The current myogenesis and myofibrillogenesis model has been based mostly on in vitro cell culture studies, and, to a lesser extent, on in situ studies in avian and mammalian embryos. While the more isolated artificial conditions of cells in culture permitted careful structural analysis, the actual in situ cellular structures have not been described in detail because the embryos are more difficult to section and manipulate. To overcome these difficulties, we used the optically clear and easy to handle embryos of the zebrafish Danio rerio. We monitored the expression of cytoskeletal and cell-adhesion proteins (actin, myosin, desmin, alpha-actinin, troponin, titin, vimentin and vinculin) using immunofluorescence microscopy and video-enhanced, background-subtracted, differential interference contrast of 24- to 48-h zebrafish embryos. In the mature myotome, the mononucleated myoblasts displayed periodic striations for all sarcomeric proteins tested. The changes in desmin distribution from aggregates to perinuclear and striated forms, although following the same sequence, occurred much faster than in other models. All desmin-positive cells were also positive for myofibrillar proteins and striated, in contrast to that which occurs in cell cultures. Vimentin appeared to be striated in mature cells...

Capillary forces arising during the evaporation of liquids from dense carbon nanotube arrays are used to reassemble the nanotubes into two-dimensional contiguous cellular foams. The stable nanotube foams can be elastically deformed, transferred to other substrates, or floated out to produce free-standing macroscopic fabrics. The lightweight cellular foams made of condensed nanotubes could have applications as shock-absorbent structural reinforcements and elastic membranes. The ability to control the length scale, orientation, and shape of the cellular structures and the simplicity of the assembly process make this a particularly attractive system for studying pattern formation in ordered media.

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.

Normal rat kidney cells infected with a Rous sarcoma virus (strain LA23) were used to study the dynamics of alpha-actinin-containing aggregates in transformed cells. Experiments were performed by microinjecting living cells with iodoacetamidotetramethylrhodamine alpha-actinin and allowing the fluorescent analogue to incorporate into cellular structures. Subsequent time-lapse recording indicated that the alpha-actinin-containing aggregates can undergo rapid formation, movement, and breakdown. In addition, experiments using the photobleaching recovery technique indicated that alpha-actinin molecules associated with the aggregates have a very high rate of exchange, whereas those associated with adhesion plaques in normal cells exchange much more slowly. The dynamic properties of alpha- actinin-containing aggregates may be closely related to the changes in cellular behavior upon oncogenic transformation.

In vivo microscopy has recently revealed the dynamic nature of many cellular organelles. The dynamic properties of several cellular structures are consistent with a role for self-organization in their formation, maintenance, and function; therefore, self-organization might be a general principle in cellular organization.

The cellular structure of Porphyridium cruentum was studied with both light and electron microscope. The photosynthetic plastid in this red alga was found to be structurally similar to that in the Chlorophyceae and higher green plants. The phycobilins, as well as the chlorophyll, seem to be associated with the lamellae of the plastid. The pyrenoid, a region of low lamellar density, contains no tubules, and does not appear to function in synthesis or storage of reserve material. Grains of floridean starch are located in the cytoplasm, outside the plastid. Typical mitochondrial organelles were not observed. The nucleus is eccentric, and contains a nucleolus located on the inner face of the nucleus, nearest the plastid. The schedule for staining the nucleus is given in detail. Other cell structures (sheath, dictyosomes, etc.) are described. Growing cells in light of intensity leads to disruption of the parallel arrangement of the lamellar characteristic of cells grown in moderate light.

Adaptive optics–optical coherence tomography (AO-OCT) permits improved imaging of microscopic retinal structures by combining the high lateral resolution of AO with the high axial resolution of OCT, resulting in the narrowest three-dimensional (3D) point-spread function (PSF) of all in vivo retinal imaging techniques. Owing to the high volumetric resolution of AO-OCT systems, it is now possible, for the first time, to acquire images of 3D cellular structures in the living retina. Thus, with AO-OCT, those retinal structures that are not visible with AO or OCT alone (e.g., bundles of retinal nerve fiber layers, 3D mosaic of photoreceptors, 3D structure of microvasculature, and detailed structure of retinal disruptions) can be visualized. Our current AO-OCT instrumentation uses spectrometer-based Fourier-domain OCT technology and two-deformable-mirror-based AO wavefront correction. We describe image processing methods that help to remove motion artifacts observed in volumetric data, followed by innovative data visualization techniques [including two-dimensional (2D) and 3D representations]. Finally, examples of microscopic retinal structures that are acquired with the University of California Davis AO-OCT system are presented.

The origins of side scattering from a fibroblast and cervical cell line were determined by comparing side-scatter images with images stained for lysosomes, nuclei, and mitochondria on a cell by cell basis. Lysosomes or nuclei are the most efficient type of scatterer depending on the cell type and incident light polarization. The relative scattering efficiencies of lysosomes and mitochondria were the same for both cell lines, while the scattering efficiencies of the nuclei differed. The percent of 90° scattering from the nucleus, mitochondria, and lysosomes as well as the group of other internal cellular objects was estimated. The nucleus was the largest contributor to side scatter in the cervical carcinoma cells. The contributions of lysosomes, mitochondria, the nucleus, and particles unstained by either Hoechst, LysoSensor or MitoTracker ranges from ∼20% to ∼30% in fibroblast cells. The contribution of lysosomes to side scatter was much stronger when the incident light was polarized perpendicular to the scattering plane than when the polarization of the side scatter laser was parallel to the scattering plane. This dependence on side scatter polarization indicates that lysosomes contain scattering structures that are much smaller than the wavelength of light used in the measurements (785 nm). In conclusion...

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.

Intracellular proteins with long lifespans have recently been linked to age-dependent defects, ranging from decreased fertility to the functional decline of neurons. Why long-lived proteins exist in metabolically active cellular environments and how they are maintained over time remains poorly understood. Here we provide a system-wide identification of proteins with exceptional lifespans in the rat brain. These proteins are inefficiently replenished despite being translated robustly throughout adulthood. Using nucleoporins as a paradigm for long-term protein persistence, we found that nuclear pore complexes (NPCs) are maintained over a cell’s life through slow but finite exchange of even its most stable subcomplexes. This maintenance is limited, however, as some nucleoporin levels decrease during aging, providing a rationale for the previously observed age-dependent deterioration of NPC function. Our identification of a long-lived proteome reveals cellular components that are at increased risk for damage accumulation, linking long-term protein persistence to the cellular aging process.

In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM.

The beta2-adrenergic receptor (β2AR) family, which is the largest family of cell surface receptors in humans. Extra attention has been focused on the human GPCRs because they have been studied as important protein targets for pharmaceutical drug development. In fact, approximately 40% of marketed drugs directly work on GPCRs. GPCRs respond to various extracellular stimuli, such as sensory signals, neurotransmitters, chemokines, and hormones, to induce structural changes at the cytoplasmic surface, activating downstream signaling pathways, primarily through interactions with heterotrimeric G proteins or through G-protein independent pathways, such as arrestin. Most GPCRs, except for rhodhopsin, which contains covalently linked 11 cis-retinal, bind to diffusible ligands, having various conformational states between inactive and active structures. The first human GPCR structure was determined using an inverse agonist bound β2AR in 2007 and since then, more than 20 distinct GPCR structures have been solved. However, most GPCR structures were solved as inactive forms, and an agonist bound fully active structure is still hard to obtain. In a structural point of view, β2AR is relatively well studied since its fully active structure as a complex with G protein as well as several inactive structures are available. The structural comparison of inactive and active states gives an important clue in understanding the activation mechanism of β2AR. In this review...

Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a “finger” domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an “open” conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a “closed” conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.; Molecular and Cellular Biology

Lipid bodies (LBs), also known as lipid droplets, are complex organelles of all eukaryotic cells linked to a variety of biological functions as well as to the development of human diseases. In cells from the immune system, such as eosinophils, neutrophils and macrophages, LBs are rapidly formed in the cytoplasm in response to inflammatory and infectious diseases and are sites of synthesis of eicosanoid lipid mediators. However, little is known about the structural organization of these organelles. It is unclear whether leukocyte LBs contain a hydrophobic core of neutral lipids as found in lipid droplets from adipocytes and how diverse proteins, including enzymes involved in eicosanoid formation, incorporate into LBs. Here, leukocyte LB ultrastructure was studied in detail by conventional transmission electron microscopy (TEM), immunogold EM and electron tomography. By careful analysis of the two-dimensional ultrastructure of LBs from human blood eosinophils under different conditions, we identified membranous structures within LBs in both resting and activated cells. Cyclooxygenase, a membrane inserted protein that catalyzes the first step in prostaglandin synthesis, was localized throughout the internum of LBs. We used fully automated dual-axis electron tomography to study the three-dimensional architecture of LBs in high resolution. By tracking 4 nm-thick serial digital sections we found that leukocyte LBs enclose an intricate system of membranes within their “cores”. After computational reconstruction...

Synaptic ribbons are presynaptic structures formed by the self-association of RIBEYE–the main structural component of ribbon synapses. RIBEYE consists of two domains: a unique N-terminal A-domain and a C-terminal B-domain that is identical to the transcription co-repressor C-terminal binding protein 2 (CtBP2). Previous studies in cell lines have shown that RIBEYE A-domain alone is sufficient to form ribbon-like aggregates and that both A- and B- domains form homo-and heterotypic interactions. As these interactions are likely the basis for synaptic-ribbon assembly and structural plasticity, we wanted to examine how zebrafish Ribeye A- and B- domains interact with synaptic ribbons in vivo. To that end, we characterized the localization of exogenously expressed Ribeye A- and B- domains and the closely related protein, CtBP1, in the hair cells of transgenic zebrafish larvae. Unexpectedly, exogenously expressed Ribeye A-domain showed variable patterns of localization in hair cells; one zebrafish paralog of A-domain failed to self-associate or localize to synaptic ribbons, while the other self-assembled but sometimes failed to localize to synaptic ribbons. By contrast, Ribeye B-domain/CtBP2 was robustly localized to synaptic ribbons. Moreover...

This course develops and applies scaling laws and the methods of continuum mechanics to biomechanical phenomena over a range of length scales. Topics include: structure of tissues and the molecular basis for macroscopic properties; chemical and electrical effects on mechanical behavior; cell mechanics, motility and adhesion; biomembranes; biomolecular mechanics and molecular motors. Experimental methods for probing structures at the tissue, cellular, and molecular levels.

Por uma escolha política e economicamente pragmática, o Brasil optou por desenvolver a telefonia celular, com intenção de abreviar uma etapa de desenvolvimento. Tomada a decisão, a implantação foi feita num ritmo explosivo a partir da década de 1990, com instalação de dezenas de milhares de estações. Apesar da disparidade de custo entre os sistemas eletrônicos e as obras civis, pouco se investiu na engenharia estrutural envolvida, resultando em projetos e construções realizados com metodologia duvidosa e na herança de uma grande quantidade de problemas estruturais. Represada por considerações de ordem ambiental e estética, a instalação indiscriminada de torres, vive-se uma nova demanda para a engenharia estrutural na análise do aproveitamento dos locais existentes para suporte de novas cargas. Nesse sentido, o que se observa, via de regra, são estruturas compostas apenas de um poste em balanço de análise enganosamente simples. O que se esquece, quase sempre, é a extraordinária esbelteza desses elementos, que ao engenheiro deveria sugerir a imediata necessidade de considerar a não-linearidade geométrica forçosamente existente. Além disso, o carregamento mais importante e dominante é o do vento, de características eminentemente dinâmicas e aleatórias...

In this paper we propose some kinds of two-dimensional square zigzag lattice structures and study their bandgaps and directional propagation of elastic waves. The band structures and the transmission spectra of the systems are calculated by using the finite element method. The effects of the geometry parameters of the 2d-zigzag lattices on the bandgaps are investigated and discussed. The mechanism of the bandgap generation is analyzed by studying the vibration modes at the bandgap edges. Multiple wide complete bandgaps are found in a wide porosity range owing to the separation of the degeneracy by introducing bending arms. The bandgaps are sensitive to the geometry parameters of the systems. The deformed displacement fields of the transient response of finite structures subjected to harmonic loads are presented to show the directional wave propagation. The research work in this paper is relevant to the practical design of cellular structures with enhanced vibro-acoustics performance.; Comment: 39 pages, 20 figures

The general description of formation the cellular structure in the system of interacting particles is proposed. Interactions between particles are presumably well-understood and the phase transition in which can be studied in the scale of particle resolution. We presented analytical results of possible cellular structures for suspension of colloidal particles, in system particles immersed in liquid crystal and gravitational system. We have shown that cellular structure formation can occur in system of interacting particles for realistic values of temperature and particles concentration.; Comment: 5 pages

Wireless Communication Networks based on Frequency Division Multiplexing (FDM in short) plays an important role in the field of communications, in which each request can be satisfied by assigning a frequency. To avoid interference, each assigned frequency must be different to the neighboring assigned frequencies. Since frequency is a scarce resource, the main problem in wireless networks is how to fully utilize the given bandwidth of frequencies. In this paper, we consider the online call control problem. Given a fixed bandwidth of frequencies and a sequence of communication requests arrive over time, each request must be either satisfied immediately after its arrival by assigning an available frequency, or rejected. The objective of call control problem is to maximize the number of accepted requests. We study the asymptotic performance of this problem, i.e., the number of requests in the sequence and the bandwidth of frequencies are very large. In this paper, we give a 7/3-competitive algorithm for call control problem in cellular network, improving the previous 2.5-competitive result. Moreover, we investigate the triangle-free cellular network, propose a 9/4-competitive algorithm and prove that the lower bound of competitive ratio is at least 5/3.; Comment: 12 pages...