Cellular Lamellae Research Articles

TomoNet: A streamlined cryogenic electron tomography software pipeline with automatic particle picking on flexible lattices.

Cryogenic electron tomography (cryoET) is capable of determining in situ biological structures of molecular complexes at near-atomic resolution by averaging half a million subtomograms. While abundant complexes/particles are often clustered in arrays, precisely locating and seamlessly averaging such particles across many tomograms present major challenges. Here, we developed TomoNet, a software package with a modern graphical user interface to carry out the entire pipeline of cryoET and subtomogram averaging to achieve high resolution. TomoNet features built-in automatic particle picking and three-dimensional (3D) classification functions and integrates commonly used packages to streamline high-resolution subtomogram averaging for structures in 1D, 2D, or 3D arrays. Automatic particle picking is accomplished in two complementary ways: one based on template matching and the other using deep learning. TomoNet's hierarchical file organization and visual display facilitate efficient data management as required for large cryoET datasets. Applications of TomoNet to three types of datasets demonstrate its capability of efficient and accurate particle picking on flexible and imperfect lattices to obtain high-resolution 3D biological structures: virus-like particles, bacterial surface layers within cellular lamellae, and membranes decorated with nuclear egress protein complexes. These results demonstrate TomoNet's potential for broad applications to various cryoET projects targeting high-resolution in situ structures.

Effects of Pectin on Intestinal Microbiota and Human Health

Gut microbes are important players in human metabolism and their genomes are abundant in genes that regulate the metabolism of SFCAs, methane, amino acids, and carbohydrates. Pectin is a water-soluble dietary fiber, an abundant heteropolysaccharide in the main and intermediate cellular lamellae of plant cell walls. Pectin is degraded by a varied group of microorganisms residing in the human gut, which depolymerize pectin by secreting both cell-bound and extracellular enzymes to produce monosaccharides. The pectin fermentation degradation pathway is the main mechanism for the degradation of carbohydrates that cannot be digested in human GIT, and the end products include SCFAs, ethanol, CO2 and H2. The makeup of intestinal microbiota is impacted by these products. Additionally, the effect of pectin on intestinal microbial fractions is related to the structure of pectin, the intestinal environment and the degree of pectin esterification. Moreover, pectin itself and pectin degradation products contribute to human health by influencing intestinal microorganisms. Pectin is effective in modifying allergies to sensitization, reducing body composition and has a preventive effect on type I diabetes.

Characterization of the rotavirus assembly pathway in situ using cryoelectron tomography

Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle.

Determining protein structures in cellular lamella at pseudo-atomic resolution by GisSPA

Cryo-electron tomography is a major tool used to study the structure of protein complexes in situ. However, the throughput of tilt-series image data collection is still quite low. Here, we show that GisSPA, a GPU accelerated program, can translationally and rotationally localize the target protein complex in cellular lamellae, as prepared with a focused ion beam, using single cryo-electron microscopy images without tilt-series, and reconstruct the protein complex at near-atomic resolution. GisSPA allows high-throughput data collection without the acquisition of tilt-series images and reconstruction of the tomogram, which is essential for high-resolution reconstruction of asymmetric or low-symmetry protein complexes. We demonstrate the power of GisSPA with 3.4-Å and 3.9-Å resolutions of resolving phycobilisome and tetrameric photosystem II complex structures in cellular lamellae, respectively. In this work, we present GisSPA as a practical tool that facilitates high-resolution in situ protein structure determination.

Stack of cellular lamellae forms a silvered cortex to conceal the opaque organ in a transparent gastropod in epipelagic habitat.

Gelatinous zooplankton in epipelagic environments often have highly transparent bodies to avoid detection by their visual predators and prey; however, the digestive systems are often exceptionally opaque even in these organisms. In a holoplanktonic gastropod, Pterotrachea coronata, the visceral nucleus is an opaque organ located at the posterior end of its alimentary system, but this organ has a mirrored surface to conceal its internal opaque tissue. Our ultrastructural observation proved that the cortex of the visceral nucleus comprised a stack of thin cellular lamellae forming a Bragg reflector, and the thickness of lamellae (0.16 µm in average) and the spaces between the lamellae (0.1 µm in average) tended to become thinner toward inner lamellae. Based on the measured values, we built virtual models of the multilamellar layer comprising 50 lamellae and spaces, and the light reflection on the models was calculated using rigorous coupled wave analysis to evaluate their properties as reflectors. Our simulation supported the idea that the layer is a reflective tissue, and the thickness of the lamella/space must be chirped to reflect sunlight as white/silver light, mostly independent of the angle of incidence. In P. coronata, the cortex of the visceral nucleus comprised multicellular lamellae that form a chirped Bragg reflector. It is distinct in structure from the intracellular Bragg structures of common iridophores. This novel Bragg reflector demonstrates the diversity and convergent evolution of reflective tissue using reflectin-like proteins in Mollusca.

Preparation and Cryo-FIB micromachining of <em>Saccharomyces cerevisiae</em> for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Cryo-Focused Ion Beam Lamella Preparation Protocol for in Situ Structural Biology.

The advances in electron cryo-microscopy have enabled high-resolution structural studies of vitrified macromolecular complexes in situ by cryo-electron tomography (cryo-ET). Since utilization of cryo-ET is generally limited to the specimens with thickness<500nm, a complex sample preparation protocol to study larger samples such as single eukaryotic cells by cryo-ET was developed and optimized over the last decade. The workflow is based on the preparation of a thin cellular lamella by cryo-focused ion beam milling (cryo-FIBM) from the vitrified cells. The sample preparation protocol is a multi-step process which includes utilization of several high-end instruments and comprises sample manipulation prone to sample deterioration. Here, we present a workflow for preparation of three different model specimens that was optimized to provide high-quality lamellae for cryo-ET or electron diffraction tomography with high reproducibility. Preparation of lamellae from large adherent mammalian cells, small suspension eukaryotic cell line, and protein crystals of intermediate size is described which represents examples of the most frequently studied samples used for cryo-FIBM in life sciences.

Assembly intermediates of orthoreovirus captured in the cell

Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected ‘single shelled’ intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is ‘collapsed’ compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.

Observations on the retina and 'optical fold' of a mesopelagic sabretooth fish, Evermanella balbo.

The 'optical fold' of Evermanella balbo covers the ventro-lateral cornea and is presumed to capture illumination that would otherwise remain undetected by the tubular eye of this mesopelagic teleost. It contains alternating bands of cellular and acellular material, running approximately perpendicular to the lateral surface of the eye. Only parts of this lamellar body lie within the eyelid-like structure. The cellular lamellae are 2-2.5μm thick centrally and composed of fibroblast-like cells. The extracellular bands (4.5-5μm thick) contain regular arrays of collagen fibrils, with layers of thin fibrils sandwiching a region of thicker fibrils. The thin fibrils are organised in alternating sheets where fibrils, although all parallel, change their orientation by 90° between each sheet. All thick fibrils are oriented parallel to the lateral surface of the 'optical fold'. In the main retina, small bundles of rod inner/outer segments are separated by the processes of the retinal pigment epithelium (rpe) laterally. Centrally, the length of tightly packed rods increases, but rpe processes no longer divide them into bundles. Medially, rod length increases further, but packing is less dense. The accessory retina is significantly thinner, and less well-developed than the main retina. Ventrally, the rods show no regular arrangement and are not grouped. Dorsally, however, rods are arranged into bundles, separated by melanosome-filled rpe processes. The thickness of the retina increases as it approaches the crystalline lens. It is on this dorsal accessory retina that light traversing the 'optical fold' most likely falls, facilitating the detection of moving objects in the ventro-lateral field of view.

RABBIT SEMINIFEROUS TUBULES

Objective: The aim of this article was to study the ultrastructure of the boundary tissue of rabbit seminiferous tubule. Design:Experimental study. Period: During July 2010 to July 2011. Material: Eight sexually mature male rabbits (Oryctolagus cuniculus) were used inthis study. Rabbit maturity was determined by age and semen samples. Method: Ultrastructure observations by electron microscopetransmission of normal rabbit testes. Setting: Samples processing were done in both university of Khartoum (Sudan) and then Assiut university(Egypt). Results: The boundary tissue is formed of three lamellae, inner fibrous, inner and outer cellular lamellae. The inner fibrous lamellaconsists of three strata; internal and external homogenous enclosing a middle stratum of collagenous fibers. The internal homogenous stratumof the inner fibrous lamella is subdivided into four layers of moderate electron density. Conclusions: The general structure of the seminiferoustubule of rabbit is similar to that of hamster and mouse, but the internal homogenous stratum is formed of four layers.

Kinectin-mediated endoplasmic reticulum dynamics supports focal adhesion growth in the cellular lamella

Focal adhesions (FAs) control cell shape and motility, which are important processes that underlie a wide range of physiological functions. FA dynamics is regulated by cytoskeleton, motor proteins and small GTPases. Kinectin is an integral endoplasmic reticulum (ER) membrane protein that extends the ER along microtubules. Here, we investigated the influence of the ER on FA dynamics within the cellular lamella by disrupting the kinectin-kinesin interaction by overexpressing the minimal kinectin-kinesin interaction domain on kinectin in cells. This perturbation resulted in a morphological change to a rounded cell shape and reduced cell spreading and migration. Immunofluorescence and live-cell imaging demonstrated a kinectin-dependent ER extension into the cellular lamella and ER colocalisation with FAs within the cellular lamella. FRAP experiments showed that ER contact with FAs was accompanied with an increase in FA protein recruitment to FAs. Disruption of the kinectin-kinesin interaction caused a reduction in FA protein recruitment to FAs. This suggests that the ER supports FA growth within the cellular lamella. Microtubule targeting to FAs is known to promote adhesion disassembly; however, ER contact increased FA size even in the presence of microtubules. Our results suggest a scenario whereby kinectin-kinesin interaction facilitates ER transport along microtubules to support FA growth.

Generalized Voronoi Tessellation as a Model of Two-dimensional Cell Tissue Dynamics

Voronoi tessellations have been used to model the geometric arrangement of cells in morphogenetic or cancerous tissues, however, so far only with flat hyper-surfaces as cell-cell contact borders. In order to reproduce the experimentally observed piecewise spherical boundary shapes, we develop a consistent theoretical framework of multiplicatively weighted distance functions, defining generalized finite Voronoi neighborhoods around cell bodies of varying radius, which serve as heterogeneous generators of the resulting model tissue. The interactions between cells are represented by adhesive and repelling force densities on the cell contact borders. In addition, protrusive locomotion forces are implemented along the cell boundaries at the tissue margin, and stochastic perturbations allow for non-deterministic motility effects. Simulations of the emerging system of stochastic differential equations for position and velocity of cell centers show the feasibility of this Voronoi method generating realistic cell shapes. In the limiting case of a single cell pair in brief contact, the dynamical nonlinear Ornstein-Uhlenbeck process is analytically investigated. In general, topologically distinct tissue conformations are observed, exhibiting stability on different time scales, and tissue coherence is quantified by suitable characteristics. Finally, an argument is derived pointing to a tradeoff in natural tissues between cell size heterogeneity and the extension of cellular lamellae.

Osteocyte lacunar size–lamellar thickness relationships in human secondary osteons

Previous investigations carried out in our laboratory on secondary osteons have shown that osteocyte lacunae decrease in size from the cement line towards the Haversian canal, and lamellar bone is made up of alternating nonosteocytic dense lamellae and osteocytic loose lamellae, all having an interwoven texture of collagen fibers. Such alternation of acellular and cellular lamellae was hypothesized to depend on osteocyte recruitment from osteogenic laminae in successive layers, assuming that the loose lamellae form because of alignment and fusion of the periosteocytic loosely arranged collagen fibers. In order to discover whether a correlation really exists between osteocyte lacunar size and lamellar thickness, as would be expected if the above-mentioned hypothesis were true, both these parameters were measured in completed secondary osteons in relation to their distance from the Haversian canal. The size of osteocyte lacunae was measured under light microscopy on undecalcified dry-mounted ground section of tibial compact bone from three adult males and three adult females not affected by metabolic bone disease. The measurement of the thickness of bony lamellae was carried out on the same samples under scanning electron microscopy. Statistical analyses of the results showed that the decrease in size of osteocytic lacunae from the outer to the inner osteonal wall is paralleled by a decrease in thickness of osteocytic loose lamellae. The fact that acellular dense lamellae do not follow such a decremental pattern, but remain of the same thickness throughout the osteonic wall, corroborates Marotti’s view that the formation of lamellar bone depends on the orderly distribution of the osteocytes in alternating planes. The topographical distribution of osteocyte lacunar size and lamellar thickness is briefly discussed in relation to secondary osteon mechanical function.

Heat shock proteins do not provide thermoprotection to normal cellular protein synthesis, alpha-amylase mRNA and endoplasmic reticulum lamellae in barley aleurone layers

Heat shock proteins do not provide thermoprotection to normal cellular protein synthesis, alpha-amylase mRNA and endoplasmic reticulum lamellae in barley aleurone layers

Dynamics of active lamellae in cultured epithelial cells: effects of expression of exogenous N-ras oncogene.

We examined the functional consequences of cellular transformation of rat IAR-2 epithelial cells, by a mutant N-ras oncogene, on the dynamics of active lamellae, structures that play an important role in cell motility, adhesion, and surface-receptor capping. Lamellar activity was assessed by measuring the rate of outer-edge pseudopodial activity and by analyzing the motility of Con A-coated beads placed on lamellar surfaces with optical tweezers. Although transformation dramatically affected the shape and size of active cellular lamellae, there was little detectable effect on either pseudopodial activity or bead movement. To investigate the potential relationship between functional lamellar activity and the microtubule cytoskeleton, lamellar activity was examined in nontransformed and transformed cells treated with the microtubule-disrupting drug nocodazole. In the absence of microtubules, transformed cells were less polarized and possessed decreased rates of pseudopodial and bead motility. On the basis of these observations, it is suggested that ras-induced transformation of epithelial cells consists of two cytoskeletal modifications: overall diminished actin cytoskeletal dynamics in lamellae and reorganization of the microtubule cytoskeleton that directs pseudopodial activity to smaller polarized lamellae.

Ultrastructure of the corpuscular nerve ending in the lymph heart of the turtle (Pseudemys scripta elegans).

Corpuscular nerve endings were found in the tunica externa of the lymph heart of a turtle (Pseudemys scripta elegans). They consisted of axon terminals and surrounding cellular lamellae. Serial thin sections of one such presumable sensory corpuscle were made and reconstructed for the three-dimensional analysis of the corpuscle, with special reference to the axon terminals. It was revealed that axon terminals ramified at several points within the corpuscle and formed varicosities of various sizes ranging from 1.0 to 3.0 microns in diameter in their courses. Such varicosities contained an abundance of mitochondria as well as clear and granular vesicles 500 to 800 A in diameter. Finger-like protrusions often projected from the axon terminals into the interlamellar spaces. The lamellae consisted of several thin cytoplasmic processes of modified Schwann cells. The corpuscle was enclosed in an incomplete capsule consisting of a thin layer of cellular processes. The corpuscular nerve endings may represent pressoreceptors in the wall of the lymph heart.

The ultrastructure of the avian Golgi tendon organ.

The ultrastructure of the avian Golgi tendon organ (GTO) is described and compared with those of mammals using transverse sections through the myo-tendinous junctions of wing muscles of adult mallard ducks. The capsule, which is continuous with the perineural epithelial sheath of the Ib afferent nerve fiber, consists of four to seven flattened cellular lamellae. Two to four muscle fibers attach to large collagen bundles which enter the GTO through a tight collar at the proximal end of the fusiform capsule. These collagen bundles divide into many smaller bundles, which run longitudinally through the lumen in compartments formed by septal cells. The septal cells contain many prominent lipid accumulations. The Ib axon divides several times, and the unmyelinated branch axons weave between the small collagen bundles. Schwann cell processes or basement membrane usually intervene between the axons and collagen bundles. The small collagen bundles regroup into larger bundles, which pass through tight capsular collars and merge with the main muscle tendon. The size of the duck GTOs was measured and found to be smaller than the GTOs of man, cat or rat.

The fine structure of the boundary tissue of the seminiferous tubule of the camel (Camelus dromedarius).

An ultrastructural study of the boundary tissue of the seminiferous tubule of the camel reveals that it consists of three lamellae; inner fibrous, inner cellular and outer cellular. The inner lamella is subdivided into two homogeneous layers enclosing a third one that contains collagenous fibres and fine filaments. The inner cellular lamella consists of several layers of myoid cells; each layer is separated from the adjacent layer by homogeneous material and varying amounts of collagen. The outer cellular lamella consists predominantly of fibrocytes together with some fibroblasts and scattered collagen.

A consideration of the processes involved in the development of the prepuce in man.

SUMMARYAs the result of the examination of thirty‐eight human fœtuses it is concluded that the prepuce results from a combination of preputial fold formation and ingrowth of a cellular lamella. These processes are closely linked with the formation and fusion of the urethral folds on the glans. This fact accounts for the formation of the frenulum in normal males and the disposition of the prepuce in females and in cases of hypospadias.