Differential nanoscale organization of excitatory synapses onto excitatory vs. inhibitory neurons.

A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters (NCs) whose precise alignment across the cleft in a transsynaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses-those forming onto excitatory principal neurons (Ex→Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex→PV synapses). As in Ex→Ex synapses, we find that in Ex→PV synapses, presynaptic Munc13-1 and postsynaptic PSD-95 both form NCs that demonstrate alignment, underscoring synaptic nanostructure and the transsynaptic nanocolumn as conserved organizational principles of excitatory synapses. Despite the general conservation of these features, we observed specific differences in the characteristics of pre- and postsynaptic Ex→PV nanostructure. Ex→PV synapses contained larger PSDs with fewer PSD-95 NCs when accounting for size than Ex→Ex synapses. Furthermore, the PSD-95 NCs were larger and denser. The identity of the postsynaptic cell was also represented in Munc13-1 organization, as Ex→PV synapses hosted larger Munc13-1 puncta that contained less dense but larger and more numerous Munc13-1 NCs. Moreover, we measured the spatial variability of transsynaptic alignment in these synapse types, revealing protein alignment in Ex→PV synapses over a distinct range of distances compared to Ex→Ex synapses. We conclude that while general principles of nanostructure and alignment are shared, cell-specific elements of nanodomain organization likely contribute to functional diversity of excitatory synapses.

Distinct SAP102 and PSD-95 Nano-organization Defines Multiple Types of Synaptic Scaffold Protein Domains at Single Synapses.

MAGUK scaffold proteins play a central role in maintaining and modulating synaptic signaling, providing a framework to retain and position receptors, signaling molecules, and other synaptic components. In particular, the MAGUKs SAP102 and PSD-95 are essential for synaptic function at distinct developmental timepoints and perform both overlapping and unique roles. While their similar structures allow for common binding partners, SAP102 is expressed earlier in synapse development and is required for synaptogenesis, whereas PSD-95 expression peaks later and is associated with synapse maturation. PSD-95 and other key synaptic proteins organize into subsynaptic nanodomains that have a significant impact on synaptic transmission, but the nanoscale organization of SAP102 is unknown. How SAP102 is organized within the synapse, and how it relates spatially to PSD-95 on a nanometer scale, could underlie its unique functions and impact how SAP102 scaffolds synaptic proteins. Here we used DNA-PAINT super-resolution microscopy to measure SAP102 nano-organization and its spatial relationship to PSD-95 at individual synapses in mixed-sex rat cultured neurons. We found that like PSD-95, SAP102 accumulates in high-density subsynaptic nanoclusters (NCs). However, SAP102 NCs were smaller and denser than PSD-95 NCs across development. Additionally, only a subset of SAP102 NCs co-organized with PSD-95, revealing MAGUK nanodomains within individual synapses containing either one or both proteins. These MAGUK nanodomain types had distinct NC properties and were differentially enriched with the presynaptic release protein Munc13-1. This organization into both shared and distinct subsynaptic nanodomains may underlie the ability of SAP102 and PSD-95 to perform both common and unique synaptic functions.

Bi-allelic Variants in METTL5 Cause Autosomal-Recessive Intellectual Disability and Microcephaly.

Intellectual disability (ID) is a genetically and clinically heterogeneous disorder, characterized by limited cognitive abilities and impaired adaptive behaviors. In recent years, exome sequencing (ES) has been instrumental in deciphering the genetic etiology of ID. Here, through ES of a large cohort of individuals with ID, we identified two bi-allelic frameshift variants in METTL5, c.344_345delGA (p.Arg115Asnfs∗19) and c.571_572delAA (p.Lys191Valfs∗10), in families of Pakistani and Yemenite origin. Both of these variants were segregating with moderate to severe ID, microcephaly, and various facial dysmorphisms, in an autosomal-recessive fashion. METTL5 is a member of the methyltransferase-like protein family, which encompasses proteins with a seven-beta-strand methyltransferase domain. We found METTL5 expression in various substructures of rodent and human brains and METTL5 protein to be enriched in the nucleus and synapses of the hippocampal neurons. Functional studies of these truncating variants in transiently transfected orthologous cells and cultured hippocampal rat neurons revealed no effect on the localization of METTL5 but alter its level of expression. Our in silico analysis and 3D modeling simulation predict disruption of METTL5 function by both variants. Finally, mettl5 knockdown in zebrafish resulted in microcephaly, recapitulating the human phenotype. This study provides evidence that biallelic variants in METTL5 cause ID and microcephaly in humans and highlights the essential role of METTL5 in brain development and neuronal function.

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.

Quantification of trans-synaptic protein alignment: A data analysis case for single-molecule localization microscopy.

Nanoscale distribution of proteins and their relative positioning within a defined subcellular region are key to their physiological functions. Thanks to the super-resolution imaging methods, especially single-molecule localization microscopy (SMLM), mapping the three-dimensional distribution of multiple proteins has been easier and more efficient than ever. Nevertheless, in spite of the many tools available for efficient localization detection and image rendering, it has been a challenge to quantitatively analyze the 3D distribution and relative positioning of proteins in these SMLM data. Here, using heterogeneously distributed synaptic proteins as examples, we describe in detail a series of analytical methods including detection of nanoscale density clusters, quantification of the trans-synaptic alignment between these protein densities, and automatic en face projection and averaging. These analyses were performed within customized Matlab routines and we make the full scripts available. The concepts behind these analytical methods and the scripts can be adapted for quantitative analysis of spatial organization of other macromolecular complexes.

Patterns of conserved gp120 epitope presentation on attached HIV-1 virions.

A complete picture of HIV antigenicity during early replication is needed to elucidate the full range of options for controlling infection. Such information is frequently gained through analyses of isolated viral envelope antigens, host CD4 receptors, and cognate antibodies. However, direct examination of viral particles and virus-cell interactions is now possible via advanced microscopy techniques and reagents. Using such methods, we recently determined that CD4-induced (CD4i) transition state epitopes in the HIV surface antigen, gp120, while not exposed on free particles, rapidly become immunoreactive upon virus-cell binding. Here, we use 3D direct stochastic optical reconstruction microscopy (dSTORM) to show that certain CD4i epitopes specific to transition state structures are exposed across the surface of cell-bound virions, thus explaining their immunoreactivity. Moreover, such structures and their marker epitopes are dispersed to regions of virions distal to CD4 contact. We further show that the appearance and positioning of distal CD4i exposures is partially dependent on Gag maturation and intact matrix-gp41 interactions within the virion. Collectively, these observations provide a unique perspective of HIV during early replication. These features may define unique insights for understanding how humoral responses target virions and for developing related antiviral countermeasures.

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.

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.

Transport along the dendritic endoplasmic reticulum mediates the trafficking of GABAB receptors.

In neurons, secretory organelles within the cell body are complemented by the dendritic endoplasmic reticulum (ER) and Golgi outposts (GOPs), whose role in neurotransmitter receptor trafficking is poorly understood. γ-aminobutyric acid (GABA) type B metabotropic receptors (GABABRs) regulate the efficacy of synaptic transmission throughout the brain. Their plasma membrane availability is controlled by mechanisms involving an ER retention motif and assembly-dependent ER export. Thus, they constitute an ideal molecular model to study ER trafficking, but the extent to which the dendritic ER participates in GABABR biosynthesis has not been thoroughly explored. Here, we show that GABAB1 localizes preferentially to the ER in dendrites and moves long distances within this compartment. Not only diffusion but also microtubule and dynein-dependent mechanisms control dendritic ER transport. GABABRs insert throughout the somatodendritic plasma membrane but dendritic post-ER carriers containing GABABRs do not fuse selectively with GOPs. This study furthers our understanding of the spatial selectivity of neurotransmitter receptors for dendritic organelles.

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.

Specific sorting and post-Golgi trafficking of dendritic potassium channels in living neurons.

Proper membrane localization of ion channels is essential for the function of neuronal cells. Particularly, the computational ability of dendrites depends on the localization of different ion channels in specific subcompartments. However, the molecular mechanisms that control ion channel localization in distinct dendritic subcompartments are largely unknown. Here, we developed a quantitative live cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels that exhibit distinct localizations: Kv2.1 in proximal dendrites and Kv4.2 in distal dendrites. Our results show that Kv2.1 and Kv4.2 channels are sorted into two distinct populations of vesicles at the Golgi apparatus. The targeting of Kv2.1 and Kv4.2 vesicles occurred by distinct mechanisms as evidenced by their requirement for specific peptide motifs, cytoskeletal elements, and motor proteins. By live cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms are crucial for their specific localizations within dendrites.

Loss of postsynaptic NMDARs drives nanoscale reorganization of Munc13-1 and PSD-95.

Nanoscale protein organization within the active zone (AZ) and post-synaptic density (PSD) influences synaptic transmission. Nanoclusters of presynaptic Munc13-1 are associated with readily releasable pool size and neurotransmitter vesicle priming, while postsynaptic PSD-95 nanoclusters coordinate glutamate receptors across from release sites to control their opening probability. Nanocluster number, size, and protein density vary between synapse types and with development and plasticity, supporting a wide range of functional states at the synapse. Whether or how the receptors themselves control this critical architecture remains unclear. One prominent PSD molecular complex is the NMDA receptor (NMDAR). NMDARs coordinate several modes of signaling within synapses, giving them the potential to influence synaptic organization through direct protein interactions or through signaling. We found that loss of NMDARs results in larger synapses that contain smaller, denser, and more numerous PSD-95 nanoclusters. Intriguingly, NMDAR loss also generates retrograde reorganization of the active zone, resulting in denser, more numerous Munc13-1 nanoclusters, more of which are aligned with PSD-95 nanoclusters. Together, these changes to synaptic nanostructure predict stronger AMPA receptor-mediated transmission in the absence of NMDARs. Notably, while prolonged antagonism of NMDAR activity increases Munc13-1 density within nanoclusters, it does not fully recapitulate these trans-synaptic effects. Thus, our results confirm that NMDARs play an important role in maintaining pre- and postsynaptic nanostructure and suggest that both decreased NMDAR expression and suppressed NMDAR activity may exert distinct effects on synaptic function, yet through unique architectural mechanisms.

Subsynaptic AMPA receptor distribution is acutely regulated by actin-driven reorganization of the postsynaptic density.

AMPA receptors (AMPARs) mediate synaptic transmission and plasticity during learning, development, and disease. Mechanisms determining subsynaptic receptor position are poorly understood but are key determinants of quantal size. We used a series of live-cell, high-resolution imaging approaches to measure protein organization within single postsynaptic densities in rat hippocampal neurons. By photobleaching receptors in synapse subdomains, we found that most AMPARs do not freely diffuse within the synapse, indicating they are embedded in a matrix that determines their subsynaptic position. However, time lapse analysis revealed that synaptic AMPARs are continuously repositioned in concert with plasticity of this scaffold matrix rather than simply by free diffusion. Using a fluorescence correlation analysis, we found that across the lateral extent of single PSDs, component proteins were differentially distributed, and this distribution was continually adjusted by actin treadmilling. The C-terminal PDZ ligand of GluA1 did not regulate its mobility or distribution in the synapse. However, glutamate receptor activation promoted subsynaptic mobility. Strikingly, subsynaptic immobility of both AMPARs and scaffold molecules remained essentially intact even after loss of actin filaments. We conclude that receptors are actively repositioned at the synapse by treadmilling of the actin cytoskeleton, an influence which is transmitted only indirectly to receptors via the pliable and surprisingly dynamic internal structure of the PSD.

Differential nanoscale organization of excitatory synapses onto excitatory vs inhibitory neurons.

A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters whose precise alignment across the cleft in a trans-synaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses - those forming onto excitatory principal neurons (Ex→Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex→PV synapses). As in Ex→Ex synapses, we find that in Ex→PV synapses presynaptic Munc13-1 and postsynaptic PSD-95 both form nanoclusters that demonstrate alignment, underscoring synaptic nanostructure and the trans-synaptic nanocolumn as conserved organizational principles of excitatory synapses. Despite the general conservation of these features, we observed specific differences in the characteristics of pre- and postsynaptic Ex→PV nanostructure. Ex→PV synapses contained larger PSDs with fewer PSD-95 NCs when accounting for size than Ex→Ex synapses. Furthermore, the PSD-95 NCs were larger and denser. The identity of the postsynaptic cell also had a retrograde impact on Munc13-1 organization, as Ex→PV synapses hosted larger Munc13-1 puncta that contained less dense but larger and more numerous Munc13-1 NCs. Moreover, we measured the spatial variability of transsynaptic alignment in these synapse types, revealing protein alignment in Ex→PV synapses over a distinct range of distances compared to Ex→Ex synapses. We conclude that while general principles of nanostructure and alignment are shared, cell-specific elements of nanodomain organization likely contribute to functional diversity of excitatory synapses. Understanding the rules of synapse nanodomain assembly, which themselves are cell-type specific, will be essential for illuminating brain network dynamics.

Distinct SAP102 and PSD-95 nano-organization defines multiple types of synaptic scaffold protein domains at single synapses.

The MAGUK family of scaffold proteins plays a central role in maintaining and modulating synaptic signaling, providing a framework to retain and position receptors, signaling molecules, and other synaptic components. Of these scaffold proteins, SAP102 and PSD-95 are essential for synaptic function at distinct developmental timepoints and perform overlapping as well as unique roles. While their similar structures allow for common binding partners, SAP102 is expressed earlier in synapse development and is required for synaptogenesis, whereas PSD-95 expression peaks later in development and is associated with synapse maturation. PSD-95 and other key synaptic proteins organize into subsynaptic nanodomains that have a significant impact on synaptic transmission, but the nanoscale organization of SAP102 is unknown. How SAP102 is organized within the synapse, and how it relates spatially to PSD-95 on a nanometer scale, could impact how SAP102 clusters synaptic proteins and underlie its ability to perform its unique functions. Here we used DNA-PAINT super-resolution microscopy to measure SAP102 nano-organization and its spatial relationship to PSD-95 at individual synapses. We found that like PSD-95, SAP102 accumulates in high-density subsynaptic nanoclusters. However, SAP102 nanoclusters were smaller and denser than PSD-95 nanoclusters across development. Additionally, only a subset of SAP102 nanoclusters co-organized with PSD-95, revealing that within individual synapses there are nanodomains that contain either one or both proteins. This organization into both shared and distinct subsynaptic nanodomains may underlie the ability of SAP102 and PSD-95 to perform both common and unique synaptic functions.

Trans-synaptic molecular context of NMDA receptor nanodomains.

Tight coordination of the spatial relationships between protein complexes is required for cellular function. In neuronal synapses, many proteins responsible for neurotransmission organize into subsynaptic nanoclusters whose trans-cellular alignment modulates synaptic signal propagation. However, the spatial relationships between these proteins and NMDA receptors (NMDARs), which are required for learning and memory, remain undefined. Here, we mapped the relationship of key NMDAR subunits to reference proteins in the active zone and postsynaptic density using multiplexed super-resolution DNA-PAINT microscopy. GluN2A and GluN2B subunits formed nanoclusters with diverse configurations that, surprisingly, were not localized near presynaptic vesicle release sites marked by Munc13-1. However, a subset of presynaptic sites was configured to maintain NMDAR activation: these were internally denser, aligned with abundant PSD-95, and associated closely with specific NMDAR nanodomains. This work reveals a new principle regulating NMDAR signaling and suggests that synaptic functional architecture depends on assembly of multiprotein nanodomains whose interior construction is conditional on trans-cellular relationships.

Molecular definition of distinct active zone protein machineries for Ca2+ channel clustering and synaptic vesicle priming.

Action potentials trigger neurotransmitter release with minimal delay. Active zones mediate this temporal precision by co-organizing primed vesicles with CaV2 Ca2+ channels. The presumed model is that scaffolding proteins directly tether primed vesicles to CaV2s. We find that CaV2 clustering and vesicle priming are executed by separate machineries. At hippocampal synapses, CaV2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins, but distinct interaction motifs independently execute these functions. In heterologous cells, Liprin-α and RIM from co-assemblies that are separate from CaV2-organizing complexes upon co-transfection. At synapses, Liprin-α1-4 knockout impairs vesicle priming, but not CaV2 clustering. The cell adhesion protein PTPσ recruits Liprin-α, RIM and Munc13 into priming complexes without co-clustering of CaV2s. We conclude that active zones consist of distinct complexes to organize CaV2s and vesicle priming, and Liprin-α and PTPσ specifically support priming site assembly.

Outer membrane targeting, ultrastructure, and single molecule localization of the enteropathogenic Escherichia coli type IV pilus secretin BfpB.

Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis.

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'.

Protein drift-diffusion dynamics and phase separation in curved cell membranes and dendritic spines: Hybrid discrete-continuum methods.

We develop methods for investigating protein drift-diffusion dynamics in heterogeneous cell membranes and the roles played by geometry, diffusion, chemical kinetics, and phase separation. Our hybrid stochastic numerical methods combine discrete particle descriptions with continuum-level models for tracking the individual protein drift-diffusion dynamics when coupled to continuum fields. We show how our approaches can be used to investigate phenomena motivated by protein kinetics within dendritic spines. The spine geometry is hypothesized to play an important biological role regulating synaptic strength, protein kinetics, and self-assembly of clusters. We perform simulation studies for model spine geometries varying the neck size to investigate how phase-separation and protein organization is influenced by different shapes. We also show how our methods can be used to study the roles of geometry in reaction-diffusion systems including Turing instabilities. Our methods provide general approaches for investigating protein kinetics and drift-diffusion dynamics within curved membrane structures.