3D cryoimaging of cell-mediated cholesterol crystal clearance in human atherosclerotic lesions.

We applied micro-computed tomography, high-resolution cryo-scanning electron microscopy (SEM) combined with cathodoluminescence, and cryo-focused ion beam Milling-SEM to perform three-dimensional imaging of human atherosclerotic tissues with tens of nanometers resolution, under hydrated, near-native conditions with minimal sample processing. The same technology was applied to cultured macrophages exposed to cholesterol crystals, and the observations made on the macrophages were compared to those made on the pathological tissue. We observed that cholesterol crystal digestion and, eventually, cholesterol crystal clearance occurs in the advanced human plaques through cellular processing.

Revisiting secretory granule fusion at vesicular pseudopodia.

Secretory granules are specialized vesicles that package large amounts of cargo, such as hormones, enzymes and mucous. They are typically stored intracellularly until an extracellular signal initiates exocytosis, characterized by fusion between the secretory vesicles and the plasma membrane, resulting in cargo release to the environment. Observations made in the 1970s suggested that some secretory granules project a pseudopodium from their surface where fusion with the cell surface occurs.

Low-Background His-Tag-Targeting Probes for Turn-On Fluorescence Detection of Cell Surface Proteins and Their Binding Interactions.

Turn-on fluorescent probes consisting of dye-ligand conjugates serve as a powerful tool for detecting cell surface proteins (CSPs) and their interactions with binding partners. However, generating such probes from protein-based ligands remains challenging. This challenge became particularly evident during the COVID-19 pandemic, which highlighted the need for assays to evaluate inhibitors of the interaction between the SARS-CoV-2 virus receptor-binding domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) receptor.

Structured RhoGEF recruitment drives myosin II organization on large exocytic vesicles.

The Rho family of GTPases plays a crucial role in cellular mechanics by regulating actomyosin contractility through the parallel induction of actin and myosin assembly and function. Using exocytosis of large vesicles in the Drosophila larval salivary gland as a model, we followed the spatiotemporal regulation of Rho1, which in turn creates distinct organization patterns of actin and myosin. After vesicle fusion, low levels of activated Rho1 reach the vesicle membrane and drive actin nucleation in an uneven, spread-out pattern.

High-throughput screening identifies broad-spectrum Coronavirus entry inhibitors.

The COVID-19 pandemic highlighted the need for antivirals against emerging coronaviruses (CoV). Inhibiting spike (S) glycoprotein-mediated viral entry is a promising strategy. To identify small molecule inhibitors that block entry downstream of receptor binding, we established a high-throughput screening (HTS) platform based on pseudoviruses.

Machine learning inference of continuous single-cell state transitions during myoblast differentiation and fusion.

Cells modify their internal organization during continuous state transitions, supporting functions from cell division to differentiation. However, tools to measure dynamic physiological states of individual transitioning cells are lacking. We combined live-cell imaging and machine learning to monitor ERK1/2-inhibited primary murine skeletal muscle precursor cells, that transition rapidly and robustly from proliferating myoblasts to post-mitotic myocytes and then fuse, forming multinucleated myotubes.

Clathrin coats partially preassemble and subsequently bend during endocytosis.

Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargo. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle. Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites.

Extracellular vesicle fusion visualized by cryo-electron microscopy.

Extracellular vesicles (EVs) transfer bioactive molecules between cells in a process reminiscent of enveloped viruses. EV cargo delivery is thought to occur by protein-mediated and pH-dependent membrane fusion of the EV and the cellular membrane. However, there is a lack of methods to identify the fusion proteins and resolve their mechanism.

Designer Liposomic Nanocarriers Are Effective Biofilm Eradicators.

Drug delivery via nanovehicles is successfully employed in several clinical settings, yet bacterial infections, forming microbial communities in the form of biofilms, present a strong challenge to therapeutic treatment due to resistance to conventional antimicrobial therapies. Liposomes can provide a versatile drug-vector strategy for biofilm treatment, but are limited by the need to balance colloidal stability with biofilm penetration. We have discovered a liposomic functionalization strategy, using membrane-embedded moieties of poly[2-(methacryloyloxy)ethyl phosphorylcholine], pMPC, that overcomes this limitation.

Genetically Engineered MRI-Trackable Extracellular Vesicles as SARS-CoV-2 Mimetics for Mapping ACE2 Binding .

The elucidation of viral-receptor interactions and an understanding of virus-spreading mechanisms are of great importance, particularly in the era of a pandemic. Indeed, advances in computational chemistry, synthetic biology, and protein engineering have allowed precise prediction and characterization of such interactions. Nevertheless, the hazards of the infectiousness of viruses, their rapid mutagenesis, and the need to study viral-receptor interactions in a complex setup call for further developments.

Ca as a coordinator of skeletal muscle differentiation, fusion and contraction.

Muscle regeneration is essential for vertebrate muscle homeostasis and recovery after injury. During regeneration, muscle stem cells differentiate into myocytes, which then fuse with pre-existing muscle fibres. Hence, differentiation, fusion and contraction must be tightly regulated during regeneration to avoid the disastrous consequences of premature fusion of myocytes to actively contracting fibres.

Malaria parasites release vesicle subpopulations with signatures of different destinations.

Malaria is the most serious mosquito-borne parasitic disease, caused mainly by the intracellular parasite Plasmodium falciparum. The parasite invades human red blood cells and releases extracellular vesicles (EVs) to alter its host responses. It becomes clear that EVs are generally composed of sub-populations.

Sample preparation and image registration for correlative cryo-FM and cryo-FIB-SEM of plunge-frozen mammalian cells.

We recently demonstrated how lipid droplets can serve as fiducials for correlating cryo-fluorescence microscopy (cryo-FM) and cryo-focused ion beam scanning electron microscopy (cryo-FIB-SEM) datasets of mammalian cells grown on grids. Here we describe a step-by-step protocol for correlative cryo-FM and cryo-FIB-SEM, starting from sample preparation of C2C12 cell line, followed by imaging with cryo-FM and cryo-FIB-SEM. Finally, we detail how to perform the 3D-correlation with sub-micron accuracy.

SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.

On 24 November 2021, the sequence of a new SARS-CoV-2 viral isolate Omicron-B.1.1.

Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.

On the 24 November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta.

ERK1/2 inhibition promotes robust myotube growth via CaMKII activation resulting in myoblast-to-myotube fusion.

Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes.

fiducial markers for 3D correlative cryo-fluorescence and FIB-SEM imaging.

Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), offering high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples.

Exocytosis by vesicle crumpling maintains apical membrane homeostasis during exocrine secretion.

Exocrine secretion commonly employs micron-scale vesicles that fuse to a limited apical surface, presenting an extreme challenge for maintaining membrane homeostasis. Using Drosophila melanogaster larval salivary glands, we show that the membranes of fused vesicles undergo actomyosin-mediated folding and retention, which prevents them from incorporating into the apical surface. In addition, the diffusion of proteins and lipids between the fused vesicle and the apical surface is limited.

50 Shades of CLEM: How to choose the right approach for you.

Correlative light and electron microscopy (CLEM) combines the strengths of light microscopy (LM) and electron microscopy (EM) to pin-point and visualize cellular or macromolecular structures. However, there are many different imaging modalities that can be combined in a CLEM workflow, creating a vast number of combinations that can overwhelm new-comers to the field. Here, we offer a conceptual framework to help guide the decision-making process for choosing the CLEM workflow that can best address your research question, based on the answer to five questions.

20S proteasomes secreted by the malaria parasite promote its growth.

Mature red blood cells (RBCs) lack internal organelles and canonical defense mechanisms, making them both a fascinating host cell, in general, and an intriguing choice for the deadly malaria parasite Plasmodium falciparum (Pf), in particular. Pf, while growing inside its natural host, the human RBC, secretes multipurpose extracellular vesicles (EVs), yet their influence on this essential host cell remains unknown. Here we demonstrate that Pf parasites, cultured in fresh human donor blood, secrete within such EVs assembled and functional 20S proteasome complexes (EV-20S).

PDZD8 interacts with Protrudin and Rab7 at ER-late endosome membrane contact sites associated with mitochondria.

Endosomes are compositionally dynamic organelles that regulate signaling, nutrient status and organelle quality by specifying whether material entering the cells will be shuttled back to the cell surface or degraded by the lysosome. Recently, membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and endosomes have emerged as important players in endosomal protein sorting, dynamics and motility. Here, we show that PDZD8, a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain-containing ER transmembrane protein, utilizes distinct domains to interact with Rab7-GTP and the ER transmembrane protein Protrudin and together these components localize to an ER-late endosome MCS.

Golgi organization is regulated by proteasomal degradation.

The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown.

IFITM3 Clusters on Virus Containing Endosomes and Lysosomes Early in the Influenza A Infection of Human Airway Epithelial Cells.

Interferon-induced transmembrane proteins (IFITMs) have been shown to strongly affect influenza A virus (IAV) infectivity in tissue culture. Moreover, polymorphisms in IFITM3 have been associated with the severity of the disease in humans. IFITM3 appears to act early in the infection, but its mechanism of action and potential interactions with incoming IAV structures are not yet defined.

Fusogens.

Segev et al. introduce fusogens - the proteins that coordinate, execute and control membrane fusion - and discuss their various physiological functions.