Teixobactin kills bacteria by a two-pronged attack on the cell envelope.

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1-3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a ß-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.

Celebrating the Birthday of AMPA Receptor Nanodomains: Illuminating the Nanoscale Organization of Excitatory Synapses with 10 Nanocandles.

A decade ago, in 2013, and over the course of 4 summer months, three separate observations were reported that each shed light independently on a new molecular organization that fundamentally reshaped our perception of excitatory synaptic transmission (Fukata et al., 2013; MacGillavry et al., 2013; Nair et al., 2013). This discovery unveiled an intricate arrangement of AMPA-type glutamate receptors and their principal scaffolding protein PSD-95, at synapses. This breakthrough was made possible, thanks to advanced super-resolution imaging techniques. It fundamentally changed our understanding of excitatory synaptic architecture and paved the way for a brand-new area of research. In this Progressions article, the primary investigators of the nanoscale organization of synapses have come together to chronicle the tale of their discovery. We recount the initial inquiry that prompted our research, the preceding studies that inspired our work, the technical obstacles that were encountered, and the breakthroughs that were made in the subsequent decade in the realm of nanoscale synaptic transmission. We review the new discoveries made possible by the democratization of super-resolution imaging techniques in the field of excitatory synaptic physiology and architecture, first by the extension to other glutamate receptors and to presynaptic proteins and then by the notion of trans-synaptic organization. After describing the organizational modifications occurring in various pathologies, we discuss briefly the latest technical developments made possible by super-resolution imaging and emerging concepts in synaptic physiology.

Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons.

Axon formation critically relies on local microtubule remodeling and marks the first step in establishing neuronal polarity. However, the function of the microtubule-organizing centrosomes during the onset of axon formation is still under debate. Here, we demonstrate that centrosomes play an essential role in controlling axon formation in human-induced pluripotent stem cell (iPSC)-derived neurons. Depleting centrioles, the core components of centrosomes, in unpolarized human neuronal stem cells results in various axon developmental defects at later stages, including immature action potential firing, mislocalization of axonal microtubule-associated Trim46 proteins, suppressed expression of growth cone proteins, and affected growth cone morphologies. Live-cell imaging of microtubules reveals that centriole loss impairs axonal microtubule reorganization toward the unique parallel plus-end out microtubule bundles during early development. We propose that centrosomes mediate microtubule remodeling during early axon development in human iPSC-derived neurons, thereby laying the foundation for further axon development and function.

VAP-SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function.

In neurons, the continuous and dynamic endoplasmic reticulum (ER) network extends throughout the axon, and its dysfunction causes various axonopathies. However, it remains largely unknown how ER integrity and remodeling modulate presynaptic function in mammalian neurons. Here, we demonstrated that ER membrane receptors VAPA and VAPB are involved in modulating the synaptic vesicle (SV) cycle. VAP interacts with secernin-1 (SCRN1) at the ER membrane via a single FFAT-like motif. Similar to VAP, loss of SCRN1 or SCRN1-VAP interactions resulted in impaired SV cycling. Consistently, SCRN1 or VAP depletion was accompanied by decreased action potential-evoked Ca2+ responses. Additionally, we found that VAP-SCRN1 interactions play an important role in maintaining ER continuity and dynamics, as well as presynaptic Ca2+ homeostasis. Based on these findings, we propose a model where the ER-localized VAP-SCRN1 interactions provide a novel control mechanism to tune ER remodeling and thereby modulate Ca2+ dynamics and SV cycling at presynaptic sites. These data provide new insights into the molecular mechanisms controlling ER structure and dynamics, and highlight the relevance of ER function for SV cycling.

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons.

The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.

A trans-synaptic nanocolumn aligns neurotransmitter release to receptors.

Synaptic transmission is maintained by a delicate, sub-synaptic molecular architecture, and even mild alterations in synapse structure drive functional changes during experience-dependent plasticity and pathological disorders. Key to this architecture is how the distribution of presynaptic vesicle fusion sites corresponds to the position of receptors in the postsynaptic density. However, while it has long been recognized that this spatial relationship modulates synaptic strength, it has not been precisely described, owing in part to the limited resolution of light microscopy. Using localization microscopy, here we show that key proteins mediating vesicle priming and fusion are mutually co-enriched within nanometre-scale subregions of the presynaptic active zone. Through development of a new method to map vesicle fusion positions within single synapses in cultured rat hippocampal neurons, we find that action-potential-evoked fusion is guided by this protein gradient and occurs preferentially in confined areas with higher local density of Rab3-interacting molecule (RIM) within the active zones. These presynaptic RIM nanoclusters closely align with concentrated postsynaptic receptors and scaffolding proteins, suggesting the existence of a trans-synaptic molecular 'nanocolumn'. Thus, we propose that the nanoarchitecture of the active zone directs action-potential-evoked vesicle fusion to occur preferentially at sites directly opposing postsynaptic receptor-scaffold ensembles. Remarkably, NMDA receptor activation triggered distinct phases of plasticity in which postsynaptic reorganization was followed by trans-synaptic nanoscale realignment. This architecture suggests a simple organizational principle of central nervous system synapses to maintain and modulate synaptic efficiency.

Temporal Quantitative Proteomics of mGluR-induced Protein Translation and Phosphorylation in Neurons.

At neuronal synapses, activation of group I metabotropic glutamate receptors (mGluR1/5) triggers a form of long-term depression (mGluR-LTD) that relies on new protein synthesis and the internalization of AMPA-type glutamate receptors. Dysregulation of these processes has been implicated in the development of mental disorders such as autism spectrum disorders and therefore merit a better understanding on a molecular level. Here, to study mGluR-induced signaling pathways, we integrated quantitative phosphoproteomics with the analyses of newly synthesized proteins via bio-orthogonal amino acids (azidohomoalanine) in a pulsed labeling strategy in cultured hippocampal neurons stimulated with DHPG, a specific agonist for group I mGluRs. We identified several kinases with important roles in DHPG-induced mGluR activation, which we confirmed using small molecule kinase inhibitors. Furthermore, changes in the AMPA receptor endocytosis pathway in both protein synthesis and protein phosphorylation were identified, whereby Intersectin-1 was validated as a novel player in this pathway. This study revealed several new insights into the molecular pathways downstream of group I mGluR activation in hippocampal neurons, and provides a rich resource for further analyses.

Functional organization of postsynaptic glutamate receptors.

Glutamate receptors are the most abundant excitatory neurotransmitter receptors in the brain, responsible for mediating the vast majority of excitatory transmission in neuronal networks. The AMPA- and NMDA-type ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the fast synaptic responses, while metabotropic glutamate receptors (mGluRs) are coupled to downstream signaling cascades that act on much slower timescales. These functionally distinct receptor sub-types are co-expressed at individual synapses, allowing for the precise temporal modulation of postsynaptic excitability and plasticity. Intriguingly, these receptors are differentially distributed with respect to the presynaptic release site. While iGluRs are enriched in the core of the synapse directly opposing the release site, mGluRs reside preferentially at the border of the synapse. As such, to understand the differential contribution of these receptors to synaptic transmission, it is important to not only consider their signaling properties, but also the mechanisms that control the spatial segregation of these receptor types within synapses. In this review, we will focus on the mechanisms that control the organization of glutamate receptors at the postsynaptic membrane with respect to the release site, and discuss how this organization could regulate synapse physiology.

Protein Crowding within the Postsynaptic Density Can Impede the Escape of Membrane Proteins.

Mechanisms regulating lateral diffusion and positioning of glutamate receptors within the postsynaptic density (PSD) determine excitatory synaptic strength. Scaffold proteins in the PSD are abundant receptor binding partners, yet electron microscopy suggests that the PSD is highly crowded, potentially restricting the diffusion of receptors regardless of binding. However, the contribution of macromolecular crowding to receptor retention remains poorly understood. We combined experimental and computational approaches to test the effect of synaptic crowding on receptor movement and positioning in Sprague Dawley rat hippocampal neurons. We modeled AMPA receptor diffusion in synapses where the distribution of scaffold proteins was determined from photoactivated localization microscopy experiments, and receptor-scaffold association and dissociation rates were adjusted to fit single-molecule tracking and fluorescence recovery measurements. Simulations predicted that variation of receptor size strongly influences the fractional synaptic area the receptor may traverse, and the proportion that may exchange in and out of the synapse. To test the model experimentally, we designed a set of novel transmembrane (TM) probes. A single-pass TM protein with one PDZ binding motif concentrated in the synapse as do AMPARs yet was more mobile there than the much larger AMPAR. Furthermore, either the single binding motif or an increase in cytoplasmic bulk through addition of a single GFP slowed synaptic movement of a small TM protein. These results suggest that both crowding and binding limit escape of AMPARs from the synapse. Moreover, tight protein packing within the PSD may modulate the synaptic dwell time of many TM proteins important for synaptic function. SIGNIFICANCE STATEMENT: Small alterations to the distribution within synapses of key transmembrane proteins, such as receptors, can dramatically change synaptic strength. Indeed, many diseases are thought to unbalance neural circuit function in this manner. Processes that regulate this in healthy synapses are unclear, however. By combining computer simulations with imaging methods that examined protein dynamics at multiple scales in space and time, we showed that both steric effects and protein-protein binding each regulate the mobility of receptors in the synapse. Our findings extend our knowledge of the synapse as a crowded environment that counteracts molecular diffusion, and support the idea that both molecular collisions and biochemical binding can be involved in the regulation of neural circuit performance.

Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons.

Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 µg of peptide starting material. The optimized workflow proved to be efficient, sensitive, and reproducible, identifying, localizing, and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications, we monitored EGF-induced signaling in hippocampal neurons, starting with only 200 000 primary cells, resulting in ∼50 µg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.

Shank-cortactin interactions control actin dynamics to maintain flexibility of neuronal spines and synapses.

The family of Shank scaffolding molecules (comprising Shank1, 2 and 3) are core components of the postsynaptic density (PSD) in neuronal synapses. Shanks link surface receptors to other scaffolding molecules within the PSD, as well as to the actin cytoskeleton. However, determining the function of Shank proteins in neurons has been complicated because the different Shank isoforms share a very high degree of sequence and domain homology. Therefore, to control Shank content while minimizing potential compensatory effects, a miRNA-based knockdown strategy was developed to reduce the expression of all synaptically targeted Shank isoforms simultaneously in rat hippocampal neurons. Using this approach, a strong (>75%) reduction in total Shank protein levels was achieved at individual dendritic spines, prompting an approximately 40% decrease in mushroom spine density. Furthermore, Shank knockdown reduced spine actin levels and increased sensitivity to the actin depolymerizing agent Latrunculin A. A SHANK2 mutant lacking the proline-rich cortactin-binding motif (SHANK2-ΔPRO) was unable to rescue these defects. Furthermore, Shank knockdown reduced cortactin levels in spines and increased the mobility of spine cortactin as measured by single-molecule tracking photoactivated localization microscopy, suggesting that Shank proteins recruit and stabilize cortactin at the synapse. Furthermore, it was found that Shank knockdown significantly reduced spontaneous remodelling of synapse morphology that could not be rescued by the SHANK2-ΔPRO mutant. It was concluded that Shank proteins are key intermediates between the synapse and the spine interior that, via cortactin, permit the actin cytoskeleton to dynamically regulate synapse morphology and function.

The internal architecture of dendritic spines revealed by super-resolution imaging: What did we learn so far?

The molecular architecture of dendritic spines defines the efficiency of signal transmission across excitatory synapses. It is therefore critical to understand the mechanisms that control the dynamic localization of the molecular constituents within spines. However, because of the small scale at which most processes within spines take place, conventional light microscopy techniques are not adequate to provide the necessary level of resolution. Recently, super-resolution imaging techniques have overcome the classical barrier imposed by the diffraction of light, and can now resolve the localization and dynamic behavior of proteins within small compartments with nanometer precision, revolutionizing the study of dendritic spine architecture. Here, we highlight exciting new findings from recent super-resolution studies on neuronal spines, and discuss how these studies revealed important new insights into how protein complexes are assembled and how their dynamic behavior shapes the efficiency of synaptic transmission.

Multiple spatial and kinetic subpopulations of CaMKII in spines and dendrites as resolved by single-molecule tracking PALM.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is essential for synaptic plasticity underlying memory formation. Some functions of CaMKII are mediated by interactions with synaptic proteins, and activity-triggered translocation of CaMKII to synapses has been heavily studied. However, CaMKII actions away from the postsynaptic density (PSD) remain poorly understood, in part because of the difficulty in discerning where CaMKII binds in live cells. We used photoactivated localization microscopy (PALM) in rat hippocampal neurons to track single molecules of CaMKIIα, mapping its spatial and kinetic heterogeneity at high resolution. We found that CaMKIIα exhibits at least three kinetic subpopulations, even within individual spines. Latrunculin application or coexpression of CaMKIIß carrying its actin-binding domain strongly modulated CaMKII diffusion, indicating that a major subpopulation is regulated by the actin cytoskeleton. CaMKII in spines was typically more slowly mobile than in dendrites, consistent with presence of a higher density of binding partners or obstacles. Importantly, NMDA receptor stimulation that triggered CaMKII activation prompted the immobilization and presumed binding of CaMKII in spines not only at PSDs but also at other points up to several hundred nanometers away, suggesting that activated kinase does not target only the PSD. Consistent with this, single endogenous activated CaMKII molecules detected via STORM immunocytochemistry were concentrated in spines both at the PSD and at points quite distant from the synapse. Together, these results indicate that CaMKII mobility within spines is determined by association with multiple interacting proteins, even outside the PSD, suggesting diverse mechanisms by which CaMKII may regulate synaptic transmission.

Axonal endoplasmic reticulum tubules control local translation via P180/RRBP1-mediated ribosome interactions.

Local mRNA translation in axons is critical for the spatiotemporal regulation of the axonal proteome. A wide variety of mRNAs are localized and translated in axons; however, how protein synthesis is regulated at specific subcellular sites in axons remains unclear. Here, we establish that the axonal endoplasmic reticulum (ER) supports axonal translation in developing rat hippocampal cultured neurons. Axonal ER tubule disruption impairs local translation and ribosome distribution. Using nanoscale resolution imaging, we find that ribosomes make frequent contacts with axonal ER tubules in a translation-dependent manner and are influenced by specific extrinsic cues. We identify P180/RRBP1 as an axonally distributed ribosome receptor that regulates local translation and binds to mRNAs enriched for axonal membrane proteins. Importantly, the impairment of axonal ER-ribosome interactions causes defects in axon morphology. Our results establish a role for the axonal ER in dynamically localizing mRNA translation, which is important for proper neuron development.

Understanding the nervous system: lessons from Frontiers in Neurophotonics.

The Frontiers in Neurophotonics Symposium is a biennial event that brings together neurobiologists and physicists/engineers who share interest in the development of leading-edge photonics-based approaches to understand and manipulate the nervous system, from its individual molecular components to complex networks in the intact brain. In this Community paper, we highlight several topics that have been featured at the symposium that took place in October 2022 in Québec City, Canada.

LLM3D: a log-linear modeling-based method to predict functional gene regulatory interactions from genome-wide expression data.

All cellular processes are regulated by condition-specific and time-dependent interactions between transcription factors and their target genes. While in simple organisms, e.g. bacteria and yeast, a large amount of experimental data is available to support functional transcription regulatory interactions, in mammalian systems reconstruction of gene regulatory networks still heavily depends on the accurate prediction of transcription factor binding sites. Here, we present a new method, log-linear modeling of 3D contingency tables (LLM3D), to predict functional transcription factor binding sites. LLM3D combines gene expression data, gene ontology annotation and computationally predicted transcription factor binding sites in a single statistical analysis, and offers a methodological improvement over existing enrichment-based methods. We show that LLM3D successfully identifies novel transcriptional regulators of the yeast metabolic cycle, and correctly predicts key regulators of mouse embryonic stem cell self-renewal more accurately than existing enrichment-based methods. Moreover, in a clinically relevant in vivo injury model of mammalian neurons, LLM3D identified peroxisome proliferator-activated receptor γ (PPARγ) as a neuron-intrinsic transcriptional regulator of regenerative axon growth. In conclusion, LLM3D provides a significant improvement over existing methods in predicting functional transcription regulatory interactions in the absence of experimental transcription factor binding data.

Recent advances and challenges in the use of CRISPR/Cas9 genome editing for understanding neuronal cell biology.

The ability to accurately map and manipulate the dynamic subcellular distribution of proteins is key for a mechanistic understanding of neuronal functioning. Current fluorescence microscopy techniques provide access to subcellular protein organization at increasing resolution but are often restricted by the availability of methods that reliably label endogenous proteins. Excitingly, recent development in CRISPR/Cas9 genome editing now allows researchers to specifically tag and visualize endogenous proteins, overcoming limitations associated with current labeling strategies. This article will discuss the progress that has been made in the last years that has led to the development of CRISPR/Cas9 genome editing tools for the reliable mapping of endogenous proteins in neurons. Furthermore, recently developed tools enable the duplex labeling of two proteins simultaneously and acute manipulation of protein distribution. Future implementations of this generation of genome editing technologies will undoubtedly drive progress in molecular and cellular neurobiology.

mGluR5 is transiently confined in perisynaptic nanodomains to shape synaptic function.

The unique perisynaptic distribution of postsynaptic metabotropic glutamate receptors (mGluRs) at excitatory synapses is predicted to directly shape synaptic function, but mechanistic insight into how this distribution is regulated and impacts synaptic signaling is lacking. We used live-cell and super-resolution imaging approaches, and developed molecular tools to resolve and acutely manipulate the dynamic nanoscale distribution of mGluR5. Here we show that mGluR5 is dynamically organized in perisynaptic nanodomains that localize close to, but not in the synapse. The C-terminal domain of mGluR5 critically controlled perisynaptic confinement and prevented synaptic entry. We developed an inducible interaction system to overcome synaptic exclusion of mGluR5 and investigate the impact on synaptic function. We found that mGluR5 recruitment to the synapse acutely increased synaptic calcium responses. Altogether, we propose that transient confinement of mGluR5 in perisynaptic nanodomains allows flexible modulation of synaptic function.

Lateral organization of the postsynaptic density.

Fast excitatory synaptic transmission is mediated by AMPA-type glutamate receptors (AMPARs). It is widely accepted that the number of AMPARs in the postsynaptic density (PSD) critically determines the efficiency of synaptic transmission, but an unappreciated aspect of synapse organization is the lateral positioning of AMPARs within the PSD, that is, their distribution across the face of a single synapse. Receptor lateral positioning is important in a number of processes, most notably because alignment with presynaptic release sites heavily influences the probability of receptor activation. In this review, we summarize current understanding of the mechanisms that dynamically control the subsynaptic positioning of AMPARs. This field is still at early stages, but the recent wave of developments in super-resolution microscopy, synapse tomography, and computational modeling now enable the study of lateral protein distribution and dynamics within the nanometer-scale boundaries of the PSD. We discuss data available measuring the lateral distribution of glutamate receptors and scaffold proteins within the PSD, and discuss potential mechanisms that might give rise to these patterns. Elucidating the mechanisms that underlie the lateral organization of the PSD will be critical to improve our understanding of synaptic processes whose disruption may be unexpectedly important in neurological disorders. This article is part of a Special Issue entitled Membrane Trafficking and Cytoskeletal Dynamics in 'Neuronal Function'.