Scanning angle interference microscopy reveals cell dynamics at the nanoscale

Emerging questions in cell biology necessitate nanoscale imaging in live cells. Here we present scanning angle interference microscopy, which is capable of localizing fluorescent objects with nanoscale precision along the optical axis in motile cellular structures. We use this approach to resolve nanotopographical features of the cell membrane and cytoskeleton as well as the temporal evolution, three-dimensional architecture and nanoscale dynamics of focal adhesion complexes.     

Imaging and manipulating the structural machinery of living cells on the micro- and nanoscale

The structure, physiology, and fate of living cells are all highly sensitive to mechanical forces in the cellular microenvironment, including stresses and strains that originate from encounters with the extracellular matrix (ECM), blood and other flowing materials, and neighbouring cells. This relationship between context and physiology bears tremendous implications for the design of cellular micro-or nanotechnologies, since any attempt to control cell behavior in a device must provide the appropriate physical microenvironment for the desired cell behavior. Cells sense, process, and respond to biophysical cues in their environment through a set of integrated, multi-scale structural complexes that span length scales from single molecules to tens of microns, including small clusters of force-sensing molecules at the cell surface, micron-sized cell-ECM focal adhesion complexes, and the cytoskeleton that permeates and defines the entire cell. This review focuses on several key technologies that have recently been developed or adapted for the study of the dynamics of structural micro-and nanosystems in living cells and how these systems contribute to spatially-and temporally-controlled changes in cellular structure and mechanics. We begin by discussing subcellular laser ablation, which permits the precise incision of nanoscale structural elements in living cells in order to discern their mechanical properties and contributions to cell structure. We then discuss fluorescence recovery after photobleaching and fluorescent speckle microscopy, two live-cell fluorescence imaging methods that enable quantitative measurement of the binding and transport properties of specific proteins in the cell. Finally, we discuss methods to manipulate cellular structural networks by engineering the extracellular environment, including microfabrication of ECM distributions of defined geometry and microdevices designed to measure cellular traction forces at micron-scale resolution. Together, these methods form a powerful arsenal that is already adding significantly to our understanding of the nanoscale architecture and mechanics of living cells and may contribute to the rational design of new cellular micro-and nanotechnologies.     

The plant cell wall: Biosynthesis,construction, and functions

The plant cell wall is composed of multiple biopolymers, representing one of the most complex structural networks in nature. Hundreds of genes are involved in building such a natural masterpiece. However, the plant cell wall is the least understood cellular structure in plants. Due to great progress in plant functional genomics,manyachievementshavebeenmadein uncovering cell wall biosynthesis, assembly, and architecture, as well as cell wall regulation and signaling. Such information has significantly advanced our understanding of the roles of the cell wall in many biological and physiological processes and has enhanced our utilization of cell wall materials. The use of cutting-edge technologies such as single-molecule imaging,nuclear magnetic resonance spectroscopy, and atomic force microscopy has provided much insight into the plant cell wall as an intricate nanoscale network, opening up unprecedented possibilities for cell wall research. In this review,we summarize the major advances made in understanding the cell wall in this era of functional genomics, including the latest findings on the biosynthesis, construction, and functions of the cell wall.     

Nanoscale Heat Transfer from Magnetic Nanoparticles and Ferritin in an Alternating Magnetic Field

Recent suggestions of nanoscale heat confinement on the surface of synthetic and biogenic magnetic nanoparticles during heating by radio frequency-alternating magnetic fields have generated intense interest because of the potential utility of this phenomenon for noninvasive control of biomolecular and cellular function. However, such confinement would represent a significant departure from the classical heat transfer theory. Here, we report an experimental investigation of nanoscale heat confinement on the surface of several types of iron oxide nanoparticles commonly used in biological research, using an all-optical method devoid of the potential artifacts present in previous studies. By simultaneously measuring the fluorescence of distinct thermochromic dyes attached to the particle surface or dissolved in the surrounding fluid during radio frequency magnetic stimulation, we found no measurable difference between the nanoparticle surface temperature and that of the surrounding fluid for three distinct nanoparticle types. Furthermore, the metalloprotein ferritin produced no temperature increase on the protein surface nor in the surrounding fluid. Experiments mimicking the designs of previous studies revealed potential sources of the artifacts. These findings inform the use of magnetic nanoparticle hyperthermia in engineered cellular and molecular systems.     

Selection and analysis of solid-binding peptides

Nature has long used peptide- and protein-based manufacturing to create structures whose remarkable mechanical, transport, optical, and even magnetic properties are determined by a fine control of composition and architecture extending from the nanoscale to the macroscale. Although there is much to learn from the tools and strategies that have been evolutionary selected for building biomaterials, accessing compositions and architectures of engineering interest is crucial to the development of the next generation of hybrid functional materials. In recent years, portable amino acid sequences selected from combinatorial libraries and supporting the assembly, nucleation, and geometrical organization of solid phases have emerged as attractive tools for bionanofabrication. Here, we review how these polypeptides are selected and progress in the understanding of their interaction with inorganic and synthetic materials.     

Magnetic nanoparticles: A multifunctional vehicle for modern theranostics

Magnetic nanoparticles provide a unique multifunctional vehicle for modern theranostics since they can be remotely and non-invasively employed as imaging probes, carrier vectors and smart actuators. Additionally, special delivery schemes beyond the typical drug delivery such as heat or mechanical stress may be magnetically triggered to promote certain cellular pathways. To start with, we need magnetic nanoparticles with several well-defined and reproducible structural, physical, and chemical features, while bio-magnetic nanoparticle design imposes several additional constraints. Except for the intrinsic requirement for high quality of magnetic properties in order to obtain the maximum efficiency with the minimum dose, the surface manipulation of the nanoparticles is a key aspect not only for transferring them from the growth medium to the biological environment but also to bind functional molecules that will undertake specific targeting, drug delivery, cell-specific monitoring and designated treatment without sparing biocompatibility and sustainability in-vivo. The ability of magnetic nanoparticles to interact with matter at the nanoscale not only provides the possibility to ascertain the molecular constituents of a disease, but also the way in which the totality of a biological function may be affected as well. The capacity to incorporate an array of structural and chemical functionalities onto the same nanoscale architecture also enables more accurate, sensitive and precise screening together with cure of diseases with significant pathological heterogeneity such as cancer. This article is part of a Special Issue entitled “Recent Advances in Bionanomaterials” Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.     

Find your coat: Using correlative light and electron microscopy to study intracellular protein coats

Protein coats, important for vesicular trafficking in eukaryotic cells, help shape membranes and package cargo. But their dynamic construction cannot be fully understood until the distinct steps of their assembly in their native intracellular context at molecular resolution can be visualized. For this, correlative light and electron microscopy (CLEM) is an essential tool. Here, we discuss how emerging CLEM techniques have been used to study the assembly of protein coats inside cells. We review how current and developing CLEM technologies are poised to answer fundamental questions of protein coat architecture at the nanoscale.     

Electron tomography of ER, Golgi and related membrane systems

A primary goal of cell biology is to uncover the mechanisms of cellular processes. A detailed structural understanding of the organelles and subcellular structures involved in these processes has often formed the foundation for the elucidation of their function. Electron tomography is a powerful technique for characterizing subcellular architecture and structural details in three dimensions. Electron tomography of cryofixed, freeze-substituted, and plastic-embedded samples allows three-dimensional visualization and display of dynamic, pleiomorphic structures at a resolution of approximately 7 nm in cell volumes up to approximately 25 microm(3). In this review, we describe the electron tomography protocols that we have employed to determine the 3D architecture of complex cellular structures, thereby gaining insights into their functional organization. We stress the need for studying specimens preserved by cryofixation methods to obtain accurate information on the geometry and size of cellular structures. We also discuss some of the challenges associated with the staining of certain types of membranes. Finally, we provide examples of how tomographic data can be analyzed, dissected, and displayed using the tools built into the IMOD software package.     

Rapid tool for cell nanoarchitecture integrity assessment

With the rapid increase and accessibility of high-resolution imaging technologies of cells, the interpretation of results relies more and more on the assumption that the three-dimensional integrity of the surrounding cellular landscape is not compromised by the experimental setup. However, the only available technology for directly probing the structural integrity of whole-cell preparations at the nanoscale is electron cryo-tomography, which is time-consuming, costly, and complex. We devised an accessible, inexpensive and reliable screening assay to quickly report on the compatibility of experimental protocols with preserving the structural integrity of whole-cell preparations at the nanoscale. Our Rapid Cell Integrity Assessment (RCIA) assay is executed at room temperature and relies solely on light microscopy imaging. Using cellular electron cryo-tomography as a benchmark, we verify that RCIA accurately unveils the adverse impact of reagents and/or protocols such as those used for virus inactivation or to arrest dynamic processes on the cellular nanoarchitecture.     

基因药物纳米给药系统的设计与研究进展

基因药物的传递面临着体内外稳定性差、缺乏靶向性、难入胞、在细胞内难以释放等一系列障碍和挑战。因此,要实现基因药物在 体内有效传递需构建能克服这些障碍的药物传递系统。随着材料科学和纳米科技的发展,大量新型的纳米载体已被用于基因药物的传递。 综述目前基因药物传递所面临的障碍和挑战,基因药物纳米给药系统的设计思路及研究进展。     

Nanoscale protein domain motion and long-range allostery in signaling proteins—a view from neutron spin echo spectroscopy

Many cellular proteins are multi-domain proteins. Coupled domain–domain interactions in these multidomain proteins are important for the allosteric relay of signals in the cellular signaling networks. We have initiated the application of neutron spin echo spectroscopy to the study of nanoscale protein domain motions on submicrosecond time scales and on nanometer length scale. Our NSE experiments reveal the activation of protein domain motions over a long distance of over more than 100 Å in a multidomain scaffolding protein NHERF1 upon binding to another protein, Ezrin. Such activation of nanoscale protein domain motions is correlated with the allosteric assembly of multi-protein complexes by NHERF1 and Ezrin. Here, we summarize the theoretical framework that we have developed, which uses simple concepts from nonequilibrium statistical mechanics to interpret the NSE data, and employs a mobility tensor to describe nanoscale protein domain motion. Extracting nanoscale protein domain motion from the NSE does not require elaborate molecular dynamics simulations, nor complex fits to rotational motion, nor elastic network models. The approach is thus more robust than multiparameter techniques that require untestable assumptions. We also demonstrate that an experimental scheme of selective deuteration of a protein subunit in a complex can highlight and amplify specific domain dynamics from the abundant global translational and rotational motions in a protein. We expect NSE to provide a unique tool to determine nanoscale protein dynamics for the understanding of protein functions, such as how signals are propagated in a protein over a long distance to a distal domain.     

Recent Progress on the Analysis of Power-law Features in Complex Cellular Networks

Complex interactions between different kinds of bio-molecules and essential nutrients are responsible for cellular functions. Rapid advances in theoretical modeling and experimental analyses have shown that drastically different biological and non-biological networks share a common architecture. That is, the probability that a selected node in the network has exactly k edges decays as a power-law. This finding has definitely opened an intense research and debate on the origin and implications of this ubiquitous pattern. In this review, we describe the recent progress on the emergence of power-law distributions in cellular networks. We first show the internal characteristics of the observed complex networks uncovered using graph theory. We then briefly review some works that have significantly contributed to the theoretical analysis of cellular networks and systems, from metabolic and protein networks to gene expression profiles. This prevalent topology observed in so many diverse biological systems suggests the existence of generic laws and organizing principles behind the cellular networks.     

The controlled display of biomolecules on nanoparticles: a challenge suited to bioorthogonal chemistry

Interest in developing diverse nanoparticle (NP)-biological composite materials continues to grow almost unabated. This is motivated primarily by the desire to simultaneously exploit the properties of both NP and biological components in new hybrid devices or materials that can be applied in areas ranging from energy harvesting and nanoscale electronics to biomedical diagnostics. The utility and effectiveness of these composites will be predicated on the ability to assemble these structures with control over NP/biomolecule ratio, biomolecular orientation, biomolecular activity, and the separation distance within the NP-bioconjugate architecture. This degree of control will be especially critical in creating theranostic NP-bioconjugates that, as a single vector, are capable of multiple functions in vivo, including targeting, image contrast, biosensing, and drug delivery. In this review, a perspective is given on current and developing chemistries that can provide improved control in the preparation of NP-bioconjugates. The nanoscale properties intrinsic to several prominent NP materials are briefly described to highlight the motivation behind their use. NP materials of interest include quantum dots, carbon nanotubes, viral capsids, liposomes, and NPs composed of gold, lanthanides, silica, polymers, or magnetic materials. This review includes a critical discussion on the design considerations for NP-bioconjugates and the unique challenges associated with chemistry at the biological-nanoscale interface-the liabilities of traditional bioconjugation chemistries being particularly prominent therein. Select bioorthogonal chemistries that can address these challenges are reviewed in detail, and include chemoselective ligations (e.g., hydrazone and Staudinger ligation), cycloaddition reactions in click chemistry (e.g., azide-alkyne cyclyoaddition, tetrazine ligation), metal-affinity coordination (e.g., polyhistidine), enzyme driven modifications (e.g., HaloTag, biotin ligase), and other site-specific chemistries. The benefits and liabilities of particular chemistries are discussed by highlighting relevant NP-bioconjugation examples from the literature. Potential chemistries that have not yet been applied to NPs are also discussed, and an outlook on future developments in this field is given.     

Function of membrane domains in rho-of-plant signaling

In a crowded environment, establishing interactions between different molecular partners can take a long time. Biological membranes have solved this issue, as they simultaneously are fluid and possess compartmentalized domains. This nanoscale organization of the membrane is often based on weak, local, and multivalent interactions between lipids and proteins. However, from local interactions at the nanoscale, different functional properties emerge at the higher scale, and these are critical to regulate and integrate cellular signaling. Rho of Plant (ROP) proteins are small guanosine triphosphate hydrolase enzymes (GTPases) involved in hormonal, biotic, and abiotic signaling, as well as fundamental cell biological properties such as polarity, vesicular trafficking, and cytoskeleton dynamics. Association with the membrane is essential for ROP function, as well as their precise targeting within micrometer-sized polar domains (i.e. microdomains) and nanometer-sized clusters (i.e. nanodomains). Here, we review our current knowledge about the formation and the maintenance of the ROP domains in membranes. Furthermore, we propose a model for ROP membrane targeting and discuss how the nanoscale organization of ROPs in membranes could determine signaling parameters like signal specificity, amplification, and integration.

The nanoscale organization of Rho of Plant proteins creates emergent properties that determine cellular signaling.     

Histone acylations and chromatin dynamics: concepts,challenges, and links to metabolism

In eukaryotic cells, DNA is tightly packed with the help of histone proteins into chromatin. Chromatin architecture can be modified by various post‐translational modifications of histone proteins. For almost 60 years now, studies on histone lysine acetylation have unraveled the contribution of this acylation to an open chromatin state with increased DNA accessibility, permissive for gene expression. Additional complexity emerged from the discovery of other types of histone lysine acylations. The acyl group donors are products of cellular metabolism, and distinct histone acylations can link the metabolic state of a cell with chromatin architecture and contribute to cellular adaptation through changes in gene expression. Currently, various technical challenges limit our full understanding of the actual impact of most histone acylations on chromatin dynamics and of their biological relevance. In this review, we summarize the state of the art and provide an overview of approaches to overcome these challenges. We further discuss the concept of subnuclear metabolic niches that could regulate local CoA availability and thus couple cellular metabolisms with the epigenome.     

Applications of STED fluorescence nanoscopy in unravelling nanoscale structure and dynamics of biological systems

Fluorescence microscopy, especially confocal microscopy, has revolutionized the field of biological imaging. Breaking the optical diffraction barrier of conventional light microscopy, through the advent of super-resolution microscopy, has ushered in the potential for a second revolution through unprecedented insight into nanoscale structure and dynamics in biological systems. Stimulated emission depletion (STED) microscopy is one such super-resolution microscopy technique which provides real-time enhanced-resolution imaging capabilities. In addition, it can be easily integrated with well-established fluorescence-based techniques such as fluorescence correlation spectroscopy (FCS) in order to capture the structure of cellular membranes at the nanoscale with high temporal resolution. In this review, we discuss the theory of STED and different modalities of operation in order to achieve the best resolution. Various applications of this technique in cell imaging, especially that of neuronal cell imaging, are discussed as well as examples of application of STED imaging in unravelling structure formation on biological membranes. Finally, we have discussed examples from some of our recent studies on nanoscale structure and dynamics of lipids in model membranes, due to interaction with proteins, as revealed by combination of STED and FCS techniques.     

Phosphoinositides in cell architecture

Inositol phospholipids have been implicated in almost all aspects of cellular physiology including spatiotemporal regulation of cellular signaling, acquisition of cellular polarity, specification of membrane identity, cytoskeletal dynamics, and regulation of cellular adhesion, motility, and cytokinesis. In this review, we examine the critical role phosphoinositides play in these processes to execute the establishment and maintenance of cellular architecture. Epithelial tissues perform essential barrier and transport functions in almost all major organs. Key to their development and function is the establishment of epithelial cell polarity. We place a special emphasis on highlighting recent studies demonstrating phosphoinositide regulation of epithelial cell polarity and how individual cells use phosphoinositides to further organize into epithelial tissues.     

Analytical tools for characterizing heterogeneity in organelle content

Heterogeneity in the content and function of subcellular organelles on the intercellular and intracellular level plays an important role in determining cell fate. These variations extend to normal-state and disease-state cellular functions and responses to environmental stimuli, such as oxidative stress and therapeutic drugs. Analytical tools to characterize variation in all types of organelles are essential to provide insights that can lead to advances in medicine, such as therapies targeted to specific subcellular regions. In this review, we discuss analytical techniques for interrogating individual intact organelles (e.g. mitochondria and synaptic vesicles) and lysates in a high-throughput manner, including a recently developed nanoscale fluorescence-activated subcellular sorter and techniques based on capillary electrophoresis with laser-induced fluorescence detection. We then highlight the advantages that droplet microfluidics offers for probing subcellular heterogeneity.