AFM in cellular and molecular microbiology.

The unique capabilities of the atomic force microscope (AFM), including super-resolution imaging, piconewton force-sensitivity, nanomanipulation and ability to work under physiological conditions, have offered exciting avenues for cellular and molecular biology research. AFM imaging has helped unravel the fine architectures of microbial cell envelopes at the nanoscale, and how these are altered by antimicrobial treatment. Nanomechanical measurements have shed new light on the elasticity, tensile strength and turgor pressure of single cells. Single-molecule and single-cell force spectroscopy experiments have revealed the forces and dynamics of receptor-ligand interactions, the nanoscale distribution of receptors on the cell surface and the elasticity and adhesiveness of bacterial pili. Importantly, recent force spectroscopy studies have demonstrated that extremely stable bonds are formed between bacterial adhesins and their cognate ligands, originating from a catch bond behaviour allowing the pathogen to reinforce adhesion under shear or tensile stress. Here, we survey how the versatility of AFM has enabled addressing crucial questions in microbiology, with emphasis on bacterial pathogens. TAKE AWAYS: AFM topographic imaging unravels the ultrastructure of bacterial envelopes. Nanomechanical mapping shows what makes cell envelopes stiff and resistant to drugs. Force spectroscopy characterises the molecular forces in pathogen adhesion. Stretching pili reveals a wealth of mechanical and adhesive responses.

Difficulties in Differentiating Coronaviruses from Subcellular Structures in Human Tissues by Electron Microscopy.

Efforts to combat the coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have placed a renewed focus on the use of transmission electron microscopy for identifying coronavirus in tissues. In attempts to attribute pathology of COVID-19 patients directly to tissue damage caused by SARS-CoV-2, investigators have inaccurately reported subcellular structures, including coated vesicles, multivesicular bodies, and vesiculating rough endoplasmic reticulum, as coronavirus particles. We describe morphologic features of coronavirus that distinguish it from subcellular structures, including particle size range (60-140 nm), intracellular particle location within membrane-bound vacuoles, and a nucleocapsid appearing in cross section as dense dots (6-12 nm) within the particles. In addition, although the characteristic spikes of coronaviruses may be visible on the virus surface, especially on extracellular particles, they are less evident in thin sections than in negative stain preparations.

Exploring the property space of periodic cellular structures based on crystal networks.

The properties of periodic cellular structures strongly depend on the regular spatial arrangement of their constituent base materials and can be controlled by changing the topology and geometry of the repeating unit cell. Recent advances in three-dimensional (3D) fabrication technologies more and more expand the limits of fabricable real-world architected materials and strengthen the need of novel microstructural topologies for applications across all length scales and fields in both fundamental science and engineering practice. Here, we systematically explore, interpret, and analyze publicly available crystallographic network topologies from a structural point of view and provide a ready-to-use unit cell catalog with more than 17,000 unique entries in total. We show that molecular crystal networks with atoms connected by chemical bonds can be interpreted as cellular structures with nodes connected by mechanical bars. By this, we identify new structures with extremal properties as well as known structures such as the octet-truss or the Kelvin cell and show how crystallographic symmetries are related to the mechanical properties of the structures. Our work provides inspiration for the discovery of novel cellular structures and paves the way for computational methods to explore and design microstructures with unprecedented properties, bridging the gap between microscopic crystal chemistry and macroscopic structural engineering.

Various dictyostelids from the environment can produce multilamellar bodies.

Multilamellar bodies (MLBs), structures composed of concentric membrane layers, are known to be produced by different protozoa, including species of ciliates, free-living amoebae, and Dictyostelium discoideum social amoebae. Initially believed to be metabolic waste, potential roles like cell communication and food storage have been suggested for D. discoideum MLBs, which could be useful for the multicellular development of social amoebae and as a food source. However, among dictyostelids, this phenomenon has only been observed with D. discoideum, and mainly with laboratory strains grown in axenic conditions. It was thought that other social amoebae may also produce MLBs. Four environmental social amoeba isolates were characterized. All strains belong to the Dictyostelium genus, including some likely to be Dictyostelium giganteum. They have distinctive phenotypes comprising their growth rate on Klebsiella aerogenes lawns and the morphology of their fruiting bodies. They all produce MLBs like those produced by a D. discoideum laboratory strain when grown on K. aerogenes lawns, as revealed by analysis using the H36 antibody in epifluorescence microscopy as well as by transmission electron microscopy. Consequently, this study shows that MLBs are produced by various dictyostelid species, which further supports a role for MLBs in the lifestyle of amoebae.

Simulation of subcellular structures.

Advances in molecular dynamics simulations have led to large increases across spatial and complexity scales, providing valuable molecular level insight into processes occurring on the subcellular level. An increasing repertoire of methods to assemble and analyse complex membrane simulations, alongside advances in structural biology methods for membrane proteins, have contributed to our increased understanding of the roles of specific lipid interactions for multiple membrane protein systems. Large scale simulations of crowded protein solutions have provided a model describing the biophysical basis for experimentally observed diffusion properties. In this review we discuss recent approaches that pave the way towards linking molecular level detail to the cellular level.

Changes in cellular structure of heat-treated Salmonella in low-moisture environments.

AIMS: Salmonella cells desiccated in an environment with low-water activity (aw ) show longer survival times and enhanced resistance to heat. However, little is known about the cellular ultrastructure of Salmonella in low-aw environment in relation to the survival and persistence during desiccation. MATERIALS AND RESULTS: In this study, Salmonella Enteritidis strain PT30 was dehydrated by exposure to air or by mixing with wheat flour (aw 0·30 at room temperature) for 7 days followed by heat treatment at 80°C for 10, 20, 60 min respectively. Transmission electron microscopy (TEM) was employed to examine and compare the ultrastructure of heat-treated S. Enteritidis cells after desiccation with the cells suspended in trypticase soy broth (TSB). Cells suspended in TSB broth showed disrupted ribosomes, congregated proteins and denatured DNA. However, no significant alterations were observed in the ultrastructure of the desiccated cells after heat treatment. The number of desiccated S. Enteritidis cells decreased by <1·5 log CFU per gram after 80°C treatment for 60 min, however, cells suspended in TSB declined more than 5 log10 CFU per mL at 80°C within 5 min. CONCLUSIONS: A drastic difference in the number of survivors and cellular ultrastructure was observed between vegetative and air or food-dried S. Enteritidis cells after subjecting to heat treatment at 80°C. No significant ultrastructure changes were observed in desiccated cells after heat treatment except for roughening and corrugating surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a direct comparison to illustrate how desiccation influences the cell ultrastructure before/after heat treatment, which will aid in better understanding of the fundamental mechanism underlying the increased thermal resistance of Salmonella cells in low-aw environment.

Label-free prediction of three-dimensional fluorescence images from transmitted-light microscopy.

Understanding cells as integrated systems is central to modern biology. Although fluorescence microscopy can resolve subcellular structure in living cells, it is expensive, is slow, and can damage cells. We present a label-free method for predicting three-dimensional fluorescence directly from transmitted-light images and demonstrate that it can be used to generate multi-structure, integrated images. The method can also predict immunofluorescence (IF) from electron micrograph (EM) inputs, extending the potential applications.

Methodologies and approaches for the analysis of cell-nanoparticle interactions.

How to study nanoparticle-cell interactions is the key question that puzzles researchers in the fields of nanomedicine as well as in nanotoxicology. In nanotoxicology, the amount of nanoparticles internalized by the cells or bound to the external surfaces of cells determines the toxic profile of those particles. In medical applications, cellular uptake and binding of medically effective nanoparticles decides their efficacy. Despite the importance of understanding the extent and mode of nanoparticle-cell interactions, these processes are underinvestigated, mainly due to the lack of suitable user-friendly methodologies. Here we discuss the advantages and limitations of currently available (and most advanced) microscopic, spectroscopic, and other bioanalytical methods that could be used to assess cell-nanoparticle interactions either qualitatively or quantitatively. Special emphasis is given to the methods that enable analysis and identification of nanoparticles at single-cell level, and allow intracellular localization and speciation analysis of nanoparticles. This article is categorized under: Nanotechnology Approaches to Biology > Cells at the Nanoscale Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials.

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells.

Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3-diaminobenzidine (DAB) and hydrogen peroxide (H2O2).

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. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resolution images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells.

From single molecules to life: microscopy at the nanoscale.

Super-resolution microscopy is the term commonly given to fluorescence microscopy techniques with resolutions that are not limited by the diffraction of light. Since their conception a little over a decade ago, these techniques have quickly become the method of choice for many biologists studying structures and processes of single cells at the nanoscale. In this review, we present the three main approaches used to tackle the diffraction barrier of ∼200 nm: stimulated-emission depletion (STED) microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM). We first present a theoretical overview of the techniques and underlying physics, followed by a practical guide to all of the facets involved in designing a super-resolution experiment, including an approachable explanation of the photochemistry involved, labeling methods available, and sample preparation procedures. Finally, we highlight some of the most exciting recent applications of and developments in these techniques, and discuss the outlook for this field. Graphical Abstract Super-resolution microscopy techniques. Working principles of the common approaches stimulated-emission depletion (STED) microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM).

Toolkit for visualization of the cellular structure and organelles in Aspergillus niger.

Aspergillus niger is a filamentous fungus that is extensively used in industrial fermentations for protein expression and the production of organic acids. Inherent biosynthetic capabilities, such as the capacity to secrete these biomolecules in high amounts, make A. niger an attractive production host. Although A. niger is renowned for this ability, the knowledge of the molecular components that underlie its production capacity, intercellular trafficking processes and secretion mechanisms is far from complete. Here, we introduce a standardized set of tools, consisting of an N-terminal GFP-actin fusion and codon optimized eforRed chromoprotein. Expression of the GFP-actin construct facilitates visualization of the actin filaments of the cytoskeleton, whereas expression of the chromoprotein construct results in a clearly distinguishable red phenotype. These experimentally validated constructs constitute the first set of standardized A. niger biomarkers, which can be used to study morphology, intercellular trafficking, and secretion phenomena.

Microcontact peeling as a new method for cell micropatterning.

Micropatterning is becoming a powerful tool for studying morphogenetic and differentiation processes of cells. Here we describe a new micropatterning technique, which we refer to as microcontact peeling. Polydimethylsiloxane (PDMS) substrates were treated with oxygen plasma, and the resulting hydrophilic layer of the surface was locally peeled off through direct contact with a peeling stamp made of aluminum, copper, or silicon. A hydrophobic layer of PDMS could be selectively exposed only at the places of the physical contact as revealed by water contact angle measurements and angle-resolved X-ray photoelectron spectroscopy, which thus enabled successful micropatterning of cells with micro-featured peeling stamps. This new micropatterning technique needs no procedure for directly adsorbing proteins to bare PDMS in contrast to conventional techniques using a microcontact printing stamp. Given the several unique characteristics, the present technique based on the peel-off of inorganic materials may become a useful option for performing cell micropatterning.

Silencing the radicals improves Click Chemistry.

Modern fluorescence imaging microscopy in living and fixed material makes use of fluorescent probes to label targeted entities. Common labelling approaches include classical immunocytochemistry, expression of chimerically tagged fluorescent protein domains, and chemical affinity-binding or covalent labelling. Of these methods, the so-called "Click Chemistry", is emerging as one of the most influential labelling chemistries introduced in recent times, offering enormous utility for bio-orthoganol attachment of fluorescent probes to biological target entities. In this issue of Biotechnology Journal, Löschberger, Niehörster and Sauer report "ClickOx", a Click Chemistry protocol that uses an enzymatic oxygen scavenger system to reduce concurrent ROS-associated damage during Click labeling.

Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx.

Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.

Quantitative analysis of F-actin redistribution in astrocytoma cells treated with candidate pharmaceuticals.

Actin fibers (F-actin) control the shape and internal organization of cells, and generate force. It has been long appreciated that these functions are tightly coupled, and in some cases drive cell behavior and cell fate. The distribution and dynamics of F-actin is different in cancer versus normal cells and in response to small molecules, including actin-targeting natural products and anticancer drugs. Therefore, quantifying actin structural changes from high resolution fluorescence micrographs is necessary for further understanding actin cytoskeleton dynamics and phenotypic consequences of drug interactions on cells. We applied an artificial neural network algorithm, which used image intensity and anisotropy measurements, to quantitatively classify F-actin subcellular features into actin along the edges of cells, actin at the protrusions of cells, internal fibers and punctate signals. The algorithm measured significant increase in F-actin at cell edges with concomitant decrease in internal punctate actin in astrocytoma cells lacking functional neurofibromin and p53 when treated with three structurally-distinct anticancer small molecules: OSW1, Schweinfurthin A (SA) and a synthetic marine compound 23'-dehydroxycephalostatin 1. Distinctly different changes were measured in cells treated with the actin inhibitor cytochalasin B. These measurements support published reports that SA acts on F-actin in NF1(-/-) neurofibromin deficient cancer cells through changes in Rho signaling. Quantitative pattern analysis of cells has wide applications for understanding mechanisms of small molecules, because many anti-cancer drugs directly or indirectly target cytoskeletal proteins. Furthermore, quantitative information about the actin cytoskeleton may make it possible to further understand cell fate decisions using mathematically testable models.

CellFinder: a cell data repository.

CellFinder (http://www.cellfinder.org) is a comprehensive one-stop resource for molecular data characterizing mammalian cells in different tissues and in different development stages. It is built from carefully selected data sets stemming from other curated databases and the biomedical literature. To date, CellFinder describes 3394 cell types and 50 951 cell lines. The database currently contains 3055 microscopic and anatomical images, 205 whole-genome expression profiles of 194 cell/tissue types from RNA-seq and microarrays and 553 905 protein expressions for 535 cells/tissues. Text mining of a corpus of >2000 publications followed by manual curation confirmed expression information on ∼900 proteins and genes. CellFinder's data model is capable to seamlessly represent entities from single cells to the organ level, to incorporate mappings between homologous entities in different species and to describe processes of cell development and differentiation. Its ontological backbone currently consists of 204 741 ontology terms incorporated from 10 different ontologies unified under the novel CELDA ontology. CellFinder's web portal allows searching, browsing and comparing the stored data, interactive construction of developmental trees and navigating the partonomic hierarchy of cells and tissues through a unique body browser designed for life scientists and clinicians.

Cell cortex composition and homeostasis resolved by integrating proteomics and quantitative imaging.

The cellular actin cortex is the cytoskeletal structure primarily responsible for the control of animal cell shape and as such plays a central role in cell division, migration, and tissue morphogenesis. Due to the lack of experimental systems where the cortex can be investigated independently from other organelles, little is known about its composition, assembly, and homeostasis. Here, we describe novel tools to resolve the composition and regulation of the cortex. We report and validate a protocol for cortex purification based on the separation of cellular blebs. Mass spectrometry analysis of purified cortices provides a first extensive list of cortical components. To assess the function of identified proteins, we design an automated imaging assay for precise quantification of cortical actomyosin assembly dynamics. We show subtle changes in cortex assembly dynamics upon depletion of the identified cortical component profilin. Our widely applicable integrated method paves the way for systems-level investigations of the actomyosin cortex and its regulation during morphogenesis.

The nuage mediates retrotransposon silencing in mouse primordial ovarian follicles.

Mobilization of endogenous retrotransposons can destabilize the genome, an imminent danger during epigenetic reprogramming of cells in the germline. The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway is known to silence retrotransposons in the mouse testes. Several piRNA pathway components localize to the unique, germline structure known as the nuage. In this study, we surveyed mouse ovaries and found, for the first time, transient appearance of nuage-like structures in oocytes of primordial follicles. Mouse vasa homolog (MVH), Piwi-like 2 (PIWIL2/MILI) and tudor domain-containing 9 (TDRD9) are present in these structures, whereas aggregates of germ cell protein with ankyrin repeats, sterile alpha motif and leucine zipper (GASZ) localize separately in the cytoplasm. Retrotransposons are silenced in primordial ovarian follicles, and de-repressed upon reduction of piRNA expression in Mvh, Mili or Gasz mutants. However, these null-mutant females, unlike their male counterparts, are fertile, uncoupling retrotransposon activation from sterility.

Imaging the invisible: resolving cellular microcompartments by superresolution microscopy techniques.

Unraveling the spatio-temporal organization of dynamic cellular microcompartments requires live cell imaging techniques capable of resolving submicroscopic structures. While the resolution of traditional far-field fluorescence imaging techniques is limited by the diffraction barrier, several fluorescence-based microscopy techniques providing sub-100 nm resolution have become available during the past decade. Here, we briefly introduce the optical principles of these techniques and compare their capabilities and limitations with respect to spatial and temporal resolution as well as live cell capabilities. Moreover, we summarize how these techniques contributed to a better understanding of plasma membrane microdomains, the dynamic nanoscale organization of neuronal synapses and the sub-compartmentation of microorganisms. Based on these applications, we highlight complementarity of these techniques and their potential to address specific challenges in the context of dynamic cellular microcompartments, as well as the perspectives to overcome current limitations of these methods.