWIR emitters have not been developed as molecular labels for microscopy applications in the life sciences, which require optimized probes that are bright, stable, and small. Here, we design and synthesize semiconductor quantum dots (QDs) with SWIR emission based on HgCdSe alloy cores red shifted to the SWIR by epitaxial deposition of thin HgCdS shells with a small band gap.

Multiple kinesins induce tension for smooth cargo transport.

How cargoes move within a crowded cell-over long distances and at speeds nearly the same as when moving on unimpeded pathway-has long been mysterious. Through an in vitro force-gliding assay, which involves measuring nanometer displacement and piconewtons of force, we show that multiple mammalian kinesin-1 (from 2 to 8) communicate in a team by inducing tension (up to 4 pN) on the cargo. Kinesins adopt two distinct states, with one-third slowing down the microtubule and two-thirds speeding it up.

Photon-separation to enhance the spatial resolution of pulsed STED microscopy.

Stimulated emission depletion microscopy (STED) is one of the pivotal super-resolution techniques. It overcomes the spatial resolution limit imposed by the diffraction by using an additional laser beam, the STED beam, intensity of which is directly related to the achievable resolution. Despite reaching nanometer resolution, much effort in recent years has been devoted to reducing the STED beam intensity because it may lead to photo-damaging effects.

A Revised View on the Role of Surface AMPAR Mobility in Tuning Synaptic Transmission: Limitations, Tools, and Alternative Views.

Calcium dynamics in presynaptic terminals regulate the response dynamics of most central excitatory synapses. However, this dogma has been challenged by the hypothesis that mobility of the postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype glutamate receptors (AMPAR) plays a role in tuning fast excitatory synaptic transmission. In this review, we reevaluate the factors regulating postsynaptic AMPAR mobility, reassess the modeling parameters, analyze the experimental tools, and end by providing alternative ideas stemming from recent results.

Delivery of Fluorescent Probes Using Streptolysin O for Fluorescence Microscopy of Living Cells.

Methods to efficiently deliver fluorophores across the cell membrane are crucial for imaging the dynamics of intracellular proteins using fluorescence. Here we describe a simple protocol for permeabilizing living cells using streptolysin O, a bacterial toxin, which allows transient uptake of fluorescent probes for labeling specific intracellular proteins. The technique is applicable for delivering different classes of fluorescent probes with a molecular weight of <150 kDa, and it is also applicable to a variety of different cell lines.

Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots.

We develop magnetic cytoskeleton affinity (MiCA) purification, which allows for rapid isolation of molecular motors conjugated to large multivalent quantum dots, in miniscule quantities, which is especially useful for single-molecule applications. When purifying labeled molecular motors, an excess of fluorophores or labels is usually used. However, large labels tend to sediment during the centrifugation step of microtubule affinity purification, a traditionally powerful technique for motor purification.

Thermal nanoimprint lithography for drift correction in super-resolution fluorescence microscopy.

Localization-based super-resolution microscopy enables imaging of biological structures with sub-diffraction-limited accuracy, but generally requires extended acquisition time. Consequently, stage drift often limits the spatial precision. Previously, we reported a simple method to correct for this by creating an array of 1 μm fiducial markers, every ~8 μm, on the coverslip, using UV-nanoimprint lithography (UV-NIL).

Structural Contributions to Hydrodynamic Diameter for Quantum Dots Optimized for Live-Cell Single-Molecule Tracking.

Quantum dots are fluorescent nanoparticles with narrow-band, size-tunable, and long-lasting emission. Typical formulations used for imaging proteins in cells are hydrodynamically much larger than the protein targets, so it is critical to assess the impact of steric effects deriving from hydrodynamic size. This report analyzes a new class of quantum dots that have been engineered for minimized size specifically for imaging receptors in narrow synaptic junctions between neurons.

Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes.

Previous studies tracking AMPA receptor (AMPAR) diffusion at synapses observed a large mobile extrasynaptic AMPAR pool. Using super-resolution microscopy, we examined how fluorophore size and photostability affected AMPAR trafficking outside of, and within, post-synaptic densities (PSDs) from rats. Organic fluorescent dyes (≈4 nm), quantum dots, either small (≈10 nm diameter; sQDs) or big (>20 nm; bQDs), were coupled to AMPARs via different-sized linkers.