Journal of cell science最新文献

Eukaryotic cells are compartmentalized into membrane-bound organelles that must coordinate their responses to stimuli. One way that organelles communicate is via membrane contact sites (MCSs), sites of close apposition between organelles used for the exchange of ions, lipids and information. In this Cell Science at a Glance article and the accompanying poster, we describe an explosion of new methods that have led to exciting progress in this area and discuss key examples of how these methods have advanced our understanding of MCSs. We discuss how diffraction-limited and super-resolution fluorescence imaging approaches have provided important insight into the biology of interorganelle communication. We also describe how the development of multiple proximity-based methods has enabled the detection of MCSs with high accuracy and precision. Finally, we assess how recent advances in electron microscopy (EM), considered the gold standard for detecting MCSs, have allowed the visualization of MCSs and associated proteins in 3D at ever greater resolution.

The unparalleled resolving power of electron microscopy is both a blessing and a curse. At 30,000× magnification, 1 µm corresponds to 3 cm in the image and the field of view is only a few micrometres or less, resulting in an inevitable reduction in the spatial data available in an image. Consequently, the gain in resolution is at the cost of loss of the contextual 'reference space', which is crucial for understanding the embedded structures of interest. This problem is particularly pronounced in immunoelectron microscopy, where the detection of a gold particle is crucial for the localisation of specific molecules. The common solution of presenting high-magnification and overview images side by side often insufficiently represents the cellular environment. To address these limitations, we propose here an interactive visualization strategy inspired by digital maps and GPS modules which enables seamless transitions between different magnifications by dynamically linking virtual low magnification overview images with primary high-resolution data. By enabling dynamic browsing, it offers the potential for a deeper understanding of cellular landscapes leading to more comprehensive analysis of the primary ultrastructural data.

Studies utilizing electron microscopy and live fluorescence microscopy have significantly enhanced our understanding of the molecular mechanisms that regulate junctional dynamics during homeostasis, development and disease. To fully grasp the enormous complexity of cell-cell adhesions, it is crucial to study the nanoscale architectures of tight junctions, adherens junctions and desmosomes. It is important to integrate these junctional architectures with the membrane morphology and cellular topography in which the junctions are embedded. In this Review, we explore new insights from studies using super-resolution and volume electron microscopy into the nanoscale organization of these junctional complexes as well as the roles of the junction-associated cytoskeleton, neighboring organelles and the plasma membrane. Furthermore, we provide an overview of junction- and cytoskeletal-related biosensors and optogenetic probes that have contributed to these advances and discuss how these microscopy tools enhance our understanding of junctional dynamics across cellular environments.

Cellular heterogeneity is a well-accepted feature of tissues, and both transcriptional and metabolic diversity have been revealed by numerous approaches, including optical imaging. However, the high magnification objective lenses needed for high-resolution imaging provides information from only small layers of tissue, which can result in poor cell statistics. There is therefore an unmet need for an imaging modality that can provide detailed molecular and cellular insight within intact tissue samples in 3D. Using GFP-tagged GLUT4 as proof of concept, we present here a novel optical mesoscopy approach that allows precise measurement of the spatial location of GLUT4 within specific anatomical structures across the myocardium in ultrathick sections (5 mm×5 mm×3 mm) of intact mouse heart. We reveal distinct GLUT4 distribution patterns across cardiac walls and highlight specific changes in GLUT4 expression levels in response to high fat diet-feeding, and we identify sex-dependent differences in expression patterns. This method is applicable to any target that can be labelled for light microscopy, and to other complex tissues when organ structure needs to be considered simultaneously with cellular detail.

Nonmuscle myosin II (NMII) generates cytoskeletal forces that drive cell division, embryogenesis, muscle contraction and many other cellular functions. However, at present there is no method that can directly measure the forces generated by myosins in living cells. Here, we describe a Förster resonance energy transfer (FRET)-based tension sensor that can detect myosin-associated force along the filamentous actin network. Fluorescence lifetime imaging microscopy (FLIM)-FRET measurements indicate that the forces generated by NMII isoform B (NMIIB) exhibit significant spatial and temporal heterogeneity as a function of donor lifetime and fluorophore energy exchange. These measurements provide a proxy for inferred forces that vary widely along the actin cytoskeleton. This initial report highlights the potential utility of myosin-based tension sensors in elucidating the roles of cytoskeletal contractility in a wide variety of contexts.

The renal glomerulus produces primary urine from blood plasma by ultrafiltration. The ultrastructure of the glomerulus is closely related to filtration function and disease development. The ultrastructure of glomeruli has mainly been evaluated using transmission electron microscopy; however, the volume that can be observed using transmission electron microscopy is extremely limited relative to the total volume of the glomerulus. Consequently, observing structures that exist in only one location in each glomerulus, such as the vascular pole, and evaluating low-density or localized lesions are challenging tasks. Array tomography (AT) is a technique used to analyze the ultrastructure of tissues and cells via scanning electron microscopy of serial sections. In this study, we present an AT workflow that is optimized for observing complete serial sections of the whole glomerulus, and we share several analytical examples that use the optimized AT workflow, demonstrating the usefulness of this approach. Overall, this AT workflow can be a powerful tool for structural and pathological evaluation of the glomerulus. This workflow is also expected to provide new insights into the ultrastructure of the glomerulus and its constituent cells.

The endomembrane system of cereal seed endosperm is a highly plastic and dynamic system reflecting the high degree of specialization of this tissue. It is capable of coping with high levels of storage protein synthesis and undergoes rapid changes to accommodate these storage proteins in newly formed storage organelles such as endoplasmic reticulum-derived protein bodies or protein storage vacuoles. The study of endomembrane morphology in cereal endosperm is challenging due to the amount of starch that cereal seeds accumulate and the progressive desiccation of the tissue. Here, we present a comprehensive study of the endomembrane system of developing barley endosperm cells, complemented by correlative light and electron microscopy (CLEM) imaging. The use of genetically fused fluorescent protein tags in combination with the high resolution of electron microscopy brings ultrastructural research to a new level and can be used to generate novel insights in cell biology in general and in cereal seed research in particular.

Environment-sensitive probes are frequently used in spectral and multi-channel microscopy to study alterations in cell homeostasis. However, the few open-source packages available for processing of spectral images are limited in scope. Here, we present VISION, a stand-alone software based on Python for spectral analysis with improved applicability. In addition to classical intensity-based analysis, our software can batch-process multidimensional images with an advanced single-cell segmentation capability and apply user-defined mathematical operations on spectra to calculate biophysical and metabolic parameters of single cells. VISION allows for 3D and temporal mapping of properties such as membrane fluidity and mitochondrial potential. We demonstrate the broad applicability of VISION by applying it to study the effect of various drugs on cellular biophysical properties. the correlation between membrane fluidity and mitochondrial potential, protein distribution in cell-cell contacts and properties of nanodomains in cell-derived vesicles. Together with the code, we provide a graphical user interface for easy adoption.

The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that have a specialized protein composition, but as the ciliary structure overlays the cell membrane and other cell parts, the identity of ciliary proteins are difficult to ascertain by conventional imaging approaches like immunofluorescence microscopy. Surface scanning electron microscopy combined with immunolabeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the three-dimensional ultrastructure of the cilium. Here, we apply immuno-SEM to specifically identify proteins on the primary cilia of mouse and human pancreatic islets, including post-translationally modified tubulin, intraflagellar transport (IFT)88, the small GTPase Arl13b, as well as subunits of axonemal dynein. Key parameters in sample preparation, immunolabeling and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.