The synaptic scaffold protein MPP2 interacts with GABAA receptors at the periphery of the postsynaptic density of glutamatergic synapses.

Recent advances in imaging technology have highlighted that scaffold proteins and receptors are arranged in subsynaptic nanodomains. The synaptic membrane-associated guanylate kinase (MAGUK) scaffold protein membrane protein palmitoylated 2 (MPP2) is a component of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-associated protein complexes and also binds to the synaptic cell adhesion molecule SynCAM 1. Using superresolution imaging, we show that-like SynCAM 1-MPP2 is situated at the periphery of the postsynaptic density (PSD). In order to explore MPP2-associated protein complexes, we used a quantitative comparative proteomics approach and identified multiple γ-aminobutyric acid (GABA)A receptor subunits among novel synaptic MPP2 interactors. In line with a scaffold function for MPP2 in the assembly and/or modulation of intact GABAA receptors, manipulating MPP2 expression had effects on inhibitory synaptic transmission. We further show that GABAA receptors are found together with MPP2 in a subset of dendritic spines and thus highlight MPP2 as a scaffold that serves as an adaptor molecule, linking peripheral synaptic elements critical for inhibitory regulation to central structures at the PSD of glutamatergic synapses.

Controlled Grafting Expansion Microscopy.

Expansion microscopy (ExM) is a recently developed technique that allows for the resolution of structures below the diffraction limit by physically enlarging a hydrogel-embedded facsimile of the biological sample. The target structure is labeled and this label must be retained in a relative position true to the original, smaller state before expansion by linking it into the gel. However, gel formation and digestion lead to a significant loss in target-delivered label, resulting in weak signal. To overcome this problem, we have here developed an agent combining targeting, fluorescent labeling and gel linkage in a single small molecule. Similar approaches in the past have still suffered from significant loss of label. Here we show that this loss is due to insufficient surface grafting of fluorophores into the hydrogel and develop a solution by increasing the amount of target-bound monomers. Overall, we obtain a significant improvement in fluorescence signal retention and our new dye allows the resolution of nuclear pores as ring-like structures, similar to STED microscopy. We furthermore provide mechanistic insight into dye retention in ExM.

Sub-membrane actin rings compartmentalize the plasma membrane.

The compartmentalization of the plasma membrane (PM) is a fundamental feature of cells. The diffusivity of membrane proteins is significantly lower in biological than in artificial membranes. This is likely due to actin filaments, but assays to prove a direct dependence remain elusive. We recently showed that periodic actin rings in the neuronal axon initial segment (AIS) confine membrane protein motion between them. Still, the local enrichment of ion channels offers an alternative explanation. Here we show, using computational modeling, that in contrast to actin rings, ion channels in the AIS cannot mediate confinement. Furthermore, we show, employing a combinatorial approach of single particle tracking and super-resolution microscopy, that actin rings are close to the PM and that they confine membrane proteins in several neuronal cell types. Finally, we show that actin disruption leads to loss of compartmentalization. Taken together, we here develop a system for the investigation of membrane compartmentalization and show that actin rings compartmentalize the PM.

Synapsin condensation controls synaptic vesicle sequestering and dynamics.

Neuronal transmission relies on the regulated secretion of neurotransmitters, which are packed in synaptic vesicles (SVs). Hundreds of SVs accumulate at synaptic boutons. Despite being held together, SVs are highly mobile, so that they can be recruited to the plasma membrane for their rapid release during neuronal activity. However, how such confinement of SVs corroborates with their motility remains unclear. To bridge this gap, we employ ultrafast single-molecule tracking (SMT) in the reconstituted system of native SVs and in living neurons. SVs and synapsin 1, the most highly abundant synaptic protein, form condensates with liquid-like properties. In these condensates, synapsin 1 movement is slowed in both at short (i.e., 60-nm) and long (i.e., several hundred-nm) ranges, suggesting that the SV-synapsin 1 interaction raises the overall packing of the condensate. Furthermore, two-color SMT and super-resolution imaging in living axons demonstrate that synapsin 1 drives the accumulation of SVs in boutons. Even the short intrinsically-disordered fragment of synapsin 1 was sufficient to restore the native SV motility pattern in synapsin triple knock-out animals. Thus, synapsin 1 condensation is sufficient to guarantee reliable confinement and motility of SVs, allowing for the formation of mesoscale domains of SVs at synapses in vivo.

Precise measurement of nanoscopic septin ring structures with deep learning-assisted quantitative superresolution microscopy.

The combination of image analysis and superresolution microscopy methods allows for unprecedented insight into the organization of macromolecular assemblies in cells. Advances in deep learning (DL)-based object recognition enable the automated processing of large amounts of data, resulting in high accuracy through averaging. However, while the analysis of highly symmetric structures of constant size allows for a resolution approaching the dimensions of structural biology, DL-based image recognition may introduce bias. This prohibits the development of readouts for processes that involve significant changes in size or shape of amorphous macromolecular complexes. Here we address this problem by using changes of septin ring structures in single molecule localization-based superresolution microscopy data as a paradigm. We identify potential sources of bias resulting from different training approaches by rigorous testing of trained models using real or simulated data covering a wide range of possible results. In a quantitative comparison of our models, we find that a trade-off exists between measurement accuracy and the range of recognized phenotypes. Using our thus verified models, we find that septin ring size can be explained by the number of subunits they are assembled from alone. Furthermore, we provide a new experimental system for the investigation of septin polymerization.

Expansion STED microscopy (ExSTED).

The recently developed expansion microscopy method (ExM) allows for the resolution of structures below the diffraction limit of light not by sophisticated instrumentation, but rather by physically expanding the molecular structure of cells. This happens by crosslinking the protein in the sample to a hydrogel that is polymerized in situ and subsequently expanded, tearing the proteins apart in a nearly isotropic manner. In the resulting, larger facsimile of the original sample, the fluorescence-labeled molecules of interest can be optically separated by conventional fluorescence microscopy since the intermolecular distances are enlarged by a factor ranging from ~4 to 20 depending on the chemistry used for the hydrogel. The achieved improvement in resolution thus corresponds to the expansion factor. Further increase in resolution beyond this value may be achieved by combining ExM with established super-resolution microscopy methods. Indeed, this is possible using structured illumination microscopy (SIM) (Halpern et al., 2017; Wang et al., 2018), single molecule localization microscopy (SMLM) (Zwettler et al., 2020) and stimulated emission depletion (STED), as we and others have shown recently (Gambarotto et al., 2019; Gao et al., 2018; Kim, Kim, Lee, & Shim, 2019; Unnersjö-Jess et al., 2016). Here, we provide a protocol, for our method, called ExSTED, which enabled us to achieve an increase in resolution of up to 30-fold compared to conventional microscopy, well beyond what is possible with conventional STED microscopy. Our protocol includes a strategy to achieve very high intensity fluorescence labeling, which is essential for optimal signal retention during the expansion process for ExSTED.

An Efficient GUI-Based Clustering Software for Simulation and Bayesian Cluster Analysis of Single-Molecule Localization Microscopy Data.

Ligand binding of membrane proteins triggers many important cellular signaling events by the lateral aggregation of ligand-bound and other membrane proteins in the plane of the plasma membrane. This local clustering can lead to the co-enrichment of molecules that create an intracellular signal or bring sufficient amounts of activity together to shift an existing equilibrium towards the execution of a signaling event. In this way, clustering can serve as a cellular switch. The underlying uneven distribution and local enrichment of the signaling cluster's constituting membrane proteins can be used as a functional readout. This information is obtained by combining single-molecule fluorescence microscopy with cluster algorithms that can reliably and reproducibly distinguish clusters from fluctuations in the background noise to generate quantitative data on this complex process. Cluster analysis of single-molecule fluorescence microscopy data has emerged as a proliferative field, and several algorithms and software solutions have been put forward. However, in most cases, such cluster algorithms require multiple analysis parameters to be defined by the user, which may lead to biased results. Furthermore, most cluster algorithms neglect the individual localization precision connected to every localized molecule, leading to imprecise results. Bayesian cluster analysis has been put forward to overcome these problems, but so far, it has entailed high computational cost, increasing runtime drastically. Finally, most software is challenging to use as they require advanced technical knowledge to operate. Here we combined three advanced cluster algorithms with the Bayesian approach and parallelization in a user-friendly GUI and achieved up to an order of magnitude faster processing than for previous approaches. Our work will simplify access to a well-controlled analysis of clustering data generated by SMLM and significantly accelerate data processing. The inclusion of a simulation mode aids in the design of well-controlled experimental assays.

Actomyosin Contractility in the Generation and Plasticity of Axons and Dendritic Spines.

Actin and non-muscle myosins have long been known to play important roles in growth cone steering and neurite outgrowth. More recently, novel functions for non-muscle myosin have been described in axons and dendritic spines. Consequently, possible roles of actomyosin contraction in organizing and maintaining structural properties of dendritic spines, the size and location of axon initial segment and axonal diameter are emerging research topics. In this review, we aim to summarize recent findings involving myosin localization and function in these compartments and to discuss possible roles for actomyosin in their function and the signaling pathways that control them.

Directed manipulation of membrane proteins by fluorescent magnetic nanoparticles.

The plasma membrane is the interface through which cells interact with their environment. Membrane proteins are embedded in the lipid bilayer of the plasma membrane and their function in this context is often linked to their specific location and dynamics within the membrane. However, few methods are available to manipulate membrane protein location at the single-molecule level. Here, we use fluorescent magnetic nanoparticles (FMNPs) to track membrane molecules and to control their movement. FMNPs allow single-particle tracking (SPT) at 10 nm and 5 ms spatiotemporal resolution, and using a magnetic needle, we pull membrane components laterally with femtonewton-range forces. In this way, we drag membrane proteins over the surface of living cells. Doing so, we detect barriers which we could localize to the submembrane actin cytoskeleton by super-resolution microscopy. We present here a versatile approach to probe membrane processes in live cells via the magnetic control of membrane protein motion.

The selective PI3Kα inhibitor BYL719 as a novel therapeutic option for neuroendocrine tumors: Results from multiple cell line models.

BACKGROUND/AIMS: The therapeutic options for metastatic neuroendocrine tumors (NETs) are limited. As PI3K signaling is often activated in NETs, we have assessed the effects of selective PI3Kp110α inhibition by the novel agent BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and pulmonary (H727) NET cell lines. METHODS: Cell viability was investigated by WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7 assay, the cell cycle by FACS, cell signaling by Western blot analysis, expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and chromogranin A secretion by ELISA. RESULTS: BYL719 dose-dependently decreased cell viability and colony formation with the highest sensitivity in BON-1, followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced apoptosis and G0/G1 cell cycle arrest associated with increased p27 expression. Western blots showed inhibition of PI3K downstream targets to a varying degree in the different cell lines, but IGF1R activation. The most sensitive BON-1 cells displayed a significant, and H727 cells a non-significant, GSK3 inhibition after BYL719 treatment, but these effects do not appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1 cells showed no GSK3 inhibition, but a modest activation, which would partially counteract the other anti-proliferative effects. Accordingly, BYL719 enhanced neuroendocrine differentiation with the strongest effect in BON-1, followed by H727 cells indicated by induction of chromogranin A and somatostatin receptor 1/2 mRNA-synthesis, but not in QGP-1 cells. In BON-1 and QGP-1 cells, the BYL719/everolimus combination was synergistic through simultaneous AKT/mTORC1 inhibition, and significantly increased somatostatin receptor 2 transcription compared to each drug separately. CONCLUSION: Our results suggest that the agent BYL719 could be a novel therapeutic approach to the treatment of NETs that may sensitize NET cells to somatostatin analogs, and that if there is resistance to its action this may be overcome by combination with everolimus.