Rescuing dendritic cell interstitial motility sustains antitumour immunity.

The dendritic cell (DC)-initiated and sustained cancer immunity cycle is indispensable for effective endogenous and therapeutically mobilized antitumour T cell responses. This necessitates the continuous migration of antigen-carrying DCs from the tumour microenvironment (TME) to the tumour draining lymph nodes (tdLNs). Here, through longitudinal analysis of human and mouse tumours, we observed a progressive decrease in migratory conventional DCs (mig-cDCs) in the tdLNs during tumour progression.

The Golgi Rim is a Precise Tetraplex of Golgin Proteins that Can Self-Assemble into Filamentous Bands.

Golgin proteins have long been suspected to be organizers of the Golgi stack. Using three-dimensional super-resolution microscopy, we comprehensively localize the human golgin family at the rim of the Golgi apparatus at 10-20 nm resolution . Unexpectedly, we find that the golgins are precisely organized into a tetraplex with four discrete layers, each containing a specific set of rim golgins.

Chemical imaging unveils mitochondria as the major site of medicinal biguanide accumulation within cells.

Metformin (MET), a commonly prescribed medication for managing type 2 diabetes, has demonstrated various beneficial effects beyond its primary anti-diabetic efficacy. However, the mechanism underlying MET activity and its distribution within organelles remain largely unknown. In this study, we integrate multiple technologies, including chemical labeling, immunostaining, and high-resolution microscopy imaging, to visualize the accumulation of MET in organelles of cultured cells.

Targeting overexpressed antigens in glioblastoma via CAR T cells with computationally designed high-affinity protein binders.

Chimeric antigen receptor (CAR) T cells targeting receptors on tumour cells have had limited success in patients with glioblastoma. Here we report the development and therapeutic performance of CAR constructs leveraging protein binders computationally designed de novo to have high affinity for the epidermal growth factor receptor (EGFR) or the tumour-associated antigen CD276, which are overexpressed in glioblastoma. With respect to T cells with a CAR using an antibody-derived single-chain variable fragment as antigen-binding domain, the designed binders on CAR T cells promoted the proliferation of the cells, the secretion of cytotoxic cytokines and their resistance to cell exhaustion, and improved antitumour performance in vitro and in vivo.

Native architecture of a human GBP1 defense complex for cell-autonomous immunity to infection.

All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, large supramolecular complexes often activate immune proteins for protection. In this work, we resolved the native structure of a massive host-defense complex that polymerizes 30,000 guanylate-binding proteins (GBPs) over the surface of gram-negative bacteria inside human cells.

Readily releasable β cells with tight Ca-exocytosis coupling dictate biphasic glucose-stimulated insulin secretion.

Biphasic glucose-stimulated insulin secretion (GSIS) is essential for blood glucose regulation, but a mechanistic model incorporating the recently identified islet β cell heterogeneity remains elusive. Here, we show that insulin secretion is spatially and dynamically heterogeneous across the islet. Using a zinc-based fluorophore with spinning-disc confocal microscopy, we reveal that approximately 40% of islet cells, which we call readily releasable β cells (RRβs), are responsible for 80% of insulin exocytosis events.

Copper assisted sequence-specific chemical protein conjugation at a single backbone amide.

Direct, site-specific methods of protein functionalization are highly desirable for biotechnology. However, such methods are challenging due to the difficulty of chemically differentiating a single site within a large protein. Herein, we propose "metal binding targeting" strategy and develop a Copper Assisted Sequence-specific conjugation Tag (CAST) method to achieve rapid (second order rate 8.

Uncovering diffusive states of the yeast membrane protein, Pma1, and how labeling method can change diffusive behavior.

We present and analyze video-microscopy-based single-particle-tracking measurements of the budding yeast (Saccharomyces cerevisiae) membrane protein, Pma1, fluorescently labeled either by direct fusion to the switchable fluorescent protein, mEos3.2, or by a novel, light-touch, labeling scheme, in which a 5 amino acid tag is directly fused to the C-terminus of Pma1, which then binds mEos3.2.

Diverse CMT2 neuropathies are linked to aberrant G3BP interactions in stress granules.

Complex diseases often involve the interplay between genetic and environmental factors. Charcot-Marie-Tooth type 2 neuropathies (CMT2) are a group of genetically heterogeneous disorders, in which similar peripheral neuropathology is inexplicably caused by various mutated genes. Their possible molecular links remain elusive.

Bip-Yorkie interaction determines oncogenic and tumor-suppressive roles of Ire1/Xbp1s activation.

Unfolded protein response (UPR) is the mechanism by which cells control endoplasmic reticulum (ER) protein homeostasis. ER proteostasis is essential to adapt to cell proliferation and regeneration in development and tumorigenesis, but mechanisms linking UPR, growth control, and cancer progression remain unclear. Here, we report that the Ire1/Xbp1s pathway has surprisingly oncogenic and tumor-suppressive roles in a context-dependent manner.

DNA-PAINT Imaging Accelerated by Machine Learning.

DNA point accumulation in nanoscale topography (DNA-PAINT) is an easy-to-implement approach for localization-based super-resolution imaging. Conventional DNA-PAINT imaging typically requires tens of thousands of frames of raw data to reconstruct one super-resolution image, which prevents its potential application for live imaging. Here, we introduce a new DNA-PAINT labeling method that allows for imaging of microtubules with both DNA-PAINT and widefield illumination.

Fluorogenic DNA-PAINT for faster, low-background super-resolution imaging.

DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution microscopy method that can acquire high-fidelity images at nanometer resolution. It suffers, however, from high background and slow imaging speed, both of which can be attributed to the presence of unbound fluorophores in solution. Here we present two-color fluorogenic DNA-PAINT, which uses improved imager probe and docking strand designs to solve these problems.

Adaptive optics in super-resolution microscopy.

Fluorescence microscopy has become a routine tool in biology for interrogating life activities with minimal perturbation. While the resolution of fluorescence microscopy is in theory governed only by the diffraction of light, the resolution obtainable in practice is also constrained by the presence of optical aberrations. The past two decades have witnessed the advent of super-resolution microscopy that overcomes the diffraction barrier, enabling numerous biological investigations at the nanoscale.

Implementation of a 4Pi-SMS super-resolution microscope.

The development of single-molecule switching (SMS) fluorescence microscopy (also called single-molecule localization microscopy) over the last decade has enabled researchers to image cell biological structures at unprecedented resolution. Using two opposing objectives in a so-called 4Pi geometry doubles the available numerical aperture, and coupling this with interferometric detection has demonstrated 3D resolution down to 10 nm over entire cellular volumes. The aim of this protocol is to enable interested researchers to establish 4Pi-SMS super-resolution microscopy in their laboratories.

Accurate 4Pi single-molecule localization using an experimental PSF model.

Interferometric single-molecule localization microscopy (iPALM, 4Pi-SMS) uses multiphase interferometry to localize single fluorophores and achieves nanometer isotropic resolution in 3D. The current data analysis workflow, however, fails to reach the theoretical resolution limit due to the suboptimal localization algorithm. Here, we develop a method to fit an experimentally derived point spread function (PSF) model to the interference 4Pi-PSF.

Palmitoylated Proteins in Plasmodium falciparum-Infected Erythrocytes: Investigation with Click Chemistry and Metabolic Labeling.

The examination of the complex cell biology of the human malaria parasite Plasmodium falciparum usually relies on the time-consuming generation of transgenic parasites. Here, metabolic labeling and click chemistry are employed as a fast transfection-independent method for the microscopic examination of protein S-palmitoylation, an important post-translational modification during the asexual intraerythrocytic replication of P. falciparum.

Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging.

Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy.

Spatial distribution of IL4 controls iNKT cell-DC crosstalk in tumors.

The spatiotemporal distribution of cytokines orchestrates immune responses in vivo, yet the underlying mechanisms remain to be explored. We showed here that the spatial distribution of interleukin-4 (IL4) in invariant natural killer T (iNKT) cells regulated crosstalk between iNKT cells and dendritic cells (DCs) and controlled iNKT cell-mediated T-helper type 1 (Th1) responses. The persistent polarization of IL4 induced by strong lipid antigens, that is, α-galactosylceramide (αGC), caused IL4 accumulation at the immunological synapse (IS), which promoted the activation of the IL4R-STAT6 (signal transducer and activator of transcription 6) pathway and production of IL12 in DCs, which enhanced interferon-γ (IFNγ) production in iNKT cells.

Dual Sensing of Physiologic pH and Calcium by EFCAB9 Regulates Sperm Motility.

Varying pH of luminal fluid along the female reproductive tract is a physiological cue that modulates sperm motility. CatSper is a sperm-specific, pH-sensitive calcium channel essential for hyperactivated motility and male fertility. Multi-subunit CatSper channel complexes organize linear Ca signaling nanodomains along the sperm tail.

3D mapping of nanoscale crosslink heterogeneities in microgels.

The majority of swollen polymer networks exhibit spatial variations in crosslink density. These spatial heterogeneities are particularly important in colloidal gel particles, or microgels, where they manifest themselves on the nanoscale and impact mechanical and transport properties. Despite their importance, the real space nanostructure of these heterogeneities at the individual particle level has remained elusive.

Dynamic nanoscale morphology of the ER surveyed by STED microscopy.

The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Superresolution microscopy recently revealed densely packed, rapidly moving ER tubules mistaken for sheets by conventional light microscopy, highlighting the importance of revisiting classical views of ER structure with high spatiotemporal resolution in living cells. In this study, we use live-cell stimulated emission depletion (STED) microscopy to survey the architecture of the ER at 50-nm resolution.

Arabidopsis Blue Light Receptor Phototropin 1 Undergoes Blue Light-Induced Activation in Membrane Microdomains.

Phototropin (phot)-mediated signaling initiated by blue light (BL) plays a critical role in optimizing photosynthetic light capture at the plasma membrane (PM) in plants. However, the mechanisms underlying the regulation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of phot1-GFP proteins we demonstrated that in the dark phot1 proteins remain in an inactive state and mostly exist as monomers.

Long time-lapse nanoscopy with spontaneously blinking membrane probes.

Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds.