RATIONALE: Three-dimensional (3D) organoid models, such as alveolospheres, are unique tools for investigating the mechanisms underlying emphysema. However, high inter-organoid heterogeneity hampers consistent results in emphysema research and drug testing. OBJECTIVES: To develop a tunable 3D alveolosphere derived from human primary type II alveolar epithelial cells (AEC2) for modeling alterations linked to cigarette smoke exposure. METHODS: AEC2 (HTII-280+) were isolated from 52 lung samples from both COPD and non-COPD patients, then cultured in 3D, comparing Matrigel to preformed photopolymerized hydrogel microwells of adjustable size and stiffness. Topological and phenotypic characterization were performed on days (D)1, 7, and 14. Lamellar bodies (LBs) were quantified using artificial intelligence (AI) analysis of transmission electron microscopy (TEM) serial block-face images. Chronic exposure to 1% or 5% cigarette smoke extract (CSE) was performed for 5 consecutive days. RESULTS: Compared to Matrigel-based spheroid cultures, alveolospheres generated in microwells display reduced heterogeneity in size. Such alveolospheres were maintained in culture for 14 days and exhibited central lumen formation from D7 to D14. Across different hydrogel stiffness, a stiffness of 5 kPa was found to best support long-term organoid maintenance. The presence of tight junctions (TEM, ZO-1 immunostaining) suggested an auto-organization. AEC1 markers (P2XR4, PDPN) increased from D1 to D14 while AEC2 markers (ABCA3, SFTPA, SFTPC) persisted over time, in qPCR. TEM indicated surfactant synthesis, and AI-driven LB quantification revealed a decrease in LB-containing cells over time. CSE exposure resulted in cell death, architectural disorganization, oxidative stress, and inflammation. Similarly, alveolospheres derived from COPD patients showed increased expression of inflammatory and cell death markers. CONCLUSION: This standardized and adjustable 3D alveolosphere model, derived from human primary AEC2, successfully reproduces key native alveolar features. Exposure to CSE provides a relevant platform for studying responses to cigarette smoke exposure.

Rationale Three-dimensional (3D) culture models such as alveolosphere, provide a unique tool to study emphysema mechanisms. Reducing the heterogeneity of alveolospheres which are currently mostly grown in Matrigel remains challenging. Objectives To develop a tunable and reproducible 3D-alveolosphere model exclusively from human primary type II alveolar epithelial cells (AEC2) for modeling and understanding emphysema. Methods AEC2 cells (HTII-280+) were isolated from 52 smoker and non-smoker lung samples and cultured in preformed photopolymerized hydrogel microwells of adjustable shape and size. Topological and phenotypic characterization were performed at Day (D)1, 7 and 14. Lamellar bodies (LB) were quantified using artificial intelligence (AI)-based image analysis of transmission electron microscopy (TEM) serial block face images. Emphysema was modeled through chronic exposure to 1 and 5% cigarette smoke extract (CSE) during 5 consecutive days. Measurements and Main Results 3D-alveolospheres were maintained in culture for 14 days with central lumen formation observed from D7 to D14. Presence of tight junctions (TEM imaging and ZO-1 immunostaining) suggested epithelial barrier formation. AEC1 markers ( p2xr4, pdpn ) appeared progressively from D1 to D14 while AEC2 markers ( abca3, sftpa, sftpc ) persisted over time. TEM images indicated surfactant synthesis (LB, lipid bodies) and AI-driven LB quantification showed a decrease in the proportion of LB-containing cells over time. CSE exposure led to cell death, architectural disorganization, oxidative stress and inflammation. Conclusion This standardized and adjustable 3D-alveolosphere model from human primary AEC2, reproduced key native alveolar features. CSE exposure provides an opportunity to appropriately study the pathophysiological pathways involved in emphysema. ### Competing Interest Statement MG CJ YB MT ES KR EL VS have nothing to disclose AP reports grants and personal fees from Chiesi, GSK HB GM FD JWD AAR PE PB LG EM GD ID has a patent (EP N 3050574). PH reports grants and non-financial support from Avad, non-financial support from Chiesi and GlaxoSmithKline, outside the submitted work. MZ reports grants and personal fees from AVAD, Boehringer Ingelheim, personal fees from Chiesi, personal fees from Sanofi, personal fees from CSLBehring, personal fees from AstraZeneca, personal fees from Menarini and personal fees from GSK, and support for attending meetings and/or travel from Chiesi, AstraZeneca and GSK outside the submitted work

Direct interactions between receptors at the neuronal surface have long been proposed to tune signaling cascades and neuronal communication in health and disease. Yet, the lack of direct investigation methods to measure, in live neurons, the interaction between different membrane receptors at the single molecule level has raised unanswered questions on the biophysical properties and biological roles of such receptor interactome. Using a multidimensional spectral single molecule-localization microscopy (MS-SMLM) approach, we monitored the interaction between two membrane receptors, i.e. glutamatergic NMDA (NMDAR) and G protein-coupled dopamine D1 (D1R) receptors. The transient interaction was randomly observed along the dendritic tree of hippocampal neurons. It was higher early in development, promoting the formation of NMDAR-D1R complexes in an mGluR5- and CK1-dependent manner, favoring NMDAR clusters and synaptogenesis in a dopamine receptor signaling-independent manner. Preventing the interaction in the neonate, and not adult, brain alters in vivo spontaneous neuronal network activity pattern in male mice. Thus, a weak and transient interaction between NMDAR and D1R plays a structural and functional role in the developing brain.

The formation of adhesive cell-cell contacts is based on the intrinsic binding properties between specific transmembrane ligand-receptor pairs. In neurons, synaptic adhesion molecules provide a physical linkage between pre- and post-synaptic compartments, but the strength and the dynamic of these complexes in their actual membrane environments remain essentially unknown. To access such information, we developed a versatile assay to measure the affinity and binding kinetics of synaptic ligand-receptor interactions, based on the immobilization of Fc-tagged ligands on micropatterned substrates combined with live imaging of fluorescently-tagged counter receptors in heterologous cells. We applied this strategy to study the heterophilic complex formed between neurexin-1β (Nrx1β) and neuroligin-1 (Nlg1), compared to the homophilic SynCAM1 complex. First, the control of ligand density combined to the measurement of steady-state receptor enrichment at micropatterns demonstrates the high specificity of the matching molecular interactions and allows for the quantification of the two-dimensional affinity of the interaction in a membrane environment. Second, long-term FRAP experiments performed on the two molecular complexes and fitted with analytical models, demonstrate a diffusion-limited regime for SynCAM1 and a reaction-limited regime for Nlg1. This analysis provides a very long bond lifetime of the Nrx1β-Nlg1 complex, which by comparison with a monomeric mutant of Nlg1, can be attributed to the constitutive dimerization of Nlg1. Finally, we used the stable Nrx1β-Nlg1 complex as a pseudo-synaptic platform to analyze the rapid binding kinetics between the scaffolding protein PSD-95 and the intracellular domain of Nlg1, dissecting the contribution of the different PDZ domains through the use of specific PSD-95 point mutants.### Competing Interest StatementThe authors have declared no competing interest.

Single molecule localization (SML) and tracking (SPT) techniques, such as (spt)PALM, (u/DNA)PAINT and quantum dot tracking, have given unprecedented insight into the nanoscale molecular organization and dynamics in living cells. They allow monitoring individual proteins with millisecond temporal resolution and high spatial resolution (<30 nm) by precisely localizing the point spread function (PSF) of individual emitters and tracking their position over time. While SPT methods have been extended to study the temporal dynamics and co-organization of multiple proteins, conventional experimental setups are restricted in the number of proteins they can probe simultaneously and usually have to tradeoff between the number of colors, the spatio-temporal resolution, and the field of view. Yet, localizing and tracking several proteins simultaneously at high spatial and temporal resolution within large field of views can provide important biological insights. By employing a dual-objective spectral imaging configuration compatible with live cell imaging combined with dedicated computation tools, we demonstrate simultaneous 3D single particle localization and tracking of multiple distinct species over large field of views to be feasible without compromising spatio-temporal resolution. The dispersive element introduced into the second optical path induces a spectrally dependent displacement, which we used to analytically separate up to five different fluorescent species of single emitters based on their emission spectra. We used commercially available microscope bodies aligned one on top of the other, offering biologists with a very ergonomic and flexible instrument covering a broad range of SMLM applications. Finally, we developed a powerful freely available software, called PALMTracer, which allows to quantitatively assess 3D + t + λ SMLM data. We illustrate the capacity of our approach by performing multi-color 3D DNA-PAINT of fixed samples, and demonstrate simultaneous tracking of multiple receptors in live fibroblast and neuron cultures.

Leucine Rich Repeat Transmembrane proteins (LRRTMs) are neuronal cell adhesion molecules involved in synapse development and plasticity. LRRTM2 is the most synaptogenic isoform of the family, and its expression is strongly restricted to excitatory synapses in mature neurons. However, the mechanisms by which LRRTM2 is trafficked and stabilized at synapses remain unknown. Here, we examine the role of LRRTM2 intracellular domain on its membrane expression and stabilization at excitatory synapses, using a knock-down strategy combined to single molecule tracking and super-resolution dSTORM microscopy. We show that LRRTM2 operates an important shift in mobility after synaptogenesis in hippocampal neurons. Knock-down of LRRTM2 during synapse formation reduced excitatory synapse density in mature neurons. Deletion of LRRTM2 C-terminal domain abolished the compartmentalization of LRRTM2 in dendrites and disrupted its synaptic enrichment. Furtheremore, we show that LRRTM2 diffusion is increased in the absence of its intracellular domain, and that the protein is more dispersed at synapses. Surprisingly, LRRTM2 confinement at synapses was strongly dependent on a YxxC motif in the C-terminal domain, but was independent of the PDZ-like binding motif ECEV. Finally, the nanoscale organization of LRRTM2 at excitatory synapses depended on its C-terminal domain, with involvement of both the PDZ-binding and YxxC motifs. Altogether, these results demonstrate that LRRTM2 trafficking and enrichment at excitatory synapses are dependent on its intracellular domain.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

The toolbox presented in article number 2000519 by Vincent Studer and co-workers provides natural hydrogel prototyping operations that are queued. With a commercialy available patterned UV light projection system, flat layers of commonplace hydrogels can be shaped and then decorated with adhesion proteins. Cells seeded on these hydrogel templates self-organize into 3D cellular models.

Accumulating evidence suggests AKT1 and DRD2-AKT-GSK3 signaling involvement in schizophrenia. AKT1 activity is also required for lithium, a GSK3 inhibitor, to modulate mood-related behaviors. Notably, GSK3 inhibitor significantly alleviates behavioral deficits in Akt1−/− female mice, whereas typical/atypical antipsychotics have no effect. In agreement with adjunctive therapy with lithium in treating schizophrenia, our data mining indicated that the average utilization rates of lithium in the Taiwan National Health Insurance Research Database from 2002 to 2013 are 10.9% and 6.63% in inpatients and outpatients with schizophrenia, respectively. Given that lithium is commonly used in clinical practice, it is of great interest to evaluate the effect of lithium on alleviating Akt1-related deficits. Taking advantage of Akt1+/− mice to mimic genetic deficiency in patients, behavioral impairments were replicated in female Akt1+/− mice but were alleviated by subchronic lithium treatment for 13 days. Lithium also effectively alleviated the observed reduction in phosphorylated GSK3α/β expression in the brains of Akt1+/− mice. Furthermore, inhibition of Akt expression using an Akt1/2 inhibitor significantly reduced neurite length in P19 cells and primary hippocampal cell cultures, which was also ameliorated by lithium. Collectively, our findings implied the therapeutic potential of lithium and the importance of the AKT1-GSK3 signaling pathway.

Physiologically relevant cell-based models require engineered microenvironments which recapitulate the topographical, biochemical, and mechanical properties encountered in vivo. In this context, hydrogels are the materials of choice. Here a light-based toolbox is able to craft such microniches out of common place materials. Extensive use of benzophenone photoinitiators and their interaction with oxygen achieves this. First, the oxygen inhibition of radicals is harnessed to photoprint hydrogel topographies. Then the chemical properties of benzophenone are exploited to crosslink and functionalize native hydrogels lacking photosensitive moieties. At last, photoscission is introduced: an oxygen-driven, benzophenone-enabled reaction that photoliquefies Matrigel and other common gels. Using these tools, soft hydrogel templates are tailored for cells to grow or self-organize into standardized structures. The described workflow emerges as an effective microniche manufacturing toolset for 3D cell culture.

Cell guidance by anchored molecules, or haptotaxis, is crucial in development, immunology and cancer. Adhesive haptotaxis, or guidance by adhesion molecules, is well established for mesenchymal cells such as fibroblasts, whereas its existence remains unreported for amoeboid cells that require less or no adhesion in order to migrate. We show that, in vitro, amoeboid human T lymphocytes develop adhesive haptotaxis mediated by densities of integrin ligands expressed by high endothelial venules. Moreover, lymphocytes orient towards increasing adhesion with VLA-4 integrins (also known as integrin α4β1), like all mesenchymal cells, but towards decreasing adhesion with LFA-1 integrins (also known as integrin αLβ4), which has not previously been observed. This counterintuitive 'reverse haptotaxis' cannot be explained by existing mechanisms of mesenchymal haptotaxis involving either competitive anchoring of cell edges under tension or differential integrin-activated growth of lamellipodia, because they both favor orientation towards increasing adhesion. The mechanisms and functions of amoeboid adhesive haptotaxis remain unclear; however, multidirectional integrin-mediated haptotaxis might operate around transmigration ports on endothelia, stromal cells in lymph nodes, and inflamed tissue where integrin ligands are spatially modulated.

Dynamic mechanical interactions between adhesion complexes and the cytoskeleton are essential for axon outgrowth and guidance. Whether planar cell polarity (PCP) proteins, which regulate cytoskeleton dynamics and appear necessary for some axon guidance, also mediate interactions with membrane adhesion is still unclear. Here we show that Vangl2 controls growth cone velocity by regulating the internal retrograde actin flow in an N-cadherin-dependent fashion. Single molecule tracking experiments show that the loss of Vangl2 decreased fast-diffusing N-cadherin membrane molecules and increased confined N-cadherin trajectories. Using optically manipulated N-cadherin-coated microspheres, we correlated this behavior to a stronger mechanical coupling of N-cadherin with the actin cytoskeleton. Lastly, we show that the spatial distribution of Vangl2 within the growth cone is selectively affected by an N-cadherin-coated substrate. Altogether, our data show that Vangl2 acts as a negative regulator of axonal outgrowth by regulating the strength of the molecular clutch between N-cadherin and the actin cytoskeleton.

Background: Compelling animal and clinical studies support the N-methyl-D-aspartate receptor (NMDAR) hypofunction hypothesis of schizophrenia and suggest promising pharmacological agents to ameliorate negative and cognitive symptoms of schizophrenia, including sarcosine, a glycine transporter-1 inhibitor. Aims and methods: It is imperative to evaluate the therapeutic potential of sarcosine in animal models, which provide indispensable tools for testing drug effects in detail and elucidating the underlying mechanisms. In this study, a series of seven experiments was conducted to investigate the effect of sarcosine in ameliorating behavioral deficits and the underlying mechanism in pharmacological (i.e., MK-801-induced) and genetic (i.e., serine racemase-null mutant (SR−/−) mice) NMDAR hypofunction models. Results: In Experiment 1, the acute administration of 500/1000 mg/kg sarcosine (i.p.) had no adverse effects on motor function and serum biochemical responses. In Experiments 2–4, sarcosine significantly alleviated MK-801-induced (0.2 mg/kg) brain abnormalities and behavioral deficits in MK-801-induced and SR−/− mouse models. In Experiment 5, the injection of sarcosine enhanced CSF levels of glycine and serine in rat brain. In Experiments 6–7, we show for the first time that sarcosine facilitated NMDAR-mediated hippocampal field excitatory postsynaptic potentials and influenced the movement of surface NMDARs at extrasynaptic sites. Conclusions: Sarcosine effectively regulated the surface trafficking of NMDARs, NMDAR-evoked electrophysiological activity, brain glycine levels and MK-801-induced abnormalities in the brain, which contributed to the amelioration of behavioral deficits in mouse models of NMDAR hypofunction.

Single particle tracking (SPT) techniques such as sptPALM, uPAINT, and quantum dot tracking have given unprecedented insight into molecular dynamics in living cells. They allow monitoring the behavior and molecular interaction of individual proteins at millisecond temporal resolution and high spatial resolution (<30 nm) by fitting the point spread function (PSF) of individual emitters and tracking their position over time. While these SPT methods have been extended to study the temporal dynamics and co-organization of multiple proteins, conventional experimental setups used to perform multicolor imaging are typically limited to two simultaneous wavelengths. Increasing the number of colors requires additional filters for specific fluorescent tags and is usually performed at the expense of spatial or temporal resolution and/or field of view. This limits the minimum diffusion coefficient that can be measured and reduces the statistics that can be gathered from a single experiment, thereby degrading the ability to differentiate between molecular diffusion regimes like immobilization and confined diffusion. Moreover, simultaneous multi-receptor tracking could also reveal specific interactions between different protein populations, which could previously only be inferred from the behavior of a single population without any knowledge of the presumed partner behavior. By employing a dual-objective imaging configuration compatible with routine live cell imaging, we will present a single molecule tracking technique that allows for simultaneous 3D single particle tracking of multiple distinct species without compromising spatio-temporal resolution. A dispersive element introduced into the second optical path induces a spectrallydependent displacement, which is used to separate numerous fluorescent species of single emitters based on their emission spectra, similar to Zhang et al. A proof of concept of the spectral separation abilities of the system will be shown via simultaneous 3D DNA-PAINT of fixed samples, where the acquisition time is significantly reduced compared to conventional sequential multicolor imaging. Lastly, we will demonstrate how the technique can be applied to track multiple receptors in live neuron cultures, and we will discuss possibilities of how advanced data analysis techniques can fully exploit the 5-dimensional data (x,y,z,t,) to extend the capabilities of conventional single particle tracking, such as the investigation of protein-protein interactions. REFERENCES 1. Zhang, Z. et al. Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy. Nature Methods 12, 935–938 (2015).

Guidance of cells by molecules anchored on a substrate, known as haptotaxis, is arguably crucial in development, immunology and cancer, however the exact cues and mechanisms driving cell orientation in vivo are hardly identified. Adhesive haptotaxis has been described in the case of mesenchymatous cells that develop strong pulling forces with their substrates and orient via a tug of war mechanism, a competition between cells pulling edges. In the case of amoeboid cells that migrate with minimal interaction with their substrate, existence of adhesive haptotaxis remains unclear. Here, we studied the crawling of human T lymphocytes on substrates with spatially modulated adhesivity, and observed haptotaxis with surface concentrations of integrin ligands found on high endothelial veinules. Overexpression of ICAM-1 and VCAM-1 molecules observed in vivo at transmigration portals can therefore promote leukocyte recruitment. Mechanistically, we show that integrin-mediated haptotaxis of lymphocytes differ both from active chemotaxis, because no mechanotransduction was detected, and from the passive tug of war mechanism of mesenchymatous cells, because different integrins support opposite phenotypes. Cells favored more adherent zones with VLA-4 and, counterintuitively, less adherent zones with LFA-1. These results reveal that integrins control differential adhesive haptotaxis behaviors without mechanotransduction, and this smart capability may support unsuspected ways for cells path selection.

Hydrogels are the material of choice to emulate the cellular micro-environment thanks to their high hydric content and tunable visco-elastic properties. In field such as cancer research, toxicology or tissue engineering, enabling technologies to control the structural and biochemical properties of hydrogels are much sought after. Indeed, structuring and functionnalizing hydrogels could unlock the design of more advanced organotypic models but existing solutions usually lack the simplicity and flexibility to adapt to the day to day flow of modern research. Here by harnessing generic chemistry with digitally processed UV widefield illumination we achieve additive or subtractive manufacturing and decoration for common hydrogels such has PEG, Matrigel, Agar or Poly-acrylamide. We grew neurons and cell lines onto chemically and topographically complex PEG gels, inside engineered Matrigel structures or within microfluidic chambers demonstrating that simple principles may unlock engineering for hydrogels that lack dedicated chemistry.

The in vitro methods to recapitulate environmental cues around cells are usually optimized to test a specific property of the environment (biochemical nature or the stiffness of the extracellular matrix (ECM), or nanotopography) for its capability to induce defined cell behaviors (lineage commitment, migration). Approaches that combine different environmental cues in 3D to assess the biological response of cells to the spatial organization of different biophysical and biochemical cues are growingly being developed. It is demonstrated how the lamination of through‐hole polymeric biofunctionalized membranes can be implemented to create complex bona fide microniches with differential 3D environmental properties using photoactive materials. The approach enables to create microniches ranging in size from single cells to cell aggregates. They are biofunctionalized in 3D simultaneously with topographical featured, protein patterns and structured ECM surrogate with 1 µm resolution. It is demonstrated how these niches extend in 3D the ability to pattern cells. It is exemplified how they can be used to standardize cells' shapes in 3D and to trigger the apicobasal polarization of single epithelial cells.