A Stereological Approach for Estimation of Cellular Immunogold Labeling and Its Spatial Distribution in Oriented Sections Using the Rotator

Particulate gold labeling applied to ultrathin sections is a powerful approach for locating cellular proteins and lipids on thin sections of cellular structures and compartments. Effective quantitative methods now allow estimation of both density and distribution of gold labeling across aggregate organelles or compartment profiles. However, current methods generally use random sections of cells and tissues, and these do not readily present the information needed for spatial mapping of cellular quantities of gold label. Yet spatial mapping of gold particle labeling becomes important when cells are polarized or show internal organization or spatial shifts in protein/lipid localization. Here we have applied a stereological approach called the rotator to estimate cellular gold label and proportions of labeling over cellular compartments at specific locations related to a chosen cell axis or chosen cellular structures. This method could be used in cell biology for mapping cell components in studies of protein translocation, cell polarity, cell cycle stages, or component cell types in tissues.

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Polymorphous endocytotic organelles in the receptor-mediated endocytosis of gold-labelled alpha 2-macroglobulin complexes by human fibroblasts

Journal of Cell Science ◽

10.1242/jcs.75.1.411 ◽

1985 ◽

Vol 75 (1) ◽

pp. 411-421

Author(s):

B. Van der Schueren ◽

D. Gasser ◽

P. Marynen ◽

F. Van Leuven ◽

G. David ◽

...

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Human Fibroblasts ◽

Cellular Structures ◽

Multivesicular Bodies ◽

Staining Reaction ◽

Human Skin Fibroblasts ◽

Thin Sections ◽

Receptor Mediated Endocytosis ◽

Gold Label

The receptor-mediated endocytosis of gold-labelled alpha 2-macroglobulin complexes with trypsin or methylamine (alpha 2M-T-Au or alpha 2M-MA-Au) was studied by electron microscopy in human skin fibroblasts. The gold label was found in coated structures and very small tubules as well as in tubulovesicular structures and in multivesicular bodies/lysosomes. Thick sections (200 nm), but especially serial thin sections, clearly showed the polymorphic character of the cellular structures involved in endocytosis. Numerous intercommunications were particularly obvious between the tubulovesicular structures, the larger vesicles and the multivesicular bodies (MVB). Continuities between MVBs and endoplasmic reticulum and interconnections between MVBs were also observed. The specificity of the staining reaction was confirmed by indirect labelling of intracellular alpha 2M by polyclonal and by monoclonal antibodies on ultracryosections. These findings are discussed in relation to observations made on epithelial cells with other ligands.

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Morphological and Ultrastructural Characterization of Hemocytes in an Insect Model, the Hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae)

Insects ◽

10.3390/insects12070640 ◽

2021 ◽

Vol 12 (7) ◽

pp. 640

Author(s):

Natalia R. Moyetta ◽

Fabián O. Ramos ◽

Jimena Leyria ◽

Lilián E. Canavoso ◽

Leonardo L. Fruttero

Keyword(s):

Cell Biology ◽

Cell Types ◽

Transmission Electron ◽

Ultrastructural Characterization ◽

Current Classification ◽

Contrast Microscopy ◽

First Time ◽

Controversial Aspect

Hemocytes, the cells present in the hemolymph of insects and other invertebrates, perform several physiological functions, including innate immunity. The current classification of hemocyte types is based mostly on morphological features; however, divergences have emerged among specialists in triatomines, the insect vectors of Chagas’ disease (Hemiptera: Reduviidae). Here, we have combined technical approaches in order to characterize the hemocytes from fifth instar nymphs of the triatomine Dipetalogaster maxima. Moreover, in this work we describe, for the first time, the ultrastructural features of D. maxima hemocytes. Using phase contrast microscopy of fresh preparations, five hemocyte populations were identified and further characterized by immunofluorescence, flow cytometry and transmission electron microscopy. The plasmatocytes and the granulocytes were the most abundant cell types, although prohemocytes, adipohemocytes and oenocytes were also found. This work sheds light on a controversial aspect of triatomine cell biology and physiology setting the basis for future in-depth studies directed to address hemocyte classification using non-microscopy-based markers.

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Electron Microscopic Immunogold Localization of Salivary Mucins MG1 and MG2 in Human Submandibular and Sublingual Glands

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540305100109 ◽

2003 ◽

Vol 51 (1) ◽

pp. 69-79 ◽

Cited By ~ 21

Author(s):

Marco Piludu ◽

Sean A. Rayment ◽

Bing Liu ◽

Gwynneth D. Offner ◽

Frank G. Oppenheim ◽

...

Keyword(s):

Salivary Glands ◽

Expression Patterns ◽

Cell Types ◽

Mucous Cells ◽

Electron Microscopic ◽

Thin Sections ◽

Dense Cores ◽

Duct Cells ◽

Rabbit Igg ◽

Lowicryl K4m

The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells.

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Desmosomal Molecules In and Out of Adhering Junctions: Normal and Diseased States of Epidermal, Cardiac and Mesenchymally Derived Cells

Dermatology Research and Practice ◽

10.1155/2010/139167 ◽

2010 ◽

Vol 2010 ◽

pp. 1-12 ◽

Cited By ~ 17

Author(s):

Sebastian Pieperhoff ◽

Mareike Barth ◽

Steffen Rickelt ◽

Werner W. Franke

Keyword(s):

Cell Biology ◽

Cell Types ◽

Cell Junctions ◽

Surface Molecules ◽

Transmembrane Glycoprotein ◽

Current Cell ◽

Plakophilin 2 ◽

Definition Of ◽

Adhering Junctions ◽

Cell Cell

Current cell biology textbooks mention only two kinds of cell-to-cell adhering junctions coated with the cytoplasmic plaques: the desmosomes (maculae adhaerentes), anchoring intermediate-sized filaments (IFs), and the actin microfilament-anchoring adherens junctions (AJs), including both punctate (puncta adhaerentia) and elongate (fasciae adhaerentes) structures. In addition, however, a series of other junction types has been identified and characterized which contain desmosomal molecules but do not fit the definition of desmosomes. Of these special cell-cell junctions containing desmosomal glycoproteins or proteins we review the composite junctions (areae compositae) connecting the cardiomyocytes of mature mammalian hearts and their importance in relation to human arrhythmogenic cardiomyopathies. We also emphasize the various plakophilin-2-positive plaques in AJs (coniunctiones adhaerentes) connecting proliferatively active mesenchymally-derived cells, including interstitial cells of the heart and several soft tissue tumor cell types. Moreover, desmoplakin has also been recognized as a constituent of the plaques of thecomplexus adhaerentesconnecting certain lymphatic endothelial cells. Finally, we emphasize the occurrence of the desmosomal transmembrane glycoprotein, desmoglein Dsg2, out of the context of any junction as dispersed cell surface molecules in certain types of melanoma cells and melanocytes. This broadening of our knowledge on the diversity of AJ structures indicates that it may still be too premature to close the textbook chapters on cell-cell junctions.

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The cell biology of touch

The Journal of Cell Biology ◽

10.1083/jcb.201006074 ◽

2010 ◽

Vol 191 (2) ◽

pp. 237-248 ◽

Cited By ~ 105

Author(s):

Ellen A. Lumpkin ◽

Kara L. Marshall ◽

Aislyn M. Nelson

Keyword(s):

Cell Biology ◽

Cell Types ◽

Evolutionary Approach ◽

Mechanical Stimuli ◽

Cellular Logic ◽

Sense Of Touch

The sense of touch detects forces that bombard the body’s surface. In metazoans, an assortment of morphologically and functionally distinct mechanosensory cell types are tuned to selectively respond to diverse mechanical stimuli, such as vibration, stretch, and pressure. A comparative evolutionary approach across mechanosensory cell types and genetically tractable species is beginning to uncover the cellular logic of touch reception.

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The Winding Road of Cardiac Regeneration—Stem Cell Omics in the Spotlight

Cells ◽

10.3390/cells7120255 ◽

2018 ◽

Vol 7 (12) ◽

pp. 255 ◽

Cited By ~ 4

Author(s):

Miruna Mihaela Micheu ◽

Alina Ioana Scarlatescu ◽

Alexandru Scafa-Udriste ◽

Maria Dorobantu

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Cell Biology ◽

Molecular Mechanisms ◽

Unmet Need ◽

Cardiac Regeneration ◽

Cell Types ◽

Great Promise ◽

Omics Data

Despite significant progress in treating ischemic cardiac disease and succeeding heart failure, there is still an unmet need to develop effective therapeutic strategies given the persistent high-mortality rate. Advances in stem cell biology hold great promise for regenerative medicine, particularly for cardiac regeneration. Various cell types have been used both in preclinical and clinical studies to repair the injured heart, either directly or indirectly. Transplanted cells may act in an autocrine and/or paracrine manner to improve the myocyte survival and migration of remote and/or resident stem cells to the site of injury. Still, the molecular mechanisms regulating cardiac protection and repair are poorly understood. Stem cell fate is directed by multifaceted interactions between genetic, epigenetic, transcriptional, and post-transcriptional mechanisms. Decoding stem cells’ “panomic” data would provide a comprehensive picture of the underlying mechanisms, resulting in patient-tailored therapy. This review offers a critical analysis of omics data in relation to stem cell survival and differentiation. Additionally, the emerging role of stem cell-derived exosomes as “cell-free” therapy is debated. Last but not least, we discuss the challenges to retrieve and analyze the huge amount of publicly available omics data.

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The organisation and functions of local Ca2+ signals

Journal of Cell Science ◽

10.1242/jcs.114.12.2213 ◽

2001 ◽

Vol 114 (12) ◽

pp. 2213-2222 ◽

Cited By ~ 5

Author(s):

Martin D. Bootman ◽

Peter Lipp ◽

Michael J. Berridge

Keyword(s):

Cell Proliferation ◽

Gene Transcription ◽

Cell Biology ◽

Building Blocks ◽

Cell Types ◽

Diverse Range ◽

Cellular Processes ◽

Different Cell Types ◽

Intracellular Messenger ◽

Sensory Mechanisms

Calcium (Ca2+) is a ubiquitous intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation. The ability of a simple ion such as Ca2+ to play a pivotal role in cell biology results from the facility that cells have to shape Ca2+ signals in space, time and amplitude. To generate and interpret the variety of observed Ca2+ signals, different cell types employ components selected from a Ca2+ signalling ‘toolkit’, which comprises an array of homeostatic and sensory mechanisms. By mixing and matching components from the toolkit, cells can obtain Ca2+ signals that suit their physiology. Recent studies have demonstrated the importance of local Ca2+ signals in defining the specificity of the interaction of Ca2+ with its targets. Furthermore, local Ca2+ signals are the triggers and building blocks for larger global signals that propagate throughout cells.

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Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

10.1101/123042 ◽

2017 ◽

Cited By ~ 1

Author(s):

Brock Roberts ◽

Amanda Haupt ◽

Andrew Tucker ◽

Tanya Grancharova ◽

Joy Arakaki ◽

...

Keyword(s):

Stem Cells ◽

Quality Control ◽

Genome Editing ◽

Cell Biology ◽

Cellular Structures ◽

Human Stem Cells ◽

Alpha Tubulin ◽

Junction Protein ◽

Endogenous Proteins ◽

Clonal Lines

AbstractWe present a CRISPR/Cas9 genome editing strategy to systematically tag endogenous proteins with fluorescent tags in human inducible pluripotent stem cells. To date we have generated multiple human iPSC lines with GFP tags for 10 proteins representing key cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, lamin B1, non-muscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome editing methodology using Cas9 ribonuclear protein electroporation and fluorescence-based enrichment of edited cells resulted in <0.1-24% HDR across all experiments. Clones were generated from each edited population and screened for precise editing. ∼25% of the clones contained precise mono-allelic edits at the targeted locus. Furthermore, 92% (36/39) of expanded clonal lines satisfied key quality control criteria including genomic stability, appropriate expression and localization of the tagged protein, and pluripotency. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. The data described here, including our editing protocol, genetic screening, quality control assays, and imaging observations, can serve as an initial resource for genome editing in cell biology and stem cell research.

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Evaluation of S1PR1/pSTAT3 and S1PR2/FOXP1 Expression in Aggressive, Mature B Cell Lymphomas

10.1101/472449 ◽

2018 ◽

Author(s):

Mustafa Al-Kawaaz ◽

Teresa Sanchez ◽

Michael J Kluk

Keyword(s):

B Cell ◽

Cell Biology ◽

Significant Proportion ◽

B Cell Lymphoma ◽

Cell Types ◽

B Cell Lymphomas ◽

Not Otherwise Specified ◽

Survival Pathways ◽

Sphingosine 1 Phosphate ◽

G Protein Coupled

AbstractAggressive, mature B-cell lymphomas represent a heterogeneous group of diseases including Burkitt Lymphoma (BL), High Grade B Cell Lymphomas (HGBL) (eg, Double-Hit B cell lymphomas (HGBL-DH: HGBL with MYC and BCL2 and/or BCL6 translocations)), HGBL, Not Otherwise Specified (HGBL, NOS) and Diffuse Large B Cell Lymphoma. The overlapping morphologic and immunohistochemical features of these lymphomas may pose diagnostic challenges in some cases, and a better understanding of potential diagnostic biomarkers and possible therapeutic targets is needed. Sphingosine 1 Phosphate Receptors (S1PR1-5) represent a family of G-protein coupled receptors that bind the sphingolipid (S1P) and influence migration and survival pathways in a variety of cell types, including lymphocytes. S1PRs are emerging as biomarkers in B cell biology and interaction between S1PR pathways and STAT3 or FOXP1 has been reported, especially in DLBCL. Our aim was to extend the understanding of the S1PR1, STAT3 and S1PR2, FOXP1 expression beyond DLBCL, into additional aggressive, mature B cell lymphomas such as BL, HGBL-DH and HGBL,NOS.Herein, we report that S1PR1 and S1PR2 showed different patterns of expression in mantle zones and follicle centers in reactive lymphoid tissue and, among the lymphomas in this study, Burkitt lymphomas showed a unique pattern of expression compared to HGBL and DLBCL. Additionally, we found that S1PR1 and S1PR2 expression was typically mutually exclusive and were expressed in a low proportion of cases (predominantly HGBL involving extranodal sites). Lastly, FOXP1 was expressed in a high proportion of the various case types and pSTAT3 was detected in a significant proportion of HGBL and DLBCL cases. Taken together, these findings provide further evidence that S1PR1, pSTAT3, S1PR2 and FOXP1 play a role in a subset of aggressive mature B cell lymphomas.

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An Algorithm for Cellular Reprogramming

10.1101/162974 ◽

2017 ◽

Cited By ~ 1

Author(s):

Scott Ronquist ◽

Geoff Patterson ◽

Markus Brown ◽

Stephen Lindsly ◽

Haiming Chen ◽

...

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Transcription Factors ◽

Cell Biology ◽

Human Fibroblasts ◽

Cell Types ◽

Approximate Model ◽

Cellular Reprogramming ◽

Biological Processes ◽

Moderate Complexity

AbstractThe day we understand the time evolution of subcellular elements at a level of detail comparable to physical systems governed by Newton’s laws of motion seems far away. Even so, quantitative approaches to cellular dynamics add to our understanding of cell biology, providing data-guided frameworks that allow us to develop better predictions about, and methods for, control over specific biological processes and system-wide cell behavior. In this paper, we describe an approach to optimizing the use of transcription factors (TFs) in the context of cellular reprogramming. We construct an approximate model for the natural evolution of a cell cycle synchronized population of human fibroblasts, based on data obtained by sampling the expression of 22,083 genes at several time points along the cell cycle. In order to arrive at a model of moderate complexity, we cluster gene expression based on the division of the genome into topologically associating domains (TADs) and then model the dynamics of the TAD expression levels. Based on this dynamical model and known bioinformatics, such as transcription factor binding sites (TFBS) and functions, we develop a methodology for identifying the top transcription factor candidates for a specific cellular reprogramming task. The approach used is based on a device commonly used in optimal control. Our data-guided methodology identifies a number of transcription factors previously validated for reprogramming and/or natural differentiation. Our findings highlight the immense potential of dynamical models, mathematics, and data-guided methodologies for improving strategies for control over biological processes.Significance StatementReprogramming the human genome toward any desirable state is within reach; application of select transcription factors drives cell types toward different lineages in many settings. We introduce the concept of data-guided control in building a universal algorithm for directly reprogramming any human cell type into any other type. Our algorithm is based on time series genome transcription and architecture data and known regulatory activities of transcription factors, with natural dimension reduction using genome architectural features. Our algorithm predicts known reprogramming factors, top candidates for new settings, and ideal timing for application of transcription factors. This framework can be used to develop strategies for tissue regeneration, cancer cell reprogramming, and control of dynamical systems beyond cell biology.

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