Current topics in membranes最新文献

Cell division, aging, and stress recovery triggers spatial reorganization of cellular components in the cytoplasm, including membrane bound organelles, with molecular changes in their compositions and structures. However, it is not clear how these events are coordinated and how they integrate with regulation of molecular crowding. We use the budding yeast Saccharomyces cerevisiae as a model system to study these questions using recent progress in optical fluorescence microscopy and crowding sensing probe technology. We used a Förster Resonance Energy Transfer (FRET) based sensor, illuminated by confocal microscopy for high throughput analyses and Slimfield microscopy for single-molecule resolution, to quantify molecular crowding. We determine crowding in response to cellular growth of both mother and daughter cells, in addition to osmotic stress, and reveal hot spots of crowding across the bud neck in the burgeoning daughter cell. This crowding might be rationalized by the packing of inherited material, like the vacuole, from mother cells. We discuss recent advances in understanding the role of crowding in cellular regulation and key current challenges and conclude by presenting our recent advances in optimizing FRET-based measurements of crowding while simultaneously imaging a third color, which can be used as a marker that labels organelle membranes. Our approaches can be combined with synchronized cell populations to increase experimental throughput and correlate molecular crowding information with different stages in the cell cycle.

Impact of different lipids on membrane structure/lipid order is critical for multiple biological processes. Laurdan microscopy provides a unique tool to assess this property in heterogeneous biological membranes. This review describes the general principles of the approach and its application in model membranes and cells. It also provides an in-depth discussion of the insights obtained using Laurdan microscopy to evaluate the differential effects of cholesterol, oxysterols and oxidized phospholipids on lipid packing of ordered and disordered domains in vascular endothelial cells.

Lipid membrane domains are supramolecular lateral heterogeneities of biological membranes. Of nanoscopic dimensions, they constitute specialized hubs used by the cell as transient signaling platforms for a great variety of biologically important mechanisms. Their property to form and dissolve in the bulk lipid bilayer endow them with the ability to engage in highly dynamic processes, and temporarily recruit subpopulations of membrane proteins in reduced nanometric compartments that can coalesce to form larger mesoscale assemblies. Cholesterol is an essential component of these lipid domains; its unique molecular structure is suitable for interacting intricately with crevices and cavities of transmembrane protein surfaces through its rough β face while "talking" to fatty acid acyl chains of glycerophospholipids and sphingolipids via its smooth α face. Progress in the field of membrane domains has been closely associated with innovative improvements in fluorescence microscopy and new fluorescence sensors. These advances enabled the exploration of the biophysical properties of lipids and their supramolecular platforms. Here I review the rationale behind the use of biosensors over the last few decades and their contributions towards elucidation of the in-plane and transbilayer topography of cholesterol-enriched lipid domains and their molecular constituents. The challenges introduced by super-resolution optical microscopy are discussed, as well as possible scenarios for future developments in the field, including virtual ("no staining") staining.

The cell membrane serves as a barrier that restricts the rate of exchange of diffusible molecules. Tension in the membrane regulates many crucial cell functions involving shape changes and motility, cell signaling, endocytosis, and mechanosensation. Tension reflects the forces contributed by the lipid bilayer, the cytoskeleton, and the extracellular matrix. With a fluid-like bilayer model, membrane tension is presumed uniform and hence propagated instantaneously. In this review, we discuss techniques to measure the mean membrane tension and how to resolve the stresses in different components and consider the role of bilayer heterogeneity.

Liquid-liquid phase separation (LLPS) is a ubiquitous process that drives the formation of membrane-less intracellular compartments. This compartmentalization contains vastly different protein/RNA/macromolecule concentrations compared to the surrounding cytosol despite the absence of a lipid boundary. Because of this, LLPS is important for many cellular signaling processes and may play a role in their dysregulation. This chapter highlights recent advances in the understanding of intracellular phase transitions along with current methods used to identify LLPS in vitro and model LLPS in situ.

Endothelial cells line the innermost layer of arterial, venous, and lymphatic vascular tree and accordingly are subject to hemodynamic, stretch, and stiffness mechanical forces. Normally quiescent, endothelial cells have a hemodynamic set point and become "activated" in response to disturbed hemodynamics, which may signal impending nutrient or gas depletion. Endothelial cells in the majority of tissue beds are normally inactivated and maintain vessel barrier functions, are anti-inflammatory, anti-coagulant, and anti-thrombotic. However, under aberrant mechanical forces, endothelial signaling transforms in response, resulting cellular changes that herald pathological diseases. Endothelial cell metabolism is now recognized as the primary intermediate pathway that undergirds cellular transformation. In this review, we discuss the various mechanical forces endothelial cells sense in the large vessels, microvasculature, and lymphatics, and how changes in environmental mechanical forces result in changes in metabolism, which ultimately influence cell physiology, cellular memory, and ultimately disease initiation and progression.

Endothelial cells (ECs) are constantly subjected to an array of mechanical cues, especially shear stress, due to their luminal placement in the blood vessels. Blood flow can regulate various aspects of endothelial biology and pathophysiology by regulating the endothelial processes at the transcriptomic, proteomic, miRNomic, metabolomics, and epigenomic levels. ECs sense, respond, and adapt to altered blood flow patterns and shear profiles by specialized mechanisms of mechanosensing and mechanotransduction, resulting in qualitative and quantitative differences in their gene expression. Chromatin-regulatory proteins can regulate transcriptional activation by modifying the organization of nucleosomes at promoters, enhancers, silencers, insulators, and locus control regions. Recent research efforts have illustrated that SWI/SNF (SWItch/Sucrose Non-Fermentable) or BRG1/BRM-associated factor (BAF) complex regulates DNA accessibility and chromatin structure. Since the discovery, the gene-regulatory mechanisms of the BAF complex associated with chromatin remodeling have been intensively studied to investigate its role in diverse disease phenotypes. Thus far, it is evident that (1) the SWI/SNF complex broadly regulates the activity of transcriptional enhancers to control lineage-specific differentiation and (2) mutations in the BAF complex proteins lead to developmental disorders and cancers. It is unclear if blood flow can modulate the activity of SWI/SNF complex to regulate EC differentiation and reprogramming. This review emphasizes the integrative role of SWI/SNF complex from a structural and functional standpoint with a special reference to cardiovascular diseases (CVDs). The review also highlights how regulation of this complex by blood flow can lead to the discovery of new therapeutic interventions for the treatment of endothelial dysfunction in vascular diseases.

Fluorescence-based sensors play a fundamental role in biological research. These sensors can be based on fluorescent proteins, fluorescent probes or they can be hybrid systems. The availability of a very large dataset of fluorescent molecules, both genetically encoded and synthetically produced, together with the structural insights on many sensing domains, allowed to rationally design a high variety of sensors, capable of monitoring both molecular and global changes in living cells or in in vitro systems. The advancements in the fluorescence-imaging field helped researchers to obtain a deeper understanding of how and where specific changes occur in a cell or in vitro by combining the readout of the fluorescent sensors with the spatial information provided by fluorescent microscopy techniques. In this review we give an overview of the state of the art in the field of fluorescent biosensors and fluorescence imaging techniques, and eventually guide the reader through the choice of the best combination of fluorescent tools and techniques to answer specific biological questions. We particularly focus on sensors for probing the bioenergetics and physicochemical status of the cell.

Endothelial cells (ECs), uniquely localized and strategically forming the inner lining of vascular wall, constitute the largest cell surface by area in the human body. The dynamic sensing and response of ECs to mechanical cues, especially shear stress, is crucial for maintenance of vascular homeostasis. It is well recognized that different flow patterns associated with atheroprotective vs atheroprone regions in the arterial tree, result in distinct EC functional phenotypes with differential transcriptome profiles. Mounting evidence has demonstrated an integrative and essential regulatory role of non-coding genome in EC biology. In particular, recent studies have begun to reveal the importance of enhancers and enhancer-derived transcripts in flow-regulated EC gene expression and function. In this minireview, we summarize studies in this area and discuss examples in support of the emerging importance of enhancers and enhancer(-derived) long non-coding RNAs (elncRNAs) in EC mechanosensing, with a focus on flow-responsive EC transcription. Finally, we will provide perspective and discuss standing questions to elucidate the role of these novel regulators in EC mechanobiology.