scholarly journals Characterization and localization of actinogelin, a Ca2+ - sensitive actin accessory protein, in nonmuscle cells.

1982 ◽  
Vol 93 (3) ◽  
pp. 899-909 ◽  
Author(s):  
N Mimura ◽  
A Asano
Keyword(s):  
Intracellular Localization ◽  
Cell Types ◽  
Cell Extract ◽  
Affinity Column ◽  
Precipitation Line ◽  
Actin Bundles ◽  
Antibody Staining ◽  
Nonmuscle Cells ◽  
Tetramethylrhodamine Isothiocyanate ◽  
Antibody Preparations

Actinogelin, which induces gelation of F-actin at Ca2+ concentrations below micromolar concentrations but not at higher concentrations, was isolated in the pure state from Ehrlich tumor cells. The protein consists of subunits of 112,000-115,000 daltons and under physiological conditions is present mostly as a dimer. Up to 1 mol of actinogelin (dimer) binds to 10-12 mol of actin monomer. The binding was slightly decreased by the presence of 50 microM Ca2+ and almost completely inhibited by 300 mM KCl. Antibodies against actinogelin giving a single precipitation line with Ehrlich cell extract and with pure actinogelin were raised in rabbits. Antibody preparations were purified before use in an affinity column containing purified actinogelin. In mouse embryo fibroblasts and 3T3 cells, staining of actin bundles by the antiactinogelin antibody usually was discontinuous or gave a striated appearance. Most of the crossing points of the actin bundles were intensively stained. In epithelial cells from mouse small intestine, actinogelin was distributed throughout the cell, with the exception of the microvilli, which were devoid of staining. In mouse peritoneal cells, the antibody staining patterns were similar to those of tetramethylrhodamine isothiocyanate-labeled heavy meromyosin, but the former usually were sharper than the latter. Intracellular localization of actinogelin was drastically altered by cytochalasin D treatment at 10 microgram/ml. We conclude that actinogelin is present in a wide variety of cell types and discuss the possible participation of actinogelin in the Ca2+-dependent regulation of microfilament distribution.

scholarly journals Nerve fibers in culture and their interactions with non-neural cells visualized by immunofluorescence.

1979 ◽  
Vol 80 (3) ◽  
pp. 629-641 ◽  
Author(s):  
H Jockusch ◽  
B M Jockusch ◽  
M M Burger
Keyword(s):  
Spinal Cord ◽  
Ganglion Cells ◽  
Cell Types ◽  
Nerve Fibers ◽  
Affinity Column ◽  
Neural Cells ◽  
Embryonic Mouse ◽  
Antibody Staining ◽  
Order Of Magnitude ◽  
Nerve Muscle

Cultures of embryonic mouse spinal cord explants, alone or in combination with rat myotubes, were stained by indirect immunofluorescence using antibodies against three structural proteins to: (a) reveal the distribution of these proteins among different cell types, and (b) test the usefulness of antibody staining to reveal the gross morphology of the neurite network in complex cultures. Affinity column purified antibodies were used against chicken gizzard actin, porcine brain tubulin, and skeletal muscle alpha-actinin. Neurites were stained intensely by anti-actin as was the stress fiber pattern of underlying fibroblasts. With anti-tubulin, the staining of neurites was an order of magnitude more intense than the staining of the microtubule pattern of background fibroblasts. Neurite cell bodies and astrocyte-like glia cells were stained with anti-tubulin and their nuclei remained unstained. Anti-tubulin could thus be used to trace even the finest extensions of nerve processes in spinal cord and spinal cord-muscle cultures. Furthermore, it could be combined with the histochemical reaction for acetylcholinesterase (AChE, EC 3.1.1.7) to demonstrate AChE-positive neurons and specialized nerve-muscle contact sites. The staining of neural elements with anti-alpha-actinin was generally much weaker than with anti-actin and anti-tubulin. Neurites were stained only moderately in comparison to myotube Z lines in the same culture. However, a distinct staining of the periphery of dorsal root ganglion cells was observed. Thus, a protein immunologically related to muscle alpha-actinin is present in the nervous system. In myotubes, Z lines were stained intensely with anti-alpha-actinin while I bands were only faintly stained with anti-actin. In isolated myofibrils, both structures were stained intensely with the same antibody preparations.

scholarly journals The mammalian 43-kD acetylcholine receptor-associated protein (RAPsyn) is expressed in some nonmuscle cells.

1989 ◽  
Vol 108 (5) ◽  
pp. 1833-1840 ◽  
Author(s):  
L S Musil ◽  
D E Frail ◽  
J P Merlie
Keyword(s):  
Skeletal Muscle ◽  
Acetylcholine Receptor ◽  
Cell Lines ◽  
Neuromuscular Junctions ◽  
Cell Types ◽  
Cardiac Cells ◽  
Mrna Levels ◽  
Receptor Associated Protein ◽  
Mouse Tissues ◽  
Nonmuscle Cells

Torpedo electric organ and vertebrate neuromuscular junctions contain the receptor-associated protein of the synapse (RAPsyn) (previously referred to as the 43K protein), a nonactin, 43,000-Mr peripheral membrane protein associated with the cytoplasmic face of postsynaptic membranes at areas of high nicotinic acetylcholine receptor (AChR) density. Although not directly demonstrated, several lines of evidence suggest that RAPsyn is involved in the synthesis and/or maintenance of such AChR clusters. Microscopic and biochemical studies had previously indicated that RAPsyn expression is restricted to differentiated, AChR-synthesizing cells. Our recent finding that RAPsyn is also produced in undifferentiated myocytes (Frail, D.E., L.S. Musil, a. Bonanno, and J.P. Merlie, 1989. Neuron. 2:1077-1086) led to to examine whether RAPsyn is synthesized in cell types that never express AChR (i.e., cells of other than skeletal muscle origin). Various primary and established rodent cell lines were metabolically labeled with [35S]methionine, and extracts were immunoprecipitated with a monospecific anti-RAPsyn serum. Analysis of these immunoprecipitates by SDS-PAGE revealed detectable RAPsyn synthesis in some (notably fibroblast and Leydig tumor cell lines and primary cardiac cells) but not all (hepatocyte- and lymphocyte-derived) cell types. These results were further substantiated by peptide mapping studies of RAPsyn immunoprecipitated from different cells and quantitation of RAPsyn-encoding mRNA levels in mouse tissues. RAPsyn synthesized in both muscle and nonmuscle cells was shown to be tightly associated with membranes. These findings demonstrate that RAPsyn is not specific to skeletal muscle-derived cells and imply that it may function in a capacity either in addition to or instead of AChR clustering.

scholarly journals Tissue factor: identification and characterization of cell types in human placentae

Blood ◽  
1990 ◽  
Vol 76 (1) ◽  
pp. 86-96 ◽  
Author(s):  
WP Faulk ◽  
CA Labarrere ◽  
SD Carson
Keyword(s):  
Connective Tissue ◽  
Tissue Factor ◽  
Cell Types ◽  
Basement Membranes ◽  
Factor Vii ◽  
Affinity Column ◽  
Chorionic Villi ◽  
Normal Term ◽  
Synthetic Polypeptide

This is an immunohistologic study of tissue factor (TF) in snap frozen, unfixed, human normal-term placentae. Antibodies to TF were a monoclonal to human brain TF purified on a factor VII-agarose affinity column, and a polyclonal to a synthetic polypeptide representing the carboxyl-terminal nine amino acids of human TF. The results detail the localization and distribution of TF and characterize the cells in which it is found. TF was not observed in trophoblast, trophoblastic basement membranes, or noncellular components of connective tissue. TF was identified in some but not all macrophages, most fibroblast-like cells, and occasionally in perivascular cells and endothelium. The most consistent and intense reactions were obtained with vimentin-positive fibroblast-like cells in loose connective tissue. TF usually was not identified in fetal stem vessel endothelial cells, but TF reactivity was found in some of these cells in chorionic villi with histologic evidence of chronic inflammation. Such areas are uncommonly found in normal-term placentae. The vast majority of TF-reactive cells did not react with antibody to factor VII and were not in contact with blood. The biologic purpose of producing relatively great amounts of TF in areas remote from circulating factor VII is not known.

Fibronectin, intercellular junctions and the sorting-out of chick embryonic tissue cells in monolayer

1982 ◽  
Vol 54 (1) ◽  
pp. 357-372
Author(s):  
A. Nicol ◽  
D.R. Garrod
Keyword(s):  
Corneal Epithelium ◽  
Fluorescent Antibody ◽  
Intercellular Junctions ◽  
Liver Parenchyma ◽  
Cell Types ◽  
Limb Bud ◽  
Antibody Staining ◽  
Intercellular Contacts ◽  
Mesenchyme Cells ◽  
Chick Embryonic Tissue

A hierarchy of relative cohesiveness in monolayer of four different embryonic chick tissues was determined in a previous study. The hierarchy is: corneal epithelium congruent to liver parenchyma greater than pigmented epithelium greater than limb bud mesenchyme. The purpose of this paper is to describe the correlation between these adhesive relationships and, firstly, the amount of the adhesive glycoprotein, fibronectin, associated with the cells and, secondly, the morphology of their intercellular contacts. Fluorescent antibody staining of the cells with anti-fibronectin antibody showed that limb bud mesenchyme cells, the most weakly cohesive, had much more fibronectin than the other cell types. Thus there was a negative correlation between the amount of fibronectin and cellular cohesiveness. Analysis of intercellular contacts by electron microscopy showed that the most strongly cohesive cell types, corneal epithelium and liver parenchyma, were also those that possessed desmosomes.

scholarly journals In vitro and in vivo characterization of four fibroblast tropomyosins produced in bacteria: TM-2, TM-3, TM-5a, and TM-5b are co-localized in interphase fibroblasts.

1992 ◽  
Vol 118 (4) ◽  
pp. 841-858 ◽  
Author(s):  
M F Pittenger ◽  
D M Helfman
Keyword(s):  
Actin Filaments ◽  
Intracellular Localization ◽  
Cell Types ◽  
Alternative Possibilities ◽  
Expression Vectors ◽  
Rat Fibroblasts ◽  
Alpha Gene ◽  
The Individual

Most cell types express several tropomyosin isoforms, the individual functions of which are poorly understood. In rat fibroblasts there are at least six isoforms; TM-1, TM-2, TM-3, TM-4, TM-5a, and TM-5b. TM-1 is the product of the beta gene. TM-4 is produced from the TM-4 gene, and TMs 2, 3, 5a, and 5b are the products of the alpha gene. To begin to study the localization and function of the isoforms in fibroblasts, cDNAs for TM isoforms 2, 3, 5a, and 5b were placed into bacterial expression vectors and used to produce TM isoforms. The bacterially produced TMs were determined to be full length by sequencing the amino- and carboxy termini. These TMs were found to bind to F-actin in vitro, with properties similar to that of skeletal muscle TM. In addition, competition experiments demonstrated that TM-5b was better than TM-5a in displacing other TM isoforms from F-actin in vitro. To investigate the intracellular localization of these fibroblast isoforms, each was derivatized with a fluorescent chromophore and microinjected into rat fibroblasts. TM-2, TM-3, TM-5a, and TM-5b were each found to associate along actin filaments. There was no preferred cellular location or subset of actin filaments for these isoforms. Furthermore, co-injection of two isoforms labeled with different fluorochromes showed identical staining. At the level of the light microscope, these isoforms from the alpha gene do not appear to achieve different functions by binding to particular subsets of actin filaments or locations in cells. Some alternative possibilities are discussed. The results show that bacterially produced TMs can be used to study in vitro and in vivo properties of the isoforms.

scholarly journals Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme

2012 ◽  
Vol 196 (6) ◽  
pp. 789-800 ◽  
Author(s):  
Anjali A. Sarkar ◽  
Irene E. Zohn
Keyword(s):  
Neural Tube ◽  
Neural Tube Defect ◽  
Intracellular Localization ◽  
Cell Types ◽  
Abnormal Behavior ◽  
Data Set ◽  
Cellular Behavior ◽  
Multiple Cell ◽  
Mesenchyme Cells ◽  
And Behavior

Hectd1 mutant mouse embryos exhibit the neural tube defect exencephaly associated with abnormal cranial mesenchyme. Cellular rearrangements in cranial mesenchyme are essential during neurulation for elevation of the neural folds. Here we investigate the molecular basis of the abnormal behavior of Hectd1 mutant cranial mesenchyme. We demonstrate that Hectd1 is a functional ubiquitin ligase and that one of its substrates is Hsp90, a chaperone protein with both intra- and extracellular clients. Extracellular Hsp90 enhances migration of multiple cell types. In mutant cranial mesenchyme cells, both secretion of Hsp90 and emigration of cells from cranial mesenchyme explants were enhanced. Importantly, we show that this enhanced emigration was highly dependent on the excess Hsp90 secreted from mutant cells. Together, our data set forth a model whereby increased secretion of Hsp90 in the cranial mesenchyme of Hectd1 mutants is responsible, at least in part, for the altered organization and behavior of these cells and provides a potential molecular mechanism underlying the neural tube defect.

scholarly journals Changes in intracellular localization of calpastatin during calpain activation

1999 ◽  
Vol 343 (2) ◽  
pp. 467-472 ◽  
Author(s):  
Roberta DE TULLIO ◽  
Mario PASSALACQUA ◽  
Monica AVERNA ◽  
Franca SALAMINO ◽  
Edon MELLONI ◽  
...  
Keyword(s):  
Intracellular Localization ◽  
Cell Types ◽  
Activation Process ◽  
Inhibitory Protein ◽  
Proteolytic System ◽  
Human Neuroblastoma ◽  
Soluble Cell ◽  
Main Components ◽  
Kinetics Of ◽  
Soluble Cell Fraction

Localization of the two main components of the Ca2+-dependent proteolytic system has been investigated in human neuroblastoma LAN-5 cells. Using a monoclonal antibody which recognizes the N-terminal calpastatin domain, it has been shown that this inhibitory protein is almost completely confined in two granule-like structures not surrounded by membranes. Similar calpastatin distribution has been found in other human and in murine cell types, indicating that aggregation of calpastatin is a general property and not an exclusive characteristic of neuronal-like cells. The existence of such calpastatin aggregates is confirmed by the kinetics of calpastatin-activity release during rat liver homogenization, which does not correspond to the rate of appearance of cytosolic proteins or to the disruption of membrane-surrounded organelles. Calpastatin distribution is affected by the intracellular increase in free Ca2+, which results in calpastatin progressively becoming a soluble protein. However, calpain is distributed in the soluble cell fraction and, in activating conditions, partially accumulates on the plasma membrane. Similar behaviour has been observed in calpastatin localization in LAN-5 cells induced with retinoic acid, suggesting that the proteolytic system is activated during the differentiation process of these cells. The involvement of calpastatin in controlling calpain activity, rather than its activation process, and the utilization of changes in calpastatin localization as a marker of activation of the system is discussed.

scholarly journals Design and Validation of a Process Based on Cationic Niosomes for Gene Delivery into Novel Urine-Derived Mesenchymal Stem Cells

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 696
Author(s):  
Yerai Vado ◽  
Gustavo Puras ◽  
Melania Rosique ◽  
Cesar Martin ◽  
Jose Luis Pedraz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Gene Delivery ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Intracellular Localization ◽  
Cell Types ◽  
Functional Studies ◽  
Cellular Models ◽  
Cationic Niosomes

Background: Mesenchymal stem cells (MSCs) are stem cells present in adult tissues. They can be cultured, have great growth capacity, and can differentiate into several cell types. The isolation of urine-derived mesenchymal stem cells (hUSCs) was recently described. hUSCs present additional benefits in the fact that they can be easily obtained noninvasively. Regarding gene delivery, nonviral vectors based on cationic niosomes have been used and are more stable and have lower immunogenicity than viral vectors. However, their transfection efficiency is low and in need of improvement. Methods: We isolated hUSCs from urine, and the cell culture was tested and characterized. Different cationic niosomes were elaborated using reverse-phase evaporation, and they were physicochemically characterized. Then, they were screened into hUSCs for transfection efficiency, and their internalization was evaluated. Results: GPxT-CQ at a lipid/DNA ratio of 5:1 (w/w) had the best transfection efficiency. Intracellular localization studies confirmed that nioplexes entered mainly via caveolae-mediated endocytosis. Conclusions: In conclusion, we established a protocol for hUSC isolation and their transfection with cationic niosomes, which could have relevant clinical applications such as in gene therapy. This methodology could also be used for creating cellular models for studying and validating pathogenic genetic variants, and even for performing functional studies. Our study increases knowledge about the internalization of tested cationic niosomes in these previously unexplored cells.

scholarly journals Machine and deep learning single-cell segmentation and quantification of multi-dimensional tissue images

2019 ◽  
Author(s):  
Eliot T McKinley ◽  
Joseph T Roland ◽  
Jeffrey L Franklin ◽  
Mary Catherine Macedonia ◽  
Paige N Vega ◽  
...  
Keyword(s):  
Deep Learning ◽  
Single Cell ◽  
Shape Descriptor ◽  
Cell Types ◽  
Colorectal Adenomas ◽  
Cell Segmentation ◽  
Tissue Imaging ◽  
Antibody Staining ◽  
Multiple Cell ◽  
Cellular Compartments

AbstractIncreasingly, highly multiplexed in situ tissue imaging methods are used to profile protein expression at the single-cell level. However, a critical limitation is a lack of robust cell segmentation tools applicable for sections of tissues with a complex architecture and multiple cell types. Using human colorectal adenomas, we present a pipeline for cell segmentation and quantification that utilizes machine learning-based pixel classification to define cellular compartments, a novel method for extending incomplete cell membranes, quantification of antibody staining, and a deep learning-based cell shape descriptor. We envision that this method can be broadly applied to different imaging platforms and tissue types.

scholarly journals Activity and intracellular localization of the human cytomegalovirus protein pp71

2002 ◽  
Vol 83 (7) ◽  
pp. 1601-1612 ◽  
Author(s):  
Ker R. Marshall ◽  
Kate V. Rowley ◽  
Angela Rinaldi ◽  
Iain P. Nicholson ◽  
Alexander M. Ishov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Intracellular Localization ◽  
Cell Types ◽  
Independent Manner ◽  
Nuclear Domain ◽  
Protein Icp0 ◽  
Simplex Virus ◽  
Hsv 1

The human cytomegalovirus (HCMV) tegument phosphoprotein pp71 activates viral immediate early (IE) transcription and thus has a role in initiating lytic infection. Protein pp71 stimulates expression from a range of promoters in a sequence-independent manner, and in this respect behaves similarly to the herpes simplex virus type 1 (HSV-1) IE protein ICP0. The intracellular localization of pp71 was investigated after its expression from transfected plasmids or from HSV-1 mutants constructed to produce pp71 transiently. The protein colocalized with the cell promyelocytic leukaemia (PML) protein at nuclear domain 10 (ND10) structures but, unlike ICP0, pp71 did not induce disruption of ND10. The activity of pp71 in mouse sensory neurons in vivo was investigated after co-inoculation of animals with pairs of HSV-1 mutants, one expressing pp71 and the second containing the E. coli lacZ gene controlled by various promoters. In this system, pp71 stimulated β-galactosidase expression from a range of viral IE promoters when mice were analysed at 4 days postinoculation. At later times, expression of pp71 resulted in a reduction in numbers of neurons containing β-galactosidase, indicating cytotoxicity or promoter shutoff. The HSV-1 latency-active promoter was not responsive to pp71, demonstrating specificity in the activity of the protein. Pp71 was as active in mice lacking both copies of the PML gene (PML−/−) as in control animals, and in PML−/− fibroblasts pp71 stimulated gene expression as effectively as in other cell types. Therefore, neither the PML protein nor the normal ND10 structure is necessary for pp71 to stimulate gene expression.

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