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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.

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.

The enrollment system is crucial in proceeding to senior high school. Online processing is now more relevant than the other way of enrollment because of the pandemic in which people were not allowed to go out. In that case, the schools have begun implementing an online process in which students can pass the requirements to the school’s website.   The researchers will use descriptive methods to derive the information needed for the system fully. The researchers will use different apps to complete a fully functional enrollment system for Bestlink College of the Philippines students. In the planning phase, the researchers conducted a Google Meet to discuss and brainstorm ideas for creating our Enrollment system. The researchers also researched and found related studies to help them create a new enrollment system before finishing and finalizing the data gathered. For the designing part, the researchers, the programmer, and the system analyst used the best experimental system to try and find the accurate program for the Enrollment system.   Furthermore, in the analysis stage, the programmer will coordinate with members like the system analyst and project manager in creating and providing codes for shaping the system. Then, in the testing stage, the researchers will test whether the system's parts are flexible and functioning. Finally, in the Implementation stage, the researchers will test the system by surveying the selected participants. With the help of the survey, the researchers will surveillance the functions, capability, and usefulness of the Enrollment system. This will also show what actions will resolve the problems encountered. In the maintenance stage, the researchers will check the survey's feedback, which will help improve the Enrollment system. The problems detected should be easily resolved and reported to the users.   Our system will provide the process you will encounter while enrolling in senior high school at Bestlink College of t

Educational attainment for rural African Americans is lower than that of urban African Americans or rural Whites. The African American Church has always had a strong role in providing educational opportunities for African American children and in community activism in general. This digest discusses partnering with rural African American churches as a strategy to improve the educational achievement and attainment of African American children in the rural South. School partnerships with African American faith communities present an opportunity to build stronger communities and improve educational outcomes for youth. Because of concerns about the separation of church and state, partnerships between schools and faith communities must focus on the common ground, the welfare of the children and the betterment of their lives, not on religious education. Guidelines developed by the Department of Education are presented for partnerships with faith communities that respect the separation of church and state. Six strategies that schools can use to develop partnerships with African American faith communities are noted. (Contains 33 endnotes.) (TD)

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.

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.

China has consolidated itself as a global economic power, and its growth has been remarkable. China’s economic influence in Latin America has significantly increased, and the country has become one of the region’s most important and relevant trade partners. Therefore, the trade relations between Latin America and China are considered “strategic.” In this context, the purpose of this study is to analyze the relationship between international trade with China and inclusive economic growth in Latin America from 2004 to 2021, using data from 13 countries in the region (Uruguay, Peru, Paraguay, Panama, Mexico, El Salvador, Ecuador, Costa Rica, Colombia, Chile, Brazil, Bolivia, and Argentina). Our research is quantitative in nature, with a non-experimental design and a correlational scope. The econometric model used panel data and the Newey-West estimator to account for first-order autocorrelation in the error. The results indicate a statistically significant and negative relationship between Latin American exports to China, which has a 10% impact on inclusive economic growth. Similarly, imports from China to Latin America show a statistically significant and negative relationship of 5% with inclusive economic growth. However, no discernible evidence was found to support a relationship between China’s foreign direct investment (FDI) in Latin American countries and inclusive economic growth., China se ha consolidado como una potencia global y su crecimiento ha sido notable. La influencia económica china en América Latina ha aumentado significativamente y el país es uno de los aliados comerciales más relevantes de la región. Por lo tanto, la relación comercial entre América Latina y China es estratégico. En este contexto, el estudio tiene como propósito analizar la relación entre el comercio internacional con China y el crecimiento económico inclusivo en América Latina entre 2004 y 2021, utilizando datos de 13 países de la región (Uruguay, Perú, Paraguay, Panamá, México, El Salvador, Ecuador, Costa Rica, Colombia, Chile, Brasil, Bolivia y Argentina). Nuestra investigación es de naturaleza cuantitativa, su diseño es no experimental y su alcance es correlacional. El modelo econométrico utilizado empleó datos de panel y el estimador Newey-West para tener en cuenta la autocorrelación de primer orden en el error. Los resultados indican una relación estadísticamente significativa y negativa entre las exportaciones latinoamericanas hacia China, lo cual tiene un impacto del 10 % en el crecimiento económico inclusivo. De manera similar, las importaciones desde China hacia América Latina muestran una relación estadísticamente significativa y negativa del 5 % con el crecimiento económico inclusivo. No se identificó evidencia que respalde una relación entre la inversión extranjera directa (IED) china en los países latinoamericanos y el crecimiento económico inclusivo

Over the past years several forms of superresolution fluorescence microscopy have been developed that offer the possibility to study cellular structures and protein distribution at a resolution well below the diffraction limit of conventional fluorescence microscopy (<200 nm). A particularly powerful superresolution technique is single-molecule localization microscopy (SMLM). SMLM enables the quantitative investigation of subcellular protein distribution at a spatial resolution up to tenfold higher than conventional imaging, even in live cells. Not surprisingly, SMLM has therefore been used in many applications in biology, including neuroscience. This chapter provides a step-by-step SMLM protocol to visualize the nanoscale organization of endogenous proteins in dissociated neurons but can be extended to image other adherent cultured cells. We outline a number of methods to visualize endogenous proteins in neurons for live-cell and fixed application, including immunolabeling, the use of intrabodies for live-cell SMLM, and endogenous tagging using CRISPR/Cas9.

At postsynaptic sites of neurons, a prominent clathrin-coated structure, the endocytic zone (EZ), controls the trafficking of glutamate receptors and is essential for synaptic plasticity. Despite its importance, little is known about how this clathrin structure is organized to mediate endocytosis. We used live-cell and super-resolution microscopy to reveal the dynamic organization of this poorly understood clathrin structure in rat hippocampal neurons. We found that a subset of endocytic proteins only transiently appeared at postsynaptic sites. In contrast, other proteins were persistently enriched and partitioned at the edge of the EZ. We found that uncoupling the EZ from the synapse led to the loss of most of these components, while disrupting interactions with the actin cytoskeleton or membrane did not alter EZ positioning. Finally, we found that plasticity-inducing stimuli promoted the reorganization of the EZ. We conclude that the EZ is a stable, highly organized molecular platform where components are differentially recruited and positioned to orchestrate the endocytosis of synaptic receptors.

The plasma membrane consists of a diverse mixture of molecules that dynamically assemble into a highly non-random organization. The formation of nanoscale domains in the membrane is of particular interest as these domains underlie critical cellular functions. Single-molecule tracking is a powerful method to detect and quantify molecular motion at high temporal and spatial resolution and has therefore been instrumental in understanding mechanisms that underlie membrane organization. In single-molecule trajectories, regions of temporal confinement can be determined that might reveal interesting biophysical interactions important for domain formation. However, analytical methods for the detection of temporal confinement in single-molecule trajectories depend on a variety of parameters that heavily depend on experimental factors and the influence of these factors on the performance of confinement detection are not well understood. Here, we present elaborate confinement analyses on simulated random walks and trajectories that display transient confined behavior to optimize the parameters for different experimental conditions. Furthermore, we demonstrate a heatmap visualization tool that allows spatial mapping of confinement hotspots relative to subcellular markers. Using these optimized tools, we reliably detected subdiffusive behavior of different membrane components and observed differences in the confinement behavior of two types of glutamate receptors in neurons. This study will help in further understanding the dynamic behavior of the complex membrane and its role in cellular functioning.

CRISPR/Cas9-mediated knock-in methods enable the labeling of individual endogenous proteins to faithfully determine their spatiotemporal distribution in cells. However, reliable multiplexing of knock-in events in neurons remains challenging because of cross talk between editing events. To overcome this, we developed conditional activation of knock-in expression (CAKE), allowing efficient, flexible, and accurate multiplex genome editing. To diminish cross talk, CAKE is based on sequential, recombinase-driven guide RNA (gRNA) expression to control the timing of genomic integration of each donor sequence. We show that CAKE is broadly applicable in rat neurons to co-label various endogenous proteins, including cytoskeletal proteins, synaptic scaffolds, ion channels and neurotransmitter receptor subunits. To take full advantage of CAKE, we resolved the nanoscale co-distribution of endogenous synaptic proteins using super-resolution microscopy, demonstrating that their coorganization correlates with synapse size. Finally, we introduced inducible dimerization modules, providing acute control over synaptic receptor dynamics in living neurons. These experiments highlight the potential of CAKE to reveal new biological insight. Altogether, CAKE is a versatile method for multiplex protein labeling that enables the detection, localization, and manipulation of endogenous proteins in neurons.

Presynaptic metabotropic glutamate receptors (mGluRs) are essential for the control of synaptic transmission. However, how the subsynaptic dynamics of these receptors is controlled and contributes to synaptic signaling remain poorly understood quantitatively. Particularly, since the affinity of individual mGluR subtypes for glutamate differs considerably, the activation of mGluR subtypes critically depends on their precise subsynaptic distribution. Here, using superresolution microscopy and single-molecule tracking, we unravel novel molecular mechanisms that control the nanoscale distribution and mobility of presynaptic mGluRs in hippocampal neurons. We demonstrate that the high-affinity group II receptor mGluR2 localizes diffusely along the axon, and is highly mobile, while the low-affinity group III receptor mGluR7 is stably anchored at the active zone. We demonstrate that intracellular interactions modulate surface diffusion of mGluR2, while immobilization of mGluR7 at the active zone relies on its extracellular domain. Receptor activation or increases in synaptic activity do not alter the surface mobility of presynaptic mGluRs. Finally, computational modeling of presynaptic mGluR activity revealed that this particular nanoscale arrangement directly impacts their ability to modulate neurotransmitter release. Altogether, this study demonstrates that distinct mechanisms control surface mobility of presynaptic mGluRs to contribute differentially to glutamatergic synaptic transmission.

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance(1-3). Teixobactin(4) represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan(5). 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 beta-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 teixobactin(4). 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.

Spirituality is generally found to have a significant but small positive association with well-being; however, the associations between spirituality and well-being vary greatly. One organizing framework for understanding the varying associations may be the degree to which spirituality and well-being measures capture connection, the pinnacle of which may be conceptualized as Oneness. The purpose of this paper is twofold: to begin exploring this hypothesis by examining the associations between beliefs in Oneness and dimensions of subjective well-being and to test possible conflating and contributing factors in the associations between beliefs in Oneness and subjective well-being. Results indicated preliminary support for the concept of Oneness as an organizing framework for understanding spirituality and well-being associations. Possible explanations for the results, as well as implications for clinical research, clinical practice, and the field of psychology more broadly, are discussed.

In this work, the formation of austenite in the intercritical interval under continuous heating and isothermal holding in ductile iron with various chemistries was investigated using high-resolution dilatometry and quantitative metallographic analysis. The study was conducted using fully ferritic and fully pearlitic matrices as initial microstructures. Subsequently, a mathematical model based on Avrami's equation, that describes the formation of austenite at the intercritical range, was proposed and adjusted to the experimental data. The results show that austenite formation at the intercritical range is faster and happens at lower temperatures when the initial microstructure is pearlitic. Additionally, the kinetic of austenite formation did not change by adding Cu and it is accelerated by adding Ni to the alloy. Finally, the Avrami's equation allowed to model the austenite formation under continuous heating followed by isothermal holding with a good adjustment to the experimental data, which contributes to the understanding of the kinetic of austenite formation in ductile iron at the intercritical range

This manual, designed to increase citizen understanding of child advocacy, offers practical suggestions on how groups of citizens, civic clubs, and fraternal organizations can become advocates for children in a community. The booklet describes child advocacy, child service agency monitoring, and possible ways to improve community services for children. Six steps for systematic monitoring are outlined: (1) the identification of agencies currently providing services, (2) the selection of agencies for monitoring, (3) the identification of data to be collected, (4) the selection of data collecting techniques, (5) the determination of scope, and (6) the summarization of the data. It is suggested that regular monitoring by child advocacy groups will stimulate self-monitoring in agencies previously unconcerned with accountability. (CS)

Impact statementSuccessful clinical tissue engineering requires functional fidelity of the cultured cell to its in vivo counterpart, but this has been elusive in renal tissue engineering. Typically, renal proximal tubule cells in culture have a flattened morphology and do not express key transporters essential to their function. In this article, we show for the first time that in vitro substrate mechanical properties dictate differentiation of cultured renal proximal tubule cells. Remarkably, this effect was only discernable after 4 weeks in culture, longer than usually reported for this cell type. These results demonstrate a new tunable parameter to optimize cell differentiation in renal tissue engineering.

The precise subsynaptic organization of proteins at the postsynaptic membrane controls synaptic transmission. In particular, postsynaptic receptor complexes are concentrated in distinct membrane nanodomains to optimize synaptic signaling. However, despite the clear functional relevance of subsynaptic receptor organization to synaptic transmission and plasticity, the mechanisms that underlie the nanoscale organization of the postsynaptic membrane remain elusive. Over the last decades, the field has predominantly focused on the role of protein-protein interactions in receptor trafficking and positioning in the synaptic membrane. In contrast, the contribution of lipids, the principal constituents of the membrane, to receptor positioning at the synapse remains poorly understood. Nevertheless, there is compelling evidence that the synaptic membrane is enriched in specific lipid species and that deregulation of lipid homeostasis in neurons severely affects synaptic functioning. In this review we focus on how lipids are organized at the synaptic membrane, with special emphasis on how current models of membrane organization could contribute to protein distribution at the synapse and synaptic transmission. Finally, we will present an outlook on how novel technical developments could be applied to study the dynamic interplay between lipids and proteins at the postsynaptic membrane.

The plethora of functions of glutamate in the brain are mediated by the complementary actions of ionotropic and metabotropic glutamate receptors (mGluRs). The ionotropic glutamate receptors carry most of the fast excitatory transmission, while mGluRs modulate transmission on longer timescales by triggering multiple intracellular signaling pathways. As such, mGluRs mediate critical aspects of synaptic transmission and plasticity. Interestingly, at synapses, mGluRs operate at both sides of the cleft, and thus bidirectionally exert the effects of glutamate. At postsynaptic sites, group I mGluRs act to modulate excitability and plasticity. At presynaptic sites, group II and III mGluRs act as auto-receptors, modulating release properties in an activity-dependent manner. Thus, synaptic mGluRs are essential signal integrators that functionally couple presynaptic and postsynaptic mechanisms of transmission and plasticity. Understanding how these receptors reach the membrane and are positioned relative to the presynaptic glutamate release site are therefore important aspects of synapse biology. In this review, we will discuss the currently known mechanisms underlying the trafficking and positioning of mGluRs at and around synapses, and how these mechanisms contribute to synaptic functioning. We will highlight outstanding questions and present an outlook on how recent technological developments will move this exciting research field forward.

Presynaptic metabotropic glutamate receptors (mGluRs) are essential for activity-dependent modulation of synaptic transmission. However, the mechanisms that control the subsynaptic dynamics of these receptors and their contribution to synaptic signaling are poorly understood. Here, using complementary super-resolution microscopy and single-molecule tracking techniques, we provide novel insights into the molecular mechanisms that control the nanoscale distribution and mobility of presynaptic mGluRs in hippocampal neurons. We demonstrate that the group II receptor mGluR2 localizes diffusely along the axon and boutons, and is highly mobile, while the group III receptor mGluR7 is stably anchored at the active zone, indicating that distinct mechanisms underlie the dynamic distribution of these receptor types. Surprisingly, using domain swapping experiments we found that intracellular interactions modulate surface diffusion of mGluR2, but not mGluR7. Instead, we found that immobilization of mGluR7 at the active zone relies on its extracellular domain. Importantly, receptor activation or increase in synaptic activity did not alter the surface mobility of presynaptic mGluRs. Finally, computational modeling of presynaptic mGluR activity revealed that the precise subsynaptic distribution of mGluRs controls their activation probability and thus directly impacts their ability to modulate neurotransmitter release. Altogether, this study demonstrates that distinct mechanisms control surface mobility of presynaptic mGluRs to differentially contribute to the regulation of glutamatergic synaptic transmission.

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.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever- increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB- Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices.

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.

The differentiation of neuronal stem cells into polarized neurons is a well-coordinated process which has mostly been studied in classical non-human model systems, but to what extent these findings are recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured hiPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomic and proteomic dynamics during these steps. The neuron transcriptome and proteome shows extensive remodeling, with differential expression profiles of ~1100 transcripts and ~2200 proteins during neuronal differentiation and polarization. We also identified a distinct axon developmental stage marked by the relocation of axon initial segment proteins and increased microtubule remodeling from the distal (stage 3a) to the proximal (stage 3b) axon. This developmental transition coincides with action potential maturation. Our comprehensive characterization and quantitative map of transcriptome and proteome dynamics provides a solid framework for studying polarization in human 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.