Nucleoid Size Scaling and Intracellular Organization of Translation across Bacteria.

The scaling of organelles with cell size is thought to be exclusive to eukaryotes. Here, we demonstrate that similar scaling relationships hold for the bacterial nucleoid. Despite the absence of a nuclear membrane, nucleoid size strongly correlates with cell size, independent of changes in DNA amount and across various nutrient conditions. This correlation is observed in diverse bacteria, revealing a near-constant ratio between nucleoid and cell size for a given species. As in eukaryotes, the nucleocytoplasmic ratio in bacteria varies greatly among species. This spectrum of nucleocytoplasmic ratios is independent of genome size, and instead it appears linked to the average population cell size. Bacteria with different nucleocytoplasmic ratios have a cytoplasm with different biophysical properties, impacting ribosome mobility and localization. Together, our findings identify new organizational principles and biophysical features of bacterial cells, implicating the nucleocytoplasmic ratio and cell size as determinants of the intracellular organization of translation.

Building the cell: design principles of cellular architecture.

The astounding structural complexity of a cell arises from the action of a relatively small number of genes, raising the question of how this complexity is achieved. Self-organizing processes combined with simple physical constraints seem to have key roles in controlling organelle size, number, shape and position, and these factors then combine to produce the overall cell architecture. By examining how these parameters are controlled in specific cell biological examples we can identify a handful of simple design principles that seem to underlie cellular architecture and assembly.

The cell biology of cell-in-cell structures.

For decades, authors have described unusual cell structures, referred to as cell-in-cell structures, in which whole cells are found in the cytoplasm of other cells. One well-characterized process that results in the transient appearance of such structures is the engulfment of apoptotic cells by phagocytosis. However, many other types of cell-in-cell structure have been described that involve viable non-apoptotic cells. Some of these structures seem to form by the invasion of one cell into another, rather than by engulfment. The mechanisms of cell-in-cell formation and the possible physiological roles of these processes will be discussed.

[Use of the Peptigel with Nanofibres in the Bone Defects Healing]. / Vyuzití peptigelu s nanovlákny k lécbe defektu kostní tkáne.

INTRODUCTION Traumatic bone injuries or pathological processes may sometimes result in very extensive bone defects. Currently, the standard procedure applied in clinical humane as well as veterinary medicine to fill a bone defect is the autogenous bone graft which, however, necessitates a more invasive procedure for the patient and in the cases of extensive defects it fails to provide adequate amount of graft. Synthetic bone replacements can be used with no further burden for the patient and can simultaneously be used as the carriers for bioactive molecules or therapeutic drugs. For clinical use, an easy and simple application is one of the requirements that have to be taken into consideration. These requirements are best satisfied by preparations in the form of gel, which may be injected into the defects of various shapes even through minimal surgical approach. MATERIAL AND METHODS Synthetic transparent PGD-AlphaProA hydro-peptide-gel was used as a basis to develop a composite hydrogel scaffold. This gel was enriched by cryogenically ground poly- -caprolactone nanofibers (PCL) in a ratio of 1 ml of gel to 16 µg of nanofibres. In experimental animals (laboratory rat Wistar, n=20), a single regular circular defect of 1.5 mm in diameter was drilled by a low speed drill machine across the whole width of distal femur diaphysis, identically in both the hind legs. In the right hindleg, this defect was filled by injection of 0.05 ml of the composite peptide gel with nanofibers (experimental defect). In the contralateral limb a similar defect was left untreated, without filling (control defect), for spontaneous healing. The group of experimental animals was subsequently divided into four sub-groups (A, B, C, D) for the purpose of further follow-up. One week after the surgical implantation, in the first group of experimental animals (Group A; n = 5) lege artis euthanasia was performed, a radiological examination of both the hind legs was carried out and a sample of the bone from both the control and experimental defect was collected for histologic examination. The other groups of experimental animals were evaluated similarly at 2, 4 and 6 weeks after the surgical procedure (Group B, C, D; n = 5). These groups of experimental animals were assessed using various histological techniques by two independent pathologists. RESULTS A difference between the control and the experimental bone defect was observed only at the healing stage at two weeks after the implantation, when a tendency for greater formation of new bone trabeculas was seen in the defect treated with the composite hydro-peptide-gel with PCL nanofibers. The results show a slightly higher angiogenesis and cellularity at the bone defect site with an increase of newly formed bone tissue and faster colonisation of lamellar bone structures by bone marrow cells at early stages of the healing process (1-2 weeks old defect). In the experimental and control groups, at the later stage of healing (4-6 weeks old defect), the process of healing and bone modelling at the defect site shows no detectable morphological differences. CONCLUSIONS The experimental use of hydro-peptide-gel with PCL nanofibers in vivo in laboratory rats shows very good applicability into the defect site and, compared to the untreated defect within two weeks after the implantation, accelerates the bone healing. This fact could be an advantage especially at the early stage of healing, and thus accelerate the healing of more extensive defects. Key words: peptide gel, polycaprolactone, PCL, replacement, bone, healing, scaffold, nanofibers, biomaterial.

Tools of the trade: podosomes as multipurpose organelles of monocytic cells.

Podosomes are adhesion and invasion structures that are particularly prominent in cells of the monocytic lineage such as macrophages, dendritic cells, and osteoclasts. They are multifunctional organelles that combine several key abilities required for cell migration and invasion. The podosome repertoire includes well-established functions such as cell-substrate adhesion, and extracellular matrix degradation, recently discovered abilities such as rigidity and topology sensing as well as antigen sampling, and also more speculative functions such as cell protrusion stabilization and transmigration. Collectively, podosomes not only enable dynamic interactions of cells with their surroundings, they also gather information about the pericellular environment, and are actively involved in its reshaping. This review presents an overview of the current knowledge on podosome composition, architecture, and regulation. We focus in particular on the growing list of podosome functions and discuss the specific properties of podosomes in macrophages, dendritic cells, and osteoclasts. Moreover, this article highlights podosome-related intracellular transport processes, the formation of podosomes in 3D environments as well as potentially podosome-associated diseases involving monocytic cells.

Killing by neutrophil extracellular traps: fact or folklore?

Neutrophil extracellular traps (NETs) are DNA structures released by dying neutrophils and claimed to constitute a new microbicidal mechanism. Killing by NET-forming cells is ascribed to these structures because it is prevented by preincubation with DNase, which has been shown to dismantle NETs, before addition of the target microorganisms. Curiously, the possibility that the microorganisms ensnared in NETs are alive has not been considered. Using Staphylococcus aureus and Candida albicans blastospores, we demonstrate that the microorganisms captured by NETs and thought to be killed are alive because they are released and recovered in cell medium by incubation with DNase. It is concluded that NETs entrap but do not kill microbes.

Toward the virtual cell: automated approaches to building models of subcellular organization "learned" from microscopy images.

We review state-of-the-art computational methods for constructing, from image data, generative statistical models of cellular and nuclear shapes and the arrangement of subcellular structures and proteins within them. These automated approaches allow consistent analysis of images of cells for the purposes of learning the range of possible phenotypes, discriminating between them, and informing further investigation. Such models can also provide realistic geometry and initial protein locations to simulations in order to better understand cellular and subcellular processes. To determine the structures of cellular components and how proteins and other molecules are distributed among them, the generative modeling approach described here can be coupled with high throughput imaging technology to infer and represent subcellular organization from data with few a priori assumptions. We also discuss potential improvements to these methods and future directions for research.

[Regulation of the Z ring positioning in bacterial cell division--a review].

The regulatory mechanism of bacterial cell division has long been a research focus. Forming a septum at the middle of the cell, the seemingly simple process is involved by multiple regulation factors. Zring (FtsZ ring) is the skeleton of the splitting complex. The locus where Z ring is formed is not only the position the septum formed but also determines the cell division site. Formation of Zring in the incorrect location results in inequality cell division. Several cell division regulation systems have been identified, including the Min system, nucleoid occlusion and the MipZ protein which effectively prevent Zring assembly by different mechanisms, ensuring formation of the fission complex at the correct position. Recent progresses about the formation process of Zring and regulation mechanism affecting the Z-ring positioning are summarized.

Synthetic chemoselective rewiring of cell surfaces: generation of three-dimensional tissue structures.

Proper cell-cell communication through physical contact is crucial for a range of fundamental biological processes including, cell proliferation, migration, differentiation, and apoptosis and for the correct function of organs and other multicellular tissues. The spatial and temporal arrangements of these cellular interactions in vivo are dynamic and lead to higher-order function that is extremely difficult to recapitulate in vitro. The development of three-dimensional (3D), in vitro model systems to investigate these complex, in vivo interconnectivities would generate novel methods to study the biochemical signaling of these processes, as well as provide platforms for tissue engineering technologies. Herein, we develop and employ a strategy to induce specific and stable cell-cell contacts in 3D through chemoselective cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. This strategy uses liposome fusion for the delivery of ketone or oxyamine groups to different populations of cells for subsequent cell assembly via oxime ligation. We demonstrate how this method can be used for several applications including, the delivery of reagents to cells for fluorescent labeling and cell-surface engineering, the formation of small, 3D spheroid cell assemblies, and the generation of large and dense, 3D multilayered tissue-like structures for tissue engineering applications.

On the possible use of exogenous histones in cell technology.

The prospect of developing transport systems using histones for site-specific delivery of therapeutic agents that have poor penetration characteristics through cellular membranes and tissue barriers has been investigated. Histones immobilized on microspheres can also be used to modify surfaces intended for cell cultivation, facilitating adhesion, proliferation and network formation by interactions of cells through contacts with several microspheres. They can be applied to three-dimensional pore matrices that are designed for producing tissue-like structures in vitro.

Lipids on the move: simulations of membrane pores, domains, stalks and curves.

In this review we describe the state-of-the-art of computer simulation studies of lipid membranes. We focus on collective lipid-lipid and lipid-protein interactions that trigger deformations of the natural lamellar membrane state, showing that many important biological processes including self-aggregation of membrane components into domains, the formation of non-lamellar phases, and membrane poration and curving, are now amenable to detailed simulation studies.

The annulus of the mouse sperm tail is required to establish a membrane diffusion barrier that is engaged during the late steps of spermiogenesis.

The annulus is a higher order septin cytoskeletal structure located between the midpiece and principal piece regions of the sperm tail. The annulus has been hypothesized to generate the diffusion barrier that exists between these two membrane domains. We tested this premise directly on septin 4 knockout mice, whose sperm are viable but lack an annulus, by following the diffusing membrane protein basigin. Basigin is normally confined to the principal piece domain on testicular and caput sperm, but undergoes relocation into the midpiece during sperm epididymal transit. On Sept4(-/-) sperm, domain confinement was lost, and basigin localized over the entire plasma membrane. Both immunofluorescence and immunoblotting further revealed reduced levels of basigin expression on sperm from the knockout. Testicular immunohistochemistry showed similar basigin expression and tail targeting in wild-type (WT) and Sept4(-/-) tubules until step 15 of spermatid development, at which point basigin was redistributed throughout the plasma membrane of Sept4(-/-) spermatids. The basigin outside of the tail was subsequently lost around the time of sperm release into the lumen. The redistribution in the knockout coincides with the time in WT sperm when the annulus completes its migration from the neck down to the midpiece-principal piece junction. We posit that basigin may not diffuse freely until after the annulus arrives at the midpiece-principal piece junction to restrict lateral movement. These results are the strongest evidence to date of a mammalian septin structure establishing a membrane diffusion barrier.

First cleavage plane of the mouse egg is not predetermined but defined by the topology of the two apposing pronuclei.

Studies of experimentally manipulated embryos have led to the long-held conclusion that the polarity of the mouse embryo remains undetermined until the blastocyst stage. However, recent studies reporting that the embryonic-abembryonic axis of the blastocyst arises perpendicular to the first cleavage plane, and hence to the animal-vegetal axis of the zygote, have led to the claim that the axis of the mouse embryo is already specified in the egg. Here we show that there is no specification of the axis in the egg. Time-lapse recordings show that the second polar body does not mark a stationary animal pole, but instead, in half of the embryos, moves towards a first cleavage plane. The first cleavage plane coincides with the plane defined by the two apposing pronuclei once they have moved to the centre of the egg. Pronuclear transfer experiments confirm that the first cleavage plane is not determined in early interphase but rather is specified by the newly formed topology of the two pronuclei. The microtubule networks that allow mixing of parental chromosomes before dividing into two may be involved in these processes.

A mercury arc lamp-based multi-color confocal real time imaging system for cellular structure and function.

Multi-point scanning confocal microscopy using a Nipkow disk enables the acquisition of fluorescent images with high spatial and temporal resolutions. Like other single-point scanning confocal systems that use Galvano meter mirrors, a commercially available Nipkow spinning disk confocal unit, Yokogawa CSU10, requires lasers as the excitation light source. The choice of fluorescent dyes is strongly restricted, however, because only a limited number of laser lines can be introduced into a single confocal system. To overcome this problem, we developed an illumination system in which light from a mercury arc lamp is scrambled to make homogeneous light by passing it through a multi-mode optical fiber. This illumination system provides incoherent light with continuous wavelengths, enabling the observation of a wide range of fluorophores. Using this optical system, we demonstrate both the high-speed imaging (up to 100 Hz) of intracellular Ca(2+) propagation, and the multi-color imaging of Ca(2+) and PKC-gamma dynamics in living cells.

Learning by structural remodeling in a class of single cell models.

Changes in neural connectivity are thought to underlie the most permanent forms of memory in the brain. We consider two models, derived from the clusteron (Mel, Adv Neural Inf Process Syst 4:35-42, 1992), to study this method of learning. The models show a direct relationship between the speed of memory acquisition and the probability of forming appropriate synaptic connections. Moreover, the strength of learned associations grows with the number of fibers that have taken part in the learning process. We provide simple and intuitive explanations of these two results by analyzing the distribution of synaptic activations. The obtained insights are then used to extend the model to perform novel tasks: feature detection, and learning spatio-temporal patterns. We also provide an analytically tractable approximation to the model to put these observations on a firm basis. The behavior of both the numerical and analytical models correlate well with experimental results of learning tasks which are thought to require a reorganization of neuronal networks.

Cell mechanics of alveolar epithelial cells (AECs) and macrophages (AMs).

Cell mechanics provides an integrated view of many biological phenomena which are intimately related to cell structure and function. Because breathing constitutes a sustained motion synonymous with life, pulmonary cells are normally designed to support permanent cyclic stretch without breaking, while receiving mechanical cues from their environment. The authors study the mechanical responses of alveolar cells, namely epithelial cells and macrophages, exposed to well-controlled mechanical stress in order to understand pulmonary cell response and function. They discuss the principle, advantages and limits of a cytoskeleton-specific micromanipulation technique, magnetic bead twisting cytometry, potentially applicable in vivo. They also compare the pertinence of various models (e.g., rheological; power law) used to extract cell mechanical properties and discuss cell stress/strain hardening properties and cell dynamic response in relation to the structural tensegrity model. Overall, alveolar cells provide a pertinent model to study the biological processes governing cellular response to controlled stress or strain.

Exciting cytoskeleton-membrane waves.

Propagating waves on the surface of cells, over many micrometers, involve active forces. We investigate here the mechanical excitation of such waves when the membrane is perturbed by an external oscillatory force. The external perturbation may trigger the propagation of such waves away from the force application. This scheme is then suggested as a method to probe the properties of the excitable medium of the cell, and learn about the mechanisms that drive the wave propagation. We then apply these ideas to a specific model of active cellular membrane waves, demonstrating how the response of the system to the external perturbation depends on the properties of the model. The most outstanding feature that we find is that the excited waves exhibit a resonance phenomenon at the frequency corresponding to the tendency of the system to develop a linear instability. Mechanical excitation of membrane waves in cells at different frequencies can therefore be used to characterize the properties of the mechanism underlying the existence of these waves.

Biological soft materials.

With one or two exceptions, biological materials are "soft", meaning that they combine viscous and elastic elements. This mechanical behavior results from self-assembled supramolecular structures that are stabilized by noncovalent interactions. It is an ongoing and profound challenge to understand the self-organization of biological materials. In many cases, concepts can be imported from soft-matter physics and chemistry, which have traditionally focused on materials such as colloids, polymers, surfactants, and liquid crystals. Using these ideas, it is possible to gain a new perspective on phenomena as diverse as DNA condensation, protein and peptide fibrillization, lipid partitioning in rafts, vesicle fusion and budding, and others, as discussed in this selective review of recent highlights from the literature.

Effects of different environmental oxygen levels on free radical processes in fish.

Changes in oxygen levels occur frequently in aquatic environments; therefore, water organisms, including fishes, evolve a wide spectrum of adaptations to both anoxia/hypoxia and hyperoxia. The review describes oxidative damage to cellular constituents by reactive oxygen species, alterations in glutathione status, and response of antioxidant enzymes to variable oxygen availability in fish. Anoxia- and hypoxia-tolerant species demonstrate an anticipatory increase of some antioxidant enzymes during low-oxygen state in order to enhance their antioxidant potential for dealing with possible oxidative stress upon return to normoxia. Under hyperoxic conditions, it seems that the glutathione system plays an important adaptive role. Most stressful conditions lead to a quick increase in lipid peroxidation products that, in turn, are detoxified rapidly by respective low- and high-molecular weight antioxidants. A scheme on possible ways of regulating antioxidant enzymes by different oxygen levels is proposed.