Super-resolved trajectory-derived nanoclustering analysis using spatiotemporal indexing.

Single-molecule localization microscopy techniques are emerging as vital tools to unravel the nanoscale world of living cells by understanding the spatiotemporal organization of protein clusters at the nanometer scale. Current analyses define spatial nanoclusters based on detections but neglect important temporal information such as cluster lifetime and recurrence in "hotspots" on the plasma membrane. Spatial indexing is widely used in video games to detect interactions between moving geometric objects. Here, we use the R-tree spatial indexing algorithm to determine the overlap of the bounding boxes of individual molecular trajectories to establish membership in nanoclusters. Extending the spatial indexing into the time dimension allows the resolution of spatial nanoclusters into multiple spatiotemporal clusters. Using spatiotemporal indexing, we found that syntaxin1a and Munc18-1 molecules transiently cluster in hotspots, offering insights into the dynamics of neuroexocytosis. Nanoscale spatiotemporal indexing clustering (NASTIC) has been implemented as a free and open-source Python graphic user interface.

Tyrosine kinases compete for growth hormone receptor binding and regulate receptor mobility and degradation.

Growth hormone (GH) acts via JAK2 and LYN to regulate growth, metabolism, and neural function. However, the relationship between these tyrosine kinases remains enigmatic. Through an interdisciplinary approach combining cell biology, structural biology, computation, and single-particle tracking on live cells, we find overlapping LYN and JAK2 Box1-Box2-binding regions in GH receptor (GHR). Our data implicate direct competition between JAK2 and LYN for GHR binding and imply divergent signaling profiles. We show that GHR exhibits distinct mobility states within the cell membrane and that activation of LYN by GH mediates GHR immobilization, thereby initiating its nanoclustering in the membrane. Importantly, we observe that LYN mediates cytokine receptor degradation, thereby controlling receptor turnover and activity, and this applies to related cytokine receptors. Our study offers insight into the molecular interactions of LYN with GHR and highlights important functions for LYN in regulating GHR nanoclustering, signaling, and degradation, traits broadly relevant to many cytokine receptors.

Axons-on-a-chip for mimicking non-disruptive diffuse axonal injury underlying traumatic brain injury.

Diffuse axonal injury (DAI) is the most severe pathological feature of traumatic brain injury (TBI). However, how primary axonal injury is induced by transient mechanical impacts remains unknown, mainly due to the low temporal and spatial resolution of medical imaging approaches. Here we established an axon-on-a-chip (AoC) model for mimicking DAI and monitoring instant cellular responses. Integrating computational fluid dynamics and microfluidic techniques, DAI was induced by injecting a precisely controlled micro-flux in the transverse direction. The clear correlation between the flow speed of injecting flux and the severity of DAI was elucidated. We next used the AoC to investigate the instant intracellular responses underlying DAI and found that the dynamic formation of focal axonal swellings (FAS) accompanied by Ca2+ surge occurs during the flux. Surprisingly, periodic axonal cytoskeleton disruption also occurs rapidly after the flux. These instant injury responses are spatially restricted to the fluxed axon, not affecting the overall viability of the neuron in the acute stage. Compatible with high-resolution live microscopy, the AoC provides a versatile system to identify early mechanisms underlying DAI, offering a platform for screening effective treatments to alleviate TBI.

Growth cone repulsion to Netrin-1 depends on lipid raft microdomains enriched in UNC5 receptors.

During brain development, Uncoordinated locomotion 5 (UNC5) receptors control axonal extension through their sensing of the guidance molecule Netrin-1. The correct positioning of receptors into cholesterol-enriched membrane raft microdomains is crucial for the efficient transduction of the recognized signals. However, whether such microdomains are required for the appropriate axonal guidance mediated by UNC5 receptors remains unknown. Here, we combine the use of confocal microscopy, live-cell FRAP analysis and single-particle tracking PALM to characterize the distribution of UNC5 receptors into raft microdomains, revealing differences in their membrane mobility properties. Using pharmacological and genetic approaches in primary neuronal cultures and brain cerebellar explants we further demonstrate that disrupting raft microdomains inhibits the chemorepulsive response of growth cones and axons against Netrin-1. Together, our findings indicate that the distribution of all UNC5 receptors into cholesterol-enriched raft microdomains is heterogeneous and that the specific localization has functional consequences for the axonal chemorepulsion against Netrin-1.

Effects of GABAA Receptor α3 Subunit Epilepsy Mutations on Inhibitory Synaptic Signaling.

Missense mutations T166M, Q242L, T336M, and Y474C in the GABAA receptor (GABAAR) α3 subunit gene are associated with epileptic seizures, dysmorphic features, intellectual disability, and developmental delay. When incorporated into GABAARs expressed in oocytes, all mutations are known to reduce GABA-evoked whole-cell currents. However, their impact on the properties of inhibitory synaptic currents (IPSCs) is unknown, largely because it is difficult to establish, much less control, the stoichiometry of GABAAR expressed in native neuronal synapses. To circumvent this problem, we employed a HEK293 cell-neuron co-culture expression system that permits the recording of IPSCs mediated by a pure population of GABAARs with a defined stoichiometry. We first demonstrated that IPSCs mediated by α3-containing GABAARs (α3ß3γ2) decay significantly slower than those mediated by α1-containing isoforms (α1ß2γ2 or α1ß3γ2). GABAAR α3 mutations did not affect IPSC peak amplitudes or 10-90% rise times, but three of the mutations affected IPSC decay. T336M significantly accelerated the IPSC decay rate whereas T166M and Y474C had the opposite effect. The acceleration of IPSC decay kinetics caused by the T366M mutation was returned to wild-type-like values by the anti-epileptic medication, midazolam. Quantification experiments in HEK293 cells revealed a significant reduction in cell-surface expression for all mutants, in agreement with previous oocyte data. Taken together, our results show that impaired surface expression and altered IPSC decay rates could both be significant factors underlying the pathologies associated with these mutations.

SAHA (Vorinostat) Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit.

The GABAA receptor (GABAAR) α1 subunit A295D epilepsy mutation reduces the surface expression of α1A295Dß2γ2 GABAARs via ER-associated protein degradation. Suberanilohydroxamic acid (SAHA, also known as Vorinostat) was recently shown to correct the misfolding of α1A295D subunits and thereby enhance the functional surface expression of α1A295Dß2γ2 GABAARs. Here we investigated whether SAHA can also restore the surface expression of γ2 GABAAR subunits that incorporate epilepsy mutations (N40S, R43Q, P44S, R138G) known to reduce surface expression via ER-associated protein degradation. As a control, we also investigated the γ2 K289M epilepsy mutation that impairs gating without reducing surface expression. Effects of mutations were evaluated on inhibitory postsynaptic currents (IPSCs) mediated by the major synaptic α1ß2γ2 GABAAR isoform. Recordings were performed in neuron-HEK293 cell artificial synapses to minimise contamination by GABAARs of undefined subunit composition. Transfection with α1ß2γ2 N40S , α1ß2γ2 R43Q , α1ß2γ2 P44S and α1ß2γ2 R138G subunits produced IPSCs with decay times slower than those of unmutated α1ß2γ2 GABAARs due to the low expression of mutant γ2 subunits and the correspondingly high expression of slow-decaying α1ß2 GABAARs. SAHA pre-treatment significantly accelerated the decay time constants of IPSCs consistent with the upregulation of mutant γ2 subunit expression. This increase in surface expression was confirmed by immunohistochemistry. SAHA had no effect on either the IPSC kinetics or surface expression levels of α1ß2γ2 K289M GABAARs, confirming its specificity for ER-retained mutant γ2 subunits. We also found that α1ß2γ2 K289M GABAARs and SAHA-treated α1ß2γ2 R43Q , α1ß2γ2 P44S and α1ß2γ2 R138G GABAARs all mediated IPSCs that decayed at significantly faster rates than wild type receptors as temperature was increased from 22 to 40°C. This may help explain why these mutations cause febrile seizures (FS). Given that SAHA is approved by therapeutic regulatory agencies for human use, we propose that it may be worth investigating as a treatment for epilepsies caused by the N40S, R43Q, P44S and R138G mutations. Although SAHA has already been proposed as a therapeutic for patients harbouring the α1A295D epilepsy mutation, the present study extends its potential utility to a new subunit and four new mutations.

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.

Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules.

Our understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulse-chase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in individual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision. We originally developed this approach for tracking single molecules of botulinum neurotoxin type A, which undergoes activity-dependent internalization and retrograde transport in autophagosomes. This method was then adapted to localize the signaling endosomes containing cholera toxin subunit-B that undergo retrograde transport in axons and to track SVs in the crowded environment of hippocampal presynapses. We describe (i) the construction of a custom-made microfluidic device that enables control over neuronal orientation; (ii) the 3D printing of a perfusion system for sdTIM experiments performed on glass-bottom dishes; (iii) the dissection, culturing and transfection of hippocampal neurons in microfluidic devices; and (iv) guidance on how to perform the pulse-chase experiments and data analysis. In addition, we describe the use of single-molecule-tracking analytical tools to reveal the average and the heterogeneous single-molecule mobility behaviors. We also discuss alternative reagents and equipment that can, in principle, be used for sdTIM experiments and describe how to adapt sdTIM to image nanocluster formation and/or tubulation in early endosomes during sorting events. The procedures described in this protocol take ∼1 week.

Inhibitory synapse deficits caused by familial α1 GABAA receptor mutations in epilepsy.

Epilepsy is a spectrum of neurological disorders with many causal factors. The GABA type-A receptor (GABAAR) is a major genetic target for heritable human epilepsies. Here we examine the functional effects of three epilepsy-causing mutations to the α1 subunit (α1T10'I, α1D192N and α1A295D) on inhibitory postsynaptic currents (IPSCs) mediated by the major synaptic GABAAR isoform, α1ß2γ2L. We employed a neuron - HEK293 cell heterosynapse preparation to record IPSCs mediated by mutant-containing GABAARs in isolation from other GABAAR isoforms. IPSCs were recorded in the presence of the anticonvulsant drugs, carbamazepine and midazolam, and at elevated temperatures (22, 37 and 40°C) to gain insight into mechanisms of febrile seizures. The mutant subunits were also transfected into cultured cortical neurons to investigate changes in synapse formation and neuronal morphology using fluorescence microscopy. We found that IPSCs mediated by α1T10'Iß2γ2L, α1D192Nß2γ2L GABAARs decayed faster than those mediated by α1ß2γ2L receptors. IPSCs mediated by α1D192Nß2γ2L and α1A295Dß2γ2L receptors also exhibited a heightened temperature sensitivity. In addition, the α1T10'Iß2γ2L GABAARs were refractory to modulation by carbamazepine or midazolam. In agreement with previous studies, we found that α1A295Dß2γ2L GABAARs were retained intracellularly in HEK293 cells and neurons. However, pre-incubation with 100nM suberanilohydroxamic acid (SAHA) induced α1A295Dß2γ2L GABAARs to mediate IPSCs that were indistinguishable in magnitude and waveform from those mediated by α1ß2γ2L receptors. Finally, mutation-specific changes to synaptic bouton size, synapse number and neurite branching were also observed. These results provide new insights into the mechanisms of epileptogenesis of α1 epilepsy mutations and suggest possible leads for improving treatments for patients harbouring these mutations.

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), ß (ß1-3) 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.

Probing the Structural Mechanism of Partial Agonism in Glycine Receptors Using the Fluorescent Artificial Amino Acid, ANAP.

The efficacy of an agonist at a pentameric ligand-gated ion channel is determined by the rate at which it induces a conformational change from the resting closed state to a preopen ("flip") state. If the ability of an agonist to promote this isomerization is sufficiently low, then it becomes a partial agonist. As partial agonists at pentameric ligand-gated ion channels show considerable promise as therapeutics, understanding the structural basis of the resting-flip-state isomerization may provide insight into therapeutic design. Accordingly, we sought to identify structural correlates of the resting-flip conformational change in the glycine receptor chloride channel. We used nonsense suppression to introduce the small, fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP), into specific sites in the extracellular and transmembrane domains. Then, under voltage-clamp conditions in Xenopus oocytes, we simultaneously quantified current and fluorescence responses induced by structurally similar agonists with high, medium, and low efficacies (glycine, ß-alanine, and taurine, respectively). Analyzing results from nine ANAP-incorporated sites, we show that glycine receptor activation by agonists with graded efficacies manifests structurally as correspondingly graded movements of the ß1-ß2 loop, the ß8-ß9 loop, and the Cys-loop from the extracellular domain and the TM2-TM3 linker in the transmembrane domain. We infer that the resting-flip transition involves an efficacy-dependent molecular reorganization at the extracellular-transmembrane domain interface that primes receptors for efficacious opening.

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

Our understanding of endocytic pathway dynamics is severely restricted by the diffraction limit of light microscopy. To address this, we implemented a novel technique based on the subdiffractional tracking of internalized molecules (sdTIM). This allowed us to image anti-green fluorescent protein Atto647N-tagged nanobodies trapped in synaptic vesicles (SVs) from live hippocampal nerve terminals expressing vesicle-associated membrane protein 2 (VAMP2)-pHluorin with 36-nm localization precision. Our results showed that, once internalized, VAMP2-pHluorin/Atto647N-tagged nanobodies exhibited a markedly lower mobility than on the plasma membrane, an effect that was reversed upon restimulation in presynapses but not in neighboring axons. Using Bayesian model selection applied to hidden Markov modeling, we found that SVs oscillated between diffusive states or a combination of diffusive and transport states with opposite directionality. Importantly, SVs exhibiting diffusive motion were relatively less likely to switch to the transport motion. These results highlight the potential of the sdTIM technique to provide new insights into the dynamics of endocytic pathways in a wide variety of cellular settings.

Ivermectin-Activated, Cation-Permeable Glycine Receptors for the Chemogenetic Control of Neuronal Excitation.

The ability to control neuronal activation is rapidly advancing our understanding of brain function and is widely viewed as having eventual therapeutic application. Although several highly effective optogenetic, optochemical genetic, and chemogenetic techniques have been developed for this purpose, new approaches may provide better solutions for addressing particular questions and would increase the number of neuronal populations that can be controlled independently. An early chemogenetic neuronal silencing method employed a glutamate receptor Cl- channel engineered for activation by 1-3 nM ivermectin. This construct has been validated in vivo. Here, we sought to develop cation-permeable ivermectin-gated receptors that were either maximally Ca2+-permeable so as to induce neuro-excitotoxic cell death or minimally Ca2+-permeable so as to depolarize neurons with minimal excitotoxic risk. Our constructs were based on the human α1 glycine receptor Cl- channel due to its high conductance, human origin, high ivermectin sensitivity (once mutated), and because pore mutations that render it permeable to Na+ alone or Na+ plus Ca2+ are well characterized. We developed a Ca2+-impermeable excitatory receptor by introducing the F207A/P-2'Δ/A-1'E/T13'V/A288G mutations and a Ca2+-permeable excitatory receptor by introducing the F207A/A-1'E/A288G mutations. The latter receptor efficiently induces cell death and strongly depolarizes neurons at nanomolar ivermectin concentrations.

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.

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.

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.

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 ß-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 3α1:2ß. 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.

A common mechanism underlies the dark fraction formation and fluorescence blinking of quantum dots.

CdSe quantum dots (QDs) are known to exhibit both power-law blinking dynamics and a dark fraction. A complete description of the mechanistic origins of these properties is still lacking. We show that a change in the pH of the QD environment systematically changes both the dark fraction and the blinking statistics. As pH is lowered, shorter "on" times and longer "off" times, as well as an increase in the permanent dark fraction, are observed. The increase in the dark fraction is preceded by a decrease in the emission intensity of a single QD. Interestingly, the form of the probability distribution function describing blinking changes when the QDs are taken from an air-exposed environment into an aqueous one. These results are used to propose a coupled role for H(+) ions by which they first reduce the intensity of the emitting state as well as affect the probabilities of the QD to switch between "on" and "off" states and eventually trap the QD in a permanent "off" state. We discuss and extend two theoretical blinking models to account for the effect of H(+) ions as well as to highlight their common principle of a diffusion-controlled mechanism governing blinking.