Cell Surface Morphology After Trypsinisation Depends on Initial Cell Shape

Cells growing in tissue culture exhibit constant variation in shape and surface morphology, particularly during the process of mitosis, where the cell rounds up exhibiting an intensely microvillous surface prior to cytokinesis. During routine subculturing, cells are induced to round up and relinquish contact with the substratum. Although the cells retain their viability throughout trypsinisation, their surface morphology demonstrates a variety of changes between finger-like microvillous projections, and spherical protruberances termed blebs.
The reaction of individual cells to cell rounding, in the presence of trypsin appears to be dependent on cell shape, which may be modulated naturally or altered by experimental agents. Cells of bipolar morphology, termed fibroblasts, produce a blebbed surface morphology in response to trypsin, whereas isometric, 'epithelioid' cells respond by the formation of a microvillous cell surface.
Blebbed cells subsequently undergo membrane reorganisation towards a more organised, and more permanent microvillous cell surface, even in the continued presence of trypsin. Naturally occurring spherical cells, for example, mitotic or suspension cultures, are microvillous and trypsin has no effect on their surface morphology. It would appear that blebs are the cells response to experimentally induced rapid change of shape of well spread cells, and thus represent a pathological response for prevention of membrane loss in conditions which produce a rapid assumption of a minimum surface area configuration, i.e. a sphere, which occurs too quickly for membrane resorption, or normal storage in the form of microvilli.     

Ectopic BASL Reveals Tissue Cell Polarity throughout Leaf Development in Arabidopsis thaliana

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The Metabolic Enzyme ManA Reveals a Link between Cell Wall Integrity and Chromosome Morphology

Synchronizing cell growth, division and DNA replication is an essential property of all living cells. Accurate coordination of these cellular events is especially crucial for bacteria, which can grow rapidly and undergo multifork replication. Here we show that the metabolic protein ManA, which is a component of mannose phosphotransferase system, participates in cell wall construction of the rod shaped bacterium Bacillus subtilis. When growing rapidly, cells lacking ManA exhibit aberrant cell wall architecture, polyploidy and abnormal chromosome morphologies. We demonstrate that these cellular defects are derived from the role played by ManA in cell wall formation. Furthermore, we show that ManA is required for maintaining the proper carbohydrate composition of the cell wall, particularly of teichoic acid constituents. This perturbed cell wall synthesis causes asynchrony between cell wall elongation, division and nucleoid segregation.     

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.     

Early B Cell Factor 1 Regulates Adipocyte Morphology and Lipolysis in White Adipose Tissue

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Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.     

Measuring Myeloperoxidase Activity in Biological Samples

Background

Enzymatic activity measurements of the highly oxidative enzyme myeloperoxidase (MPO), which is implicated in many diseases, are widely used in the literature, but often suffer from nonspecificity and lack of uniformity. Thus, validation and standardization are needed to establish a robust method that is highly specific, sensitive, and reproducible for assaying MPO activity in biological samples.

Principal findings

We found conflicting results between in vivo molecular MR imaging of MPO, which measures extracellular activity, and commonly used in vitro MPO activity assays. Thus, we established and validated a protocol to obtain extra- and intracellular MPO from murine organs. To validate the MPO activity assays, three different classes of MPO activity assays were used in spike and recovery experiments. However, these assay methods yielded inconsistent results, likely because of interfering substances and other peroxidases present in tissue extracts. To circumvent this, we first captured MPO with an antibody. The MPO activity of the resultant samples was assessed by ADHP and validated against samples from MPO-knockout mice in murine disease models of multiple sclerosis, steatohepatitis, and myocardial infarction. We found the measurements performed using this protocol to be highly specific and reproducible, and when performed using ADHP, to be highly sensitive over a broad range. In addition, we found that intracellular MPO activity correlated well with tissue neutrophil content, and can be used as a marker to assess neutrophil infiltration in the tissue.

Conclusion

We validated a highly specific and sensitive assay protocol that should be used as the standard method for all MPO activity assays in biological samples. We also established a method to obtain extra- and intracellular MPO from murine organs. Extracellular MPO activity gives an estimate of the oxidative stress in inflammatory diseases, while intracellular MPO activity correlates well with tissue neutrophil content. A detailed step-by-step protocol is provided.     

Neuronal Morphology: Shape Characteristics and Models

This paper is focused on quantification (morphometry) and modeling of neuronal morphological complexity. First, computer-aided methods for reconstruction, processing, and analysis of raw morphological data are reviewed. Then, topological and metrical measures are touched upon. Fractal measures (together with the extension of multiscale fractal dimension) are presented more explicitly. Models of neuronal arborizations are differentiated between reconstruction models and growth models (stochastic or mechanistic). The growth model approach is discussed in more detail. The methods presented are applied to several types of neurons and shown to have considerable discriminative power. Recent developments stress the importance of these methods for optimizing virtual neuronal trees in view of functional characteristics of the neurons. Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 366–372, July–August, 2008.     

The Photosynthesis and Cell Shape Rhythms can be Naturally Uncoupled from the Biological Clock in Euglena gracilis

A naturally occurring population of Euglena (Klebs Strain Z)cells, with unusual biological clock properties, has been isolated.The photosynthesis reactions, which are usually controlled bythe biological clock, are uncoupled from the clock in the newpopulation. The rate of oxygen evolution is influenced predominantlyby the environmental growth parameters instead of the biologicalclock. In addition, the rhythm in cellular shape has a differenttiming from the control population and can be temporarily uncoupledfrom the clock by lowering the light intensity used for growth Key words: Biological clock, photosynthesis, Euglena     

Use of a Tissue Sectioner to Expose Internal Structures of Biological Samples for Scanning Electron Microscopy

A procedure yielding sections of unembedded biological samples for observation by scanning electron microscopy is described. Sections of samples, fixed and hardened in OsO₄, were obtained in quantity with a tissue sectioner. Subsequent treatments to osmium-coat cut surfaces were employed prior to critical point drying. The procedure yields cleanly cut surfaces through cells and cytoplasmic organelles which are retained in their normal position. Sections of apple leaf and mouse kidney are illustrated. Sections can be readily cut in a desired plane with less structural damage than is typically encountered by other sectioning or dissection techniques.     

X-Ray Fluorescence Microscopy Reveals the Role of Selenium in Spermatogenesis

Selenium (Se) is a trace element with important roles in human health. Several selenoproteins have essential functions in development. However, the cellular and tissue distribution of Se remains largely unknown because of the lack of analytical techniques that image this element with sufficient sensitivity and resolution. Herein, we report that X-ray fluorescence microscopy (XFM) can be used to visualize and quantify the tissue, cellular, and subcellular topography of Se. We applied this technique to characterize the role of Se in spermatogenesis and identified a dramatic Se enrichment specifically in late spermatids, a pattern that was not seen in any other elemental maps. This enrichment was due to elevated levels of the mitochondrial form of glutathione peroxidase 4 and was fully dependent on the supplies of Se by selenoprotein P. High-resolution scans revealed that Se concentrated near the lumen side of elongating spermatids, where structural components of sperm are formed. During spermatogenesis, maximal Se associated with decreased phosphorus, whereas Zn did not change. In sperm, Se was primarily in the midpiece and colocalized with Cu and Fe. XFM allowed quantification of Se in the midpiece (0.8 fg) and head (0.2 fg) of individual sperm cells, revealing the ability of sperm cells to handle the amounts of this element well above its toxic levels. Overall, the use of XFM allowed visualization of tissue and cellular Se and provided important insights in the role of this and other trace elements in spermatogenesis.     

Site-directed Fluorescence Labeling Reveals a Revised N-terminal Membrane Topology and Functional Periplasmic Residues in the Escherichia coli Cell Division Protein FtsK

In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsK_N) is essential for division, and the C terminus (FtsK_C) is a well characterized DNA translocase. Although the function of FtsK_N is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsK_N and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsK_N is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsK_N was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsK_N also provides an important platform for future studies on essential interactions required for this process.     

Two-color Fluorescence Labeling in Acrolein-fixed Brain Tissue

Acrolein is a potent fixative that provides both excellent preservation of ultrastructural morphology and retention of antigenicity, thus it is frequently used for immunocytochemical detection of antigens at the electron microscopic level. However, acrolein is not commonly used for fluorescence microscopy because of concerns about possible autofluorescence and destruction of the luminosity of fluorescent dyes. Here we describe a simple protocol that allows fine visualization of two fluorescent markers in 40-μm sections from acrolein-perfused rat brain. Autofluorescence was removed by pretreatment with 1% sodium borohydride for 30 min, and subsequent incubation in a 50% ethanol solution containing 0.3% hydrogen peroxide enhanced fluorescence labeling. Thus, fluorescence labeling can be used for high-quality detection of markers in tissue perfused with acrolein. Furthermore, adjacent acrolein-fixed sections from a single experiment can be processed to produce high-quality results for electron microscopy or fluorescence labeling. (J Histochem Cytochem 58:359–368, 2010)     

Quantifying Shape Changes and Tissue Deformation in Leaf Development

The analysis of biological shapes has applications in many areas of biology, and tools exist to quantify organ shape and detect shape differences between species or among variants. However, such measurements do not provide any information about the mechanisms of shape generation. Quantitative data on growth patterns may provide insights into morphogenetic processes, but since growth is a complex process occurring in four dimensions, growth patterns alone cannot intuitively be linked to shape outcomes. Here, we present computational tools to quantify tissue deformation and surface shape changes over the course of leaf development, applied to the first leaf of Arabidopsis (Arabidopsis thaliana). The results show that the overall leaf shape does not change notably during the developmental stages analyzed, yet there is a clear upward radial deformation of the leaf tissue in early time points. This deformation pattern may provide an explanation for how the Arabidopsis leaf maintains a relatively constant shape despite spatial heterogeneities in growth. These findings highlight the importance of quantifying tissue deformation when investigating the control of leaf shape. More generally, experimental mapping of deformation patterns may help us to better understand the link between growth and shape in organ development.The analysis of biological shapes is a field of broad interest, with diverse applications ranging from medical imaging to comparative anatomy and botany (Lestrel, 2011). Within plant biology, the wide variation of leaf shapes among species has long intrigued evolutionary biologists, physiologists, and developmental biologists alike. Leaves typically develop into flat structures that maximize photosynthetic surface, with variations that may help to facilitate gas exchange, offset water loss, improve convective cooling, increase mechanical support, or reduce resistance to physical environmental forces, for example (for review, see Tsukaya, 2006; Cronk, 2009). The processes controlling leaf shape development, therefore, are important to plant survival and biomass accumulation and hence have important agricultural implications.Understanding how leaf shape is controlled typically involves studying shape variation among related species and in shape mutants and requires tools to quantify phenotypic differences. In recent years, several sophisticated semiautomatic methods have been developed to analyze leaf shapes in terms of their two-dimensional (2D) profiles. Analyses range from simple length and width measurements and allometric ratios to statistical analysis of outline coordinates (Langlade et al., 2005; Bylesjö et al., 2008; Weight et al., 2008; Backhaus et al., 2010).Leaf shape can also be quantified in terms of a three-dimensional (3D) surface, which can range from flat to curved or ruffled, as observed in nature and in many leaf shape mutants. Approaches to 3D shape analysis based on flattened and unflattened leaf dimensions in the proximodistal and mediolateral axes have been presented by Liu et al. (2010) and Wu et al. (2007). Kaminuma et al. (2004) developed a technique for obtaining coordinates of the leaf surface in vivo, from which they measured the angle of the surface across the leaf and fit curves along the leaf proximodistal and mediolateral axes to characterize blade epinasty.These existing methods for leaf shape analysis are very useful for detecting and quantitatively describing shape differences, but they do not provide information about the underlying mechanisms that give rise to those shape differences. For example, the correspondence between evenly spaced outline points (Langlade et al., 2005; Bylesjö et al., 2008; Weight et al., 2008; Backhaus et al., 2010) is arbitrary. Furthermore, existing work has not provided an analysis of leaf shape in terms of both 2D and 3D phenotypic features together, and with the exception of Kaminuma et al. (2004), the methods are destructive and cannot be used to measure changes in shape in vivo.Differences in shapes between two organs arise through differences in how the organs grow. It is impossible, however, to ascertain from differences in final organ shape what differences in growth gave rise to them, as the possible combinations of spatial and temporal alterations in growth patterns that could be responsible for the final shape are endless. By the same token, it can be difficult to conceptualize from growth patterns how the interconnected tissues will deform and, thus, how overall organ shape will change.To address this issue, Kennaway et al. (2011) recently proposed a simulation modeling framework to investigate how a shape may locally and globally deform under the control of spatially distributed growth- and polarity-regulating substances. This framework was used to hypothesize how growth and shape deformation may be controlled in flowers (Green et al., 2010; Sauret-Güeto et al., 2013) and leaves (Kuchen et al., 2012). However, to date, shape deformation patterns have not been quantified experimentally. Here, we use data collected for a study on leaf growth (Remmler and Rolland-Lagan, 2012) to develop a method for describing 3D surface shape and shape deformations during leaf development. In particular, we generate experimental maps of tissue deformation, which offer a new approach to investigating shape differences and uncovering the link between growth and shape during development.     

Synthesis of the Neurotoxin Quinolinic Acid in Apoptotic Tissue from Suberites domuncula: Cell Biological, Molecular Biological, and Chemical Analyses

Sessile marine animals, such as sponges, are prone to infection by prokaryotic as well as by eukaryotic attacking organisms. Using the sponge Suberites domuncula we document for the first time that in its apoptotic tissue a toxic compound is produced that very likely controls the elimination of the dying tissue. Apoptosis was induced by exposing the sponges to 2,2'-dipyridyl or by maintaining them under nonaeration conditions. After that treatment at least one eukaryotic epibiont (Bittium sp.) could be found grazing on apoptotic tissue. Cell proliferation assays demonstrated that aqueous extracts from unaffected sponge tissue displayed no cytotoxicity. However, addition of an extract from apoptotic tissue to neuronal cells from rat brain exerted strong toxicity. The underlying compound was identified as quinolinic acid; quantitative determination showed that quinolinic acid is present only in apoptotic tissue (4.8 mg/g dry wet weight). The complementary DNA encoding the key enzyme of the quinolinic acid pathway, 3-hydroxyanthranilate 3,4-dioxygenase, was cloned and characterized. The expression of this gene is up-regulated in apoptotic tissue. These data suggest that a complex molecular network controls apoptotic elimination of sponge tissue, which results in the synthesis of the bioactive compound quinolinic acid that controls the elimination of the tissue, perhaps via differential effects on grazing epibionts.     

Fluorescence and Absorption Spectra of Biological Dyes

Data include charts and tables of absorbance and fluorescence of 4 coupling agents for immunity reactions, 34 typical fluorescent dyes and reagents, and 12 dyes or reagents having no fluorescence in solution but which may be fluorescent in the dry state. Since this report consists largely of reference data, the original must be consulted for details. Reprint source: The Perkin-Elmer Corporation, Norwalk, Connecticut, U. S. A.