Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold.

Correlative light and electron microscopy (CLEM) combines light microscopy (LM) of fluorescent samples to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between LM and EM modalities. Post-embedding CLEM enables precise registration of structures directly on EM sections, but requires fluorescent markers withstanding EM sample preparation, especially osmium tetroxide fixation, dehydration and EPON embedding.

Resolving exit strategies of mycobacteria in Dictyostelium discoideum by combining high-pressure freezing with 3D-correlative light and electron microscopy.

The infection course of Mycobacterium tuberculosis is highly dynamic and comprises sequential stages that require damaging and crossing of several membranes to enable the translocation of the bacteria into the cytosol or their escape from the host. Many important breakthroughs such as the restriction of mycobacteria by the autophagy pathway and the recruitment of sophisticated host repair machineries to the Mycobacterium-containing vacuole have been gained in the Dictyostelium discoideum/M. marinum system.

Formation of amyloid fibrils from ovalbumin under Ohmic heating.

Ohmic heating (OH) is an alternative sustainable heating technology that has demonstrated its potential to modify protein structures and aggregates. Furthermore, certain protein aggregates, namely amyloid fibrils (AF), are associated with an enhanced protein functionality, such as gelation. This study evaluates how Ohmic heating (OH) influences the formation of AF structures from ovalbumin source under two electric field strength levels, 8.

Human skeletal muscle organoids model fetal myogenesis and sustain uncommitted PAX7 myogenic progenitors.

In vitro culture systems that structurally model human myogenesis and promote PAX7 myogenic progenitor maturation have not been established. Here we report that human skeletal muscle organoids can be differentiated from induced pluripotent stem cell lines to contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures reassembling neural plate border and dermomyotome. Culture conditions instigate neural lineage arrest and promote fetal hypaxial myogenesis toward limb axial anatomical identity, with generation of sustainable uncommitted PAX7 myogenic progenitors and fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from the second trimester of human gestation.

Loss of respiratory complex I subunit NDUFB10 affects complex I assembly and supercomplex formation.

The orchestrated activity of the mitochondrial respiratory or electron transport chain (ETC) and ATP synthase convert reduction power (NADH, FADH) into ATP, the cell's energy currency in a process named oxidative phosphorylation (OXPHOS). Three out of the four ETC complexes are found in supramolecular assemblies: complex I, III, and IV form the respiratory supercomplexes (SC). The plasticity model suggests that SC formation is a form of adaptation to changing conditions such as energy supply, redox state, and stress.

Early Endosomes Act as Local Exocytosis Hubs to Repair Endothelial Membrane Damage.

The plasma membrane of a cell is subject to stresses causing ruptures that must be repaired immediately to preserve membrane integrity and ensure cell survival. Yet, the spatio-temporal membrane dynamics at the wound site and the source of the membrane required for wound repair are poorly understood. Here, it is shown that early endosomes, previously only known to function in the uptake of extracellular material and its endocytic transport, are involved in plasma membrane repair in human endothelial cells.

A mesh microelectrode array for non-invasive electrophysiology within neural organoids.

Organoids are emerging in vitro models of human physiology. Neural models require the evaluation of functional activity of single cells and networks, which is commonly measured by microelectrode arrays. The characteristics of organoids clash with existing in vitro or in vivo microelectrode arrays.

Single molecule analyses reveal dynamics of Salmonella translocated effector proteins in host cell endomembranes.

The facultative intracellular pathogen Salmonella enterica remodels the host endosomal system for survival and proliferation inside host cells. Salmonella resides within the Salmonella-containing vacuole (SCV) and by Salmonella-induced fusions of host endomembranes, the SCV is connected with extensive tubular structures termed Salmonella-induced filaments (SIF). The intracellular lifestyle of Salmonella critically depends on effector proteins translocated into host cells.

Inner mitochondrial membrane structure and fusion dynamics are altered in senescent human iPSC-derived and primary rat cardiomyocytes.

Dysfunction of the aging heart is a major cause of death in the human population. Amongst other tasks, mitochondria are pivotal to supply the working heart with ATP. The mitochondrial inner membrane (IMM) ultrastructure is tailored to meet these demands and to provide nano-compartments for specific tasks.

Plasticity and therapeutic potential of cAMP and cGMP-specific phosphodiesterases in .

is a common zoonotic protozoan pathogen adapted to intracellular parasitism in many host cells of diverse organisms. Our previous work has identified 18 cyclic nucleotide phosphodiesterase (PDE) proteins encoded by the parasite genome, of which 11 are expressed during the lytic cycle of its acutely-infectious tachyzoite stage in human cells. Here, we show that ten of these enzymes are promiscuous dual-specific phosphodiesterases, hydrolyzing cAMP and cGMP.

Identification of a Thyroid Hormone Binding Site in Hsp90 with Implications for Its Interaction with Thyroid Hormone Receptor Beta.

While many proteins are known clients of heat shock protein 90 (Hsp90), it is unclear whether the transcription factor, thyroid hormone receptor beta (TRb), interacts with Hsp90 to control hormonal perception and signaling. Higher Hsp90 expression in mouse fibroblasts was elicited by the addition of triiodothyronine (T3). T3 bound to Hsp90 and enhanced adenosine triphosphate (ATP) binding of Hsp90 due to a specific binding site for T3, as identified by molecular docking experiments.

Preparation of Drosophila Tissues and Organs for Transmission Electron Microscopy.

Transmission electron microscopy (TEM) is the method of choice to image the ultrastructure of cells or tissues. TEM allows the visualization of molecular complexes up to an atomic resolution. Thus, TEM data have led to important conclusions about cellular processes and supported findings obtained by functional analyses.

Deciphering the Emulsification Process to Create an Albumin-Perfluorocarbon-(o/w) Nanoemulsion with High Shelf Life and Bioresistivity.

This work aimed at the development of a stable albumin-perfluorocarbon (o/w) emulsion as an artificial oxygen carrier suitable for clinical application. So far, albumin-perfluorocarbon-(o/w) emulsions have been successfully applied in preclinical trials. Cross-linking a variety of different physical and chemical methods for the characterization of an albumin-perfluorocarbon (PFC)-(o/w) emulsion was necessary to gain a deep understanding of its specific emulsification processes during high-pressure homogenization.

Landmark-based retrieval of inflamed skin vessels enabled by 3D correlative intravital light and volume electron microscopy.

The nanometer spatial resolution of electron microscopy imaging remains an advantage over light microscopy, but the restricted field of view that can be inspected and the inability to visualize dynamic cellular events are definitely drawbacks of standard transmission electron microscopy (TEM). Several methods have been developed to overcome these limitations, mainly by correlating the light microscopical image to the electron microscope with correlative light and electron microscopy (CLEM) techniques. Since there is more than one method to obtain the region of interest (ROI), the workflow must be adjusted according to the research question and biological material addressed.

Confocal Real-Time Analysis of Cutaneous Platelet Recruitment during Immune Complex‒Mediated Inflammation.

Platelets preserve vascular integrity during immune complex‒mediated skin inflammation by preventing neutrophil-provoked hemorrhage. However, the single-cell dynamics of this hemostatic process have never been studied in real-time. To monitor the onset of thrombocytopenia-associated hemorrhages and analyze platelet recruitment, we developed a confocal microscopy‒based video-imaging platform for the dorsal skinfold chamber in living mice.

SARS-CoV-2 infects and replicates in photoreceptor and retinal ganglion cells of human retinal organoids.

Several studies have pointed to retinal involvement in COVID-19, yet many questions remain regarding the ability of SARS-CoV-2 to infect and replicate in retinal cells and its effects on the retina. Here, we have used human pluripotent stem cell-derived retinal organoids to study retinal infection by SARS-CoV-2. Indeed, SARS-CoV-2 can infect and replicate in retinal organoids, as it is shown to infect different retinal lineages, such as retinal ganglion cells and photoreceptors.

Ischemic tolerance and cardiac repair in the spiny mouse (Acomys).

Ischemic heart disease and by extension myocardial infarction is the primary cause of death worldwide, warranting regenerative therapies to restore heart function. Current models of natural heart regeneration are restricted in that they are not of adult mammalian origin, precluding the study of class-specific traits that have emerged throughout evolution, and reducing translatability of research findings to humans. Here, we present the spiny mouse (Acomys spp.

Lipid Dynamics in Diisobutylene-Maleic Acid (DIBMA) Lipid Particles in Presence of Sensory Rhodopsin II.

Amphiphilic diisobutylene/maleic acid (DIBMA) copolymers extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding nanosized, discoidal DIBMA lipid particles (DIBMALPs). Depending on the DIBMA/lipid ratio, the size of DIBMALPs can be broadly varied which makes them suitable for the incorporation of proteins of different sizes. Here, we examine the influence of the DIBMALP sizes and the presence of protein on the dynamics of encased lipids.

Evaluation of DIBMA nanoparticles of variable size and anionic lipid content as tools for the structural and functional study of membrane proteins.

Amphiphilic maleic acid-containing polymers allow for the direct extraction of membrane proteins into stable, homogenous, water-soluble copolymer/lipid nanoparticles without the use of detergents. By adjusting the polymer/lipid ratio, the size of the nanoparticles can be tuned at convenience for the incorporation of protein complexes of different size. However, an increase in the size of the lipid nanoparticles may correlate with increased sample heterogeneity, thus hampering their application to spectroscopic and structural techniques where highly homogeneous samples are desirable.

Three-Dimensional Interfacing of Cells with Hierarchical Silicon Nano/Microstructures for Midinfrared Interrogation of In Situ Captured Proteins.

Label-free optical detection of biomolecules is currently limited by a lack of specificity rather than sensitivity. To exploit the much more characteristic refractive index dispersion in the mid-infrared (IR) regime, we have engineered three-dimensional IR-resonant silicon micropillar arrays (Si-MPAs) for protein sensing. By exploiting the unique hierarchical nano- and microstructured design of these Si-MPAs attained by CMOS-compatible silicon-based microfabrication processes, we achieved an optimized interrogation of surface protein binding.

Inhibition of the mitochondrial ATPase function by IF1 changes the spatiotemporal organization of ATP synthase.

• Mitochondrial FF ATP synthase is the key enzyme for mitochondrial bioenergetics. Dimeric FF-ATP synthase, is preferentially located at the edges of the cristae and its oligomerization state determines mitochondrial ultrastructure. The ATP synthase inhibitor protein IF1 modulates not only ATP synthase activity but also regulates both the structure and function of mitochondria.

A trimeric metazoan Rab7 GEF complex is crucial for endocytosis and scavenger function.

Endosome biogenesis in eukaryotic cells is critical for nutrient uptake and plasma membrane integrity. Early endosomes initially contain Rab5, which is replaced by Rab7 on late endosomes prior to their fusion with lysosomes. Recruitment of Rab7 to endosomes requires the Mon1-Ccz1 guanine-nucleotide-exchange factor (GEF).

The spatio-temporal organization of mitochondrial FF ATP synthase in cristae depends on its activity mode.

FF ATP synthase, also known as complex V, is a key enzyme of mitochondrial energy metabolism that can synthesize and hydrolyze ATP. It is not known whether the ATP synthase and ATPase function are correlated with a different spatio-temporal organisation of the enzyme. In order to analyze this, we tracked and localized single ATP synthase molecules in situ using live cell microscopy.

An integrated genome-wide multi-omics analysis of gene expression dynamics in the preimplantation mouse embryo.

Early mouse embryos have an atypical translational machinery that consists of cytoplasmic lattices and is poorly competent for translation. Hence, the impact of transcriptomic changes on the operational level of proteins is predicted to be relatively modest. To investigate this, we performed liquid chromatography-tandem mass spectrometry and mRNA sequencing at seven developmental stages, from the mature oocyte to the blastocyst, and independently validated our data by immunofluorescence and qPCR.

Drosophila pericardial nephrocyte ultrastructure changes during ageing.

Here we show that a labyrinth channel compartment and slit diaphragms, which are the histological structures enabling insect nephrocytes ultrafiltration, are established during embryogenesis first by the garland nephrocytes (GCNs). The later pericardial nephrocytes, which represent the majority of functional nephrocytes in larvae and adults, lack these characteristic features at the embryonic stage. During larval development, a subpopulation of the pericardial cells survives and matures into functional nephrocytes (PCNs) displaying a fully differentiated slit diaphragm and a labyrinth channel compartment.