On-Demand, Reversible, Ultrasensitive Polymer Membrane Based on Molecular Imprinting Polymer.

The development of in vivo, longitudinal, real-time monitoring devices is an essential step toward continuous, precision health monitoring. Molecularly imprinted polymers (MIPs) are popular sensor capture agents that are more robust than antibodies and have been used for sensors, drug delivery, affinity separations, assays, and solid-phase extraction. However, MIP sensors are typically limited to one-time use due to their high binding affinity (>107 M-1) and slow-release kinetics (<10-4 µM/sec). To overcome this challenge, current research has focused on stimuli-responsive MIPs (SR-MIPs), which undergo a conformational change induced by external stimuli to reverse molecular binding, requiring additional chemicals or outside stimuli. Here, we demonstrate fully reversible MIP sensors based on electrostatic repulsion. Once the target analyte is bound within a thin film MIP on an electrode, a small electrical potential successfully releases the bound molecules, enabling repeated, accurate measurements. We demonstrate an electrostatically refreshed dopamine sensor with a 760 pM limit of detection, linear response profile, and accuracy even after 30 sensing-release cycles. These sensors could repeatedly detect <1 nM dopamine released from PC-12 cells in vitro, demonstrating they can longitudinally measure low concentrations in complex biological environments without clogging. Our work provides a simple and effective strategy for enhancing the use of MIPs-based biosensors for all charged molecules in continuous, real-time health monitoring and other sensing applications.

Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection.

Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating the measurement of expression kinetics in both isolated cells and whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a foundation for the development and expansion of transgenic techniques in this unique model system.

The Exocyst Component Exo70 Modulates Dendrite Arbor Formation, Synapse Density, and Spine Maturation in Primary Hippocampal Neurons.

Neurons are highly polarized cells displaying an elaborate architectural morphology. The design of their dendritic arborization and the distribution of their synapses contribute importantly to information processing in the brain. The growth and complexity of dendritic arbors are driven by the formation of synapses along their lengths. Synaptogenesis is augmented by the secretion of factors, like BDNF, Reelin, BMPs, or Wnts. Exo70 is a component of the exocyst complex, a protein complex that guides membrane addition and polarized exocytosis. While it has been linked to cytokinesis and the establishment of cell polarity, its role in synaptogenesis is poorly understood. In this report, we show that Exo70 plays a role in the arborization of dendrites and the development of synaptic connections between cultured hippocampal neurons. Specifically, while the overexpression of Exo70 increases dendritic arborization, synapse number, and spine density, the inhibition of Exo70 expression reduces secondary and tertiary dendrite formation and lowers synapse density. Moreover, increasing Exo70 expression augmented synaptic vesicle recycling as evaluated by FM4-64 dye uptake and the inverse was observed with downregulation of endogenous Exo70. Monitoring the formation of dendritic spines by super-resolution microscopy, we also observed that mRFP-Exo70 accumulates at the tip of EGFP-ß-actin-positive filopodia. Together, these results suggest that Exo70 is essentially involved in the formation of synapses and neuronal dendritic morphology.

Shank and Zinc Mediate an AMPA Receptor Subunit Switch in Developing Neurons.

During development, pyramidal neurons undergo dynamic regulation of AMPA receptor (AMPAR) subunit composition and density to help drive synaptic plasticity and maturation. These normal developmental changes in AMPARs are particularly vulnerable to risk factors for Autism Spectrum Disorders (ASDs), which include loss or mutations of synaptic proteins and environmental insults, such as dietary zinc deficiency. Here, we show how Shank2 and Shank3 mediate a zinc-dependent regulation of AMPAR function and subunit switch from GluA2-lacking to GluA2-containing AMPARs. Over development, we found a concomitant increase in Shank2 and Shank3 with GluA2 at synapses, implicating these molecules as potential players in AMPAR maturation. Since Shank activation and function require zinc, we next studied whether neuronal activity regulated postsynaptic zinc at glutamatergic synapses. Zinc was found to increase transiently and reversibly with neuronal depolarization at synapses, which could affect Shank and AMPAR localization and activity. Elevated zinc induced multiple functional changes in AMPAR, indicative of a subunit switch. Specifically, zinc lengthened the decay time of AMPAR-mediated synaptic currents and reduced their inward rectification in young hippocampal neurons. Mechanistically, both Shank2 and Shank3 were necessary for the zinc-sensitive enhancement of AMPAR-mediated synaptic transmission and act in concert to promote removal of GluA1 while enhancing recruitment of GluA2 at pre-existing Shank puncta. These findings highlight a cooperative local dynamic regulation of AMPAR subunit switch controlled by zinc signaling through Shank2 and Shank3 to shape the biophysical properties of developing glutamatergic synapses. Given the zinc sensitivity of young neurons and its dependence on Shank2 and Shank3, genetic mutations and/or environmental insults during early development could impair synaptic maturation and circuit formation that underlie ASD etiology.

Functionalisation of Detonation Nanodiamond for Monodispersed, Soluble DNA-Nanodiamond Conjugates Using Mixed Silane Bead-Assisted Sonication Disintegration.

Nanodiamonds have many attractive properties that make them suitable for a range of biological applications, but their practical use has been limited because nanodiamond conjugates tend to aggregate in solution during or after functionalisation. Here we demonstrate the production of DNA-detonation nanodiamond (DNA-DND) conjugates with high dispersion and solubility using an ultrasonic, mixed-silanization chemistry protocol based on the in situ Bead-Assisted Sonication Disintegration (BASD) silanization method. We use two silanes to achieve these properties: (1) 3-(trihydroxysilyl)propyl methylphosphonate (THPMP); a negatively charged silane that imparts high zeta potential and solubility in solution; and (2) (3-aminopropyl)triethoxysilane (APTES); a commonly used functional silane that contributes an amino group for subsequent bioconjugation. We target these amino groups for covalent conjugation to thiolated, single-stranded DNA oligomers using the heterobifunctional crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The resulting DNA-DND conjugates are the smallest reported to date, as determined by Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). The functionalisation method we describe is versatile and can be used to produce a wide variety of soluble DND-biomolecule conjugates.

Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring.

Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation.

Bassoon Controls Presynaptic Autophagy through Atg5.

Mechanisms regulating the surveillance and clearance of synaptic proteins are not well understood. Intriguingly, the loss of the presynaptic active zone proteins Piccolo and Bassoon triggers the loss of synaptic vesicles (SVs) and compromises synaptic integrity. Here we report that the destruction of SVs in boutons lacking Piccolo and Bassoon was associated with the induction of presynaptic autophagy, a process that depended on poly-ubiquitination, but not the E3 ubiquitin ligase Siah1. Surprisingly, gain or loss of function (LOF) of Bassoon alone suppressed or enhanced presynaptic autophagy, respectively, implying a fundamental role for Bassoon in the local regulation of presynaptic autophagy. Mechanistically, Bassoon was found to interact with Atg5, an E3-like ligase essential for autophagy, and to inhibit the induction of autophagy in heterologous cells. Importantly, Atg5 LOF as well as targeting an Atg5-binding peptide derived from Bassoon inhibited presynaptic autophagy in boutons lacking Piccolo and Bassoon, providing insights into the molecular mechanisms regulating presynaptic autophagy.

Piccolo Directs Activity Dependent F-Actin Assembly from Presynaptic Active Zones via Daam1.

The dynamic assembly of filamentous (F) actin plays essential roles in the assembly of presynaptic boutons, the fusion, mobilization and recycling of synaptic vesicles (SVs), and presynaptic forms of plasticity. However, the molecular mechanisms that regulate the temporal and spatial assembly of presynaptic F-actin remain largely unknown. Similar to other F-actin rich membrane specializations, presynaptic boutons contain a set of molecules that respond to cellular cues and trans-synaptic signals to facilitate activity-dependent assembly of F-actin. The presynaptic active zone (AZ) protein Piccolo has recently been identified as a key regulator of neurotransmitter release during SV cycling. It does so by coordinating the activity-dependent assembly of F-Actin and the dynamics of key plasticity molecules including Synapsin1, Profilin and CaMKII. The multidomain structure of Piccolo, its exquisite association with the AZ, and its ability to interact with a number of actin-associated proteins suggest that Piccolo may function as a platform to coordinate the spatial assembly of F-actin. Here we have identified Daam1, a Formin that functions with Profilin to drive F-actin assembly, as a novel Piccolo binding partner. We also found that within cells Daam1 activation promotes Piccolo binding, an interaction that can spatially direct the polymerization of F-Actin. Moreover, similar to Piccolo and Profilin, Daam1 loss of function impairs presynaptic-F-actin assembly in neurons. These data suggest a model in which Piccolo directs the assembly of presynaptic F-Actin from the AZ by scaffolding key actin regulatory proteins including Daam1.

Plasma membrane and actin cytoskeleton as synergistic barriers to nanowire cell penetration.

Nanowires are a rapidly emerging platform for manipulation of and material delivery directly into the cell cytosol. These high aspect ratio structures can breach the lipid membrane; however, the yield of penetrant structures is low, and the mechanism is largely unknown. In particular, some nanostructures appear to defeat the membrane transiently, while others can retain long-term access. Here, we examine if local dissolution of the lipid membrane, actin cytoskeleton, or both can enhance nanowire penetration. It is possible that, during cell contact, membrane rupture occurs; however, if the nanostructures do not penetrate the cytoskeleton, the membrane may reclose over a relatively short time frame. We show with quantitative analysis of the number of penetrating nanowires that the lipid bilayer and actin cytoskeleton are synergistic barriers to nanowire cell access, yet chemical poration through both is still insufficient to increase long-term access for adhered cells.

In vivo knockdown of Piccolino disrupts presynaptic ribbon morphology in mouse photoreceptor synapses.

Piccolo is the largest known cytomatrix protein at active zones of chemical synapses. A growing number of studies on conventional chemical synapses assign Piccolo a role in the recruitment and integration of molecules relevant for both endo- and exocytosis of synaptic vesicles, the dynamic assembly of presynaptic F-actin, as well as the proteostasis of presynaptic proteins, yet a direct function in the structural organization of the active zone has not been uncovered in part due to the expression of multiple alternatively spliced isoforms. We recently identified Piccolino, a Piccolo splice variant specifically expressed in sensory ribbon synapses of the eye and ear. Here we down regulated Piccolino in vivo via an adeno-associated virus-based RNA interference approach and explored the impact on the presynaptic structure of mouse photoreceptor ribbon synapses. Detailed immunocytochemical light and electron microscopical analysis of Piccolino knockdown in photoreceptors revealed a hitherto undescribed photoreceptor ribbon synaptic phenotype with striking morphological changes of synaptic ribbon ultrastructure.

Quantification of nanowire penetration into living cells.

High-aspect ratio nanostructures such as nanowires and nanotubes are a powerful new tool for accessing the cell interior for delivery and sensing. Controlling and optimizing cellular access is a critical challenge for this new technology, yet even the most basic aspect of this process, whether these structures directly penetrate the cell membrane, is still unknown. Here we report the first quantification of hollow nanowires-nanostraws-that directly penetrate the membrane by observing dynamic ion delivery from each 100-nm diameter nanostraw. We discover that penetration is a rare event: 7.1±2.7% of the nanostraws penetrate the cell to provide cytosolic access for an extended period for an average of 10.7±5.8 penetrations per cell. Using time-resolved delivery, the kinetics of the first penetration event are shown to be adhesion dependent and coincident with recruitment of focal adhesion-associated proteins. These measurements provide a quantitative basis for understanding nanowire-cell interactions, and a means for rapidly assessing membrane penetration.

Nanostraw-electroporation system for highly efficient intracellular delivery and transfection.

Nondestructive introduction of genes, proteins, and small molecules into mammalian cells with high efficiency is a challenging, yet critical, process. Here we demonstrate a simple nanoelectroporation platform to achieve highly efficient molecular delivery and high transfection yields with excellent uniformity and cell viability. The system is built on alumina nanostraws extending from a track-etched membrane, forming an array of hollow nanowires connected to an underlying microfluidic channel. Cellular engulfment of the nanostraws provides an intimate contact, significantly reducing the necessary electroporation voltage and increasing homogeneity over a large area. Biomolecule delivery is achieved by diffusion through the nanostraws and enhanced by electrophoresis during pulsing. The system was demonstrated to offer excellent spatial, temporal, and dose control for delivery, as well as providing high-yield cotransfection and sequential transfection.

Bassoon and Piccolo maintain synapse integrity by regulating protein ubiquitination and degradation.

The presynaptic active zone (AZ) is a specialized microdomain designed for the efficient and repetitive release of neurotransmitter. Bassoon and Piccolo are two high molecular weight components of the AZ, with hypothesized roles in its assembly and structural maintenance. However, glutamatergic synapses lacking either protein exhibit relatively minor defects, presumably due to their significant functional redundancy. In the present study, we have used interference RNAs to eliminate both proteins from glutamatergic synapses, and find that they are essential for maintaining synaptic integrity. Loss of Bassoon and Piccolo leads to the aberrant degradation of multiple presynaptic proteins, culminating in synapse degeneration. This phenotype is mediated in part by the E3 ubiquitin ligase Siah1, an interacting partner of Bassoon and Piccolo whose activity is negatively regulated by their conserved zinc finger domains. Our findings demonstrate a novel role for Bassoon and Piccolo as critical regulators of presynaptic ubiquitination and proteostasis.

Formation of Golgi-derived active zone precursor vesicles.

Vesicular trafficking of presynaptic and postsynaptic components is emerging as a general cellular mechanism for the delivery of scaffold proteins, ion channels, and receptors to nascent and mature synapses. However, the molecular mechanisms leading to the selection of cargos and their differential transport to subneuronal compartments are not well understood, in part because of the mixing of cargos at the plasma membrane and/or within endosomal compartments. In the present study, we have explored the cellular mechanisms of active zone precursor vesicle assembly at the Golgi in dissociated hippocampal neurons of Rattus norvegicus. Our studies show that Piccolo, Bassoon, and ELKS2/CAST exit the trans-Golgi network on a common vesicle that requires Piccolo and Bassoon for its proper assembly. In contrast, Munc13 and synaptic vesicle proteins use distinct sets of Golgi-derived transport vesicles, while RIM1α associates with vesicular membranes in a post-Golgi compartment. Furthermore, Piccolo and Bassoon are necessary for ELKS2/CAST to leave the Golgi in association with vesicles, and a core domain of Bassoon is sufficient to facilitate formation of these vesicles. While these findings support emerging principles regarding active zone differentiation, the cellular and molecular analyses reported here also indicate that the Piccolo-Bassoon transport vesicles leaving the Golgi may undergo further changes in protein composition before arriving at synaptic sites.

Piccolo regulates the dynamic assembly of presynaptic F-actin.

Filamentous (F)-actin is a known regulator of the synaptic vesicle (SV) cycle, with roles in SV mobilization, fusion, and endocytosis. However, the molecular pathways that regulate its dynamic assembly within presynaptic boutons remain unclear. In this study, we have used shRNA-mediated knockdown to demonstrate that Piccolo, a multidomain protein of the active zone cytomatrix, is a key regulator of presynaptic F-actin assembly. Boutons lacking Piccolo exhibit enhanced activity-dependent Synapsin1a dispersion and SV exocytosis, and reduced F-actin polymerization and CaMKII recruitment. These phenotypes are rescued by stabilizing F-actin filaments and mimicked by knocking down Profilin2, another regulator of presynaptic F-actin assembly. Importantly, we find that mice with a targeted deletion of exon 14 from the Pclo gene, reported to lack >95% of Piccolo, continue to express multiple Piccolo isoforms. Furthermore, neurons cultured from these mice exhibit no defects in presynaptic F-actin assembly due to the expression of these isoforms at presynaptic boutons. These data reveal that Piccolo regulates neurotransmitter release by facilitating activity-dependent F-actin assembly and the dynamic recruitment of key signaling molecules into presynaptic boutons, and highlight the need for new genetic models with which to study Piccolo loss of function.

v-SNARE composition distinguishes synaptic vesicle pools.

Synaptic vesicles belong to two distinct pools, a recycling pool responsible for the evoked release of neurotransmitter and a resting pool unresponsive to stimulation. The uniform appearance of synaptic vesicles has suggested that differences in location or cytoskeletal association account for these differences in function. We now find that the v-SNARE tetanus toxin-insensitive vesicle-associated membrane protein (VAMP7) differs from other synaptic vesicle proteins in its distribution to the two pools, providing evidence that they differ in molecular composition. We also find that both resting and recycling pools undergo spontaneous release, and when activated by deletion of the longin domain, VAMP7 influences the properties of release. Further, the endocytosis that follows evoked and spontaneous release differs in mechanism, and specific sequences confer targeting to the different vesicle pools. The results suggest that different endocytic mechanisms generate synaptic vesicles with different proteins that can endow the vesicles with distinct properties.

SAP97 directs NMDA receptor spine targeting and synaptic plasticity.

SAP97 is a multidomain scaffold protein implicated in the forward trafficking and synaptic localization of NMDA- and AMPA-type glutamate receptors. Alternative splicing of SAP97 transcripts gives rise to palmitoylated αSAP97 and L27-domain containing ßSAP97 isoforms that differentially regulate the subsynaptic localization of GluR1 subunits of AMPA receptors. Here, we examined whether SAP97 isoforms regulate the mechanisms underlying long-term potentiation (LTP) and depression (LTD) and find that both α- and ß-forms of SAP97 impair LTP but enhance LTD via independent isoform-specific mechanisms. Live imaging of α- and ßSAP97 revealed that the altered synaptic plasticity was not due to activity-dependent changes in SAP97 localization or exchange kinetics. However, by recording from pairs of synaptically coupled hippocampal neurons, we show that αSAP97 occludes LTP by enhancing the levels of postsynaptic AMPA receptors, while ßSAP97 blocks LTP by reducing the synaptic localization of NMDA receptors. Examination of the surface pools of AMPA and NMDA receptors indicates that αSAP97 selectively regulates the synaptic pool of AMPA receptors, whereas ßSAP97 regulates the extrasynaptic pools of both AMPA and NMDA receptors. Knockdown of ßSAP97 increases the synaptic localization of both AMPA and NMDA receptors, showing that endogenous ßSAP97 restricts glutamate receptor expression at excitatory synapses. This isoform-dependent differential regulation of synaptic versus extrasynaptic pools of glutamate receptors will determine how many receptors are available for the induction and the expression of synaptic plasticity. Our data support a model wherein SAP97 isoforms can regulate the ability of synapses to undergo plasticity by controlling the surface distribution of AMPA and NMDA receptors.

A bicistronic lentiviral vector based on the 1D/2A sequence of foot-and-mouth disease virus expresses proteins stoichiometrically.

Classic IRES sequences are notorious for exerting biased expression in favor of upstream coding regions when placed into polycistronic vectors. Here, we report the development of a bicistronic lentiviral system based on the 1D/2A sequence from the foot-and-mouth disease virus that is able to maintain tightly balanced control of upstream and downstream protein expression for several days at a stoichiometry very closely approaching 1.0. Our results suggest that the 1D/2A sequence can be optimized in an FUGW lentiviral setting to coordinate expression of multiple polypeptides, presenting a potentially valuable tool to signaling network researchers and to the gene therapy community.

Rapid assembly of functional presynaptic boutons triggered by adhesive contacts.

CNS synapse assembly typically follows after stable contacts between "appropriate" axonal and dendritic membranes are made. We show that presynaptic boutons selectively form de novo following neuronal fiber adhesion to beads coated with poly-d-lysine (PDL), an artificial cationic polypeptide. As demonstrated by atomic force and live confocal microscopy, functional presynaptic boutons self-assemble as rapidly as 1 h after bead contact, and are found to contain a variety of proteins characteristic of presynaptic endings. Interestingly, presynaptic compartment assembly does not depend on the presence of a biological postsynaptic membrane surface. Rather, heparan sulfate proteoglycans, including syndecan-2, as well as others possibly adsorbed onto the bead matrix or expressed on the axon surface, are required for assembly to proceed by a mechanism dependent on the dynamic reorganization of F-actin. Our results indicate that certain (but not all) nonspecific cationic molecules like PDL, with presumably electrostatically mediated adhesive properties, can effectively bypass cognate and natural postsynaptic ligands to trigger presynaptic assembly in the absence of specific target recognition. In contrast, we find that postsynaptic compartment assembly depends on the prior presence of a mature presynaptic ending.