scholarly journals Enzymatic dissection of embryonic cell adhesive mechanisms.

1980 ◽  
Vol 85 (3) ◽  
pp. 766-776 ◽  
Author(s):  
G B Grunwald ◽  
R L Geller ◽  
J Lilien
Keyword(s):  
Protein Synthesis ◽  
Single Cells ◽  
Cluster Formation ◽  
Embryonic Cell ◽  
Embryonic Chick ◽  
Adhesive Systems ◽  
Kinetic Assay ◽  
Tissue Dissociation ◽  
Morphogenetic Potential ◽  
Two Parameters

In this paper we describe a kinetic assay for cell adhesion which measures the formation of cell clusters. Cluster formation is dependent on both calcium and protein synthesis, two parameters essential for the formation of histotypic aggregates. We also describe modifications of the stndard method for trypsinization of tissues which result in populations of single cells that appear to bear intact and functional cell surface adhesive systems. These modifications involve the use of chymotrypsin and the inclusion of calcium during enzyme digestion of tissues with trypsin and chymotrypsin. Using the cluster formation assay and the modified tissue dissociation techniques, we demonstrate the presence of two functionally distinct adhesive systems operating among embryonic chick neural retina cells. These two systems differ in proteolytic sensitivity, protection by calcium against proteolysis, dependence on calcium for function and morphogenetic potential. Cells possessing one of these intact adhesive systems are capable of extensive morphogenetic interactions in the absence of protein synthesis.

Nanofiber curvature with Rho GTPase activity increases mouse embryonic fibroblast random migration velocity

2022 ◽  
Author(s):  
Daniel T Bowers ◽  
Justin L Brown
Keyword(s):  
Stem Cell ◽  
Flat Surface ◽  
Rho Gtpase ◽  
Kinase Inhibitor ◽  
Single Cells ◽  
Stem Cell Differentiation ◽  
Migration Velocity ◽  
Embryonic Cell ◽  
Small Gtpase ◽  
Curved Surface

Abstract Mechanotransduction arises from information encoded in the shape of materials such as curvature. It induces activation of small GTPase signaling affecting cell phenotypes including differentiation. We carried out a set of preliminary experiments to test the hypothesis that curvature (1/radius) would also affect cell motility due to signal pathway crosstalk. High molecular weight poly (methyl methacrylate) straight nanofibers were electrospun with curvature ranging from 41 to 1 μm−1 and collected on a passivated glass substrate. The fiber curvature increased mouse mesenchymal stem cell aspect ratio (P < 0.02) and decreased cell area (P < 0.01). Despite little effect on some motility patterns such as polarity and persistence, we found selected fiber curvatures can increase normalized random fibroblastic mouse embryonic cell (MEF) migration velocity close to 2.5 times compared with a flat surface (P < 0.001). A maximum in the velocity curve occurred near 2.5 μm−1 and may vary with the time since initiation of attachment to the surface (range of 0–20 h). In the middle range of fiber curvatures, the relative relationship to curvature was similar regardless of treatment with Rho-kinase inhibitor (Y27632) or cdc42 inhibitor (ML141), although it was decreased on most curvatures (P < 0.05). However, below a critical curvature threshold MEFs may not be able to distinguish shallow curvature from a flat surface, while still being affected by contact guidance. The preliminary data in this manuscript suggested the large low curvature fibers were interpreted in a manner similar to a non-curved surface. Thus, curvature is a biomaterial construct design parameter that should be considered when specific biological responses are desired. Statement of integration, innovation, and insight  Replacement of damaged or diseased tissues that cannot otherwise regenerate is transforming modern medicine. However, the extent to which we can rationally design materials to affect cellular outcomes remains low. Knowing the effect of material stiffness and diameter on stem cell differentiation, we investigated cell migration and signaling on fibrous scaffolds. By investigating diameters across orders of magnitude (50–2000 nm), we identified a velocity maximum of ~800 nm. Furthermore, the results suggest large fibers may not be interpreted by single cells as a curved surface. This work presents insight into the design of constructs for engineering tissues.

Zygotic expression of the pebble locus is required for cytokinesis during the postblastoderm mitoses of Drosophila

Development ◽  
1992 ◽  
Vol 114 (1) ◽  
pp. 165-171 ◽  
Author(s):  
G. Hime ◽  
R. Saint
Keyword(s):  
Drosophila Melanogaster ◽  
Developmental Regulation ◽  
Single Cells ◽  
Embryonic Cell ◽  
Cell Cycles ◽  
Drosophila Embryogenesis ◽  
Zygotic Transcription ◽  
Homozygous Mutant ◽  
Mitotic Figures

Mutations at the pebble locus of Drosophila melanogaster result in embryonic lethality. Examination of homozygous mutant embryos at the end of embryogenesis revealed the presence of fewer and larger cells which contained enlarged nuclei. Characterization of the embryonic cell cycles using DAPI, propidium iodide, anti-tubulin and anti-spectrin staining showed that the first thirteen rapid syncytial nuclear divisions proceeded normally in pebble mutant embryos. Following cellularization, the postblastoderm nuclear divisions occurred (mitoses 14, 15 and 16), but cytokinesis was never observed. Multinucleate cells and duplicate mitotic figures were seen within single cells at the time of the cycle 15 mitoses. We conclude that zygotic expression of the pebble gene is required for cytokinesis following cellularization during Drosophila embryogenesis. We postulate that developmental regulation of zygotic transcription of the pebble gene is a consequence of the transition from syncytial to cellular mitoses during cycle 14 of embryogenesis.

scholarly journals CELL ELONGATION IN THE CULTURED EMBRYONIC CHICK LENS EPITHELIUM WITH AND WITHOUT PROTEIN SYNTHESIS

1972 ◽  
Vol 55 (1) ◽  
pp. 82-92 ◽  
Author(s):  
Joram Piatigorsky ◽  
Henry deF. Webster ◽  
Miriam Wollberg
Keyword(s):  
Protein Synthesis ◽  
Cell Elongation ◽  
Time Course ◽  
De Novo ◽  
Lens Epithelium ◽  
Cell Length ◽  
Continuous Exposure ◽  
Embryonic Chick ◽  
The Mean ◽  
Chick Lens

Previous studies have shown that cells in the 6-day old embryonic chick lens epithelium elongate in tissue culture. In the present study, the time course of elongation during the 1st day of cultivation has been examined histologically. Cultured epithelia were also treated with cycloheximide or colchicine in order to determine if cell elongation depends on new protein synthesis and on the utilization of microtubules, respectively. In the first 5 hr of culture, the mean cell length increased from 11 µ to 21 µ. Subsequently, elongation was slower; the mean cell length was 28 µ after 24 hr in culture. Continuous exposure to cycloheximide did not inhibit the initial doubling of cell length, but did prevent further elongation. By contrast, colchicine inhibited elongation almost immediately. When added after the cell length had doubled, cycloheximide and colchicine each inhibited further elongation; the treated cells remained columnar. Radioautographic and electrophoretic tests showed that protein synthesis was not appreciably affected by colchicine, but was suppressed by cycloheximide. Electron microscopic examination revealed that microtubules oriented along surface membranes were present in epithelia cultured with serum alone and with cycloheximide, but not in those incubated with colchicine. These results indicate that the early stages of cell elongation in the cultured lens epithelium require an initial assembly and organization of preexisting microtubular elements and that continued elongation depends, in addition, on the de novo synthesis of protein, possibly microtubule protein.

scholarly journals Dicodon monitoring of protein synthesis (DiCoMPS) reveals levels of synthesis of a viral protein in single cells

2013 ◽  
Vol 41 (18) ◽  
pp. e177-e177 ◽  
Author(s):  
Sima Barhoom ◽  
Ian Farrell ◽  
Ben Shai ◽  
Dvir Dahary ◽  
Barry S. Cooperman ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Viral Protein ◽  
Single Cells

scholarly journals TISSUE-SPECIFIC CELL-SURFACE ANTIGENS IN EMBRYONIC CELLS

1972 ◽  
Vol 53 (2) ◽  
pp. 435-449 ◽  
Author(s):  
Irving Goldschneider ◽  
A. A. Moscona
Keyword(s):  
Cell Surface ◽  
Surface Antigens ◽  
Embryonic Cell ◽  
Specific Cell ◽  
Embryonic Chick ◽  
Embryonic Cells ◽  
Cell Surfaces ◽  
Cell Surface Antigens ◽  
Tissue Specific ◽  
High Degree

With the use of antisera prepared in rabbits against suspensions of live embryonic chick tissue cells, qualitative differences in cell surface antigens were demonstrated on cells from different embryonic chick tissues by immune agglutination and immunofluorescence. Unabsorbed antisera reacted with both homologous and nonhomologous cells; thorough absorption of the antisera with heterologous tissues removed cross-reacting antibodies, and the antisera acquired a high degree of tissue specificity. Thus, antiretina cell serum absorbed with nonretina cells or tissues, agglutinated only neural retina cells, and was shown by immunofluorescence tests to react specifically with the surface of retina cells, both in cell suspensions and in frozen tissue sections. Comparable results with antisera against cells from embryonic liver and other tissues demonstrated the existence of tissue-specific, phenotypic disparities in the antigenicities of embryonic cell surfaces, in addition to the presence of cell-surface antigens shared by certain classes of cells, and of antigens common to all cells in the embryo. The results are discussed in terms of the possible involvement of such phenotypic determinants in the specification of cell surfaces, in relation to cell recognition and developmental interactions.

scholarly journals The Electrical Activity of Embryonic Chick Heart Cells Isolated in Tissue Culture Singly or in Interconnected Cell Sheets

1968 ◽  
Vol 52 (3) ◽  
pp. 643-665 ◽  
Author(s):  
Robert L. DeHaan ◽  
Sheldon H. Gottlieb
Keyword(s):  
Action Potential ◽  
Single Cells ◽  
Electrical Contact ◽  
Resting Potential ◽  
Heart Cells ◽  
Embryonic Chick ◽  
Irreversible Damage ◽  
Diastolic Depolarization ◽  
Chick Heart ◽  
Embryonic Chick Heart

Embryonic chick heart cells were cultured on a plastic surface in sparse sheets of 2–50 cells mutually in contact, or isolated as single cells. Conditions are described which permitted conjoint cells to be impaled with recording microelectrodes with 75% success, and isolated single cells with 8% success. It is proposed that cells in electrical contact with neighbors are protected from irreversible damage by the penetrating electrode, by a flow of ions or other substances from connected cells across low-impedance intercellular junctions. Action potentials recorded from conjoint and isolated single cells were similar in form and amplitude. The height or shape of the action potential thus appears not to depend upon spatial relationships of one cell to another. As the external potassium concentration was increased from 1.3 mM to 6 mM, cells became hyperpolarized while the afterhyperpolarization was reduced. At higher potassium levels, the afterhyperpolarization disappeared, the slope of the slow diastolic depolarization decreased, and resting potential fell along a linear curve with a slope of 61 mv per 10-fold increase in potassium. In pacemaker cells the diastolic depolarization consists of two phases: (a) recovery from the afterpotential of the previous action potential and (b) the pacemaker potential. These phases are separated by a point of inflection, and represent manifestations of different mechanisms. Evidence is presented that it is the point of inflection (PBA) rather than the point of maximal diastolic potential, that should be taken as the resting potential.

scholarly journals Preparation of Tissues and Heterogeneous Cellular Samples for Single-Cell Analysis

2021 ◽  
Author(s):  
E. Celeste Welch ◽  
Anubhav Tripathi
Keyword(s):  
Sample Preparation ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Cell Analysis ◽  
Processing Times ◽  
Tissue Dissociation ◽  
Emerging Trends ◽  
Recent Developments ◽  
Preparation Techniques

While sample preparation techniques for the chemical and biochemical analysis of tissues are fairly well advanced, the preparation of complex, heterogenous samples for single-cell analysis can be difficult and challenging. Nevertheless, there is growing interest in preparing complex cellular samples, particularly tissues, for analysis via single-cell resolution techniques such as single-cell sequencing or flow cytometry. Recent microfluidic tissue dissociation approaches have helped to expedite the preparation of single cells from tissues through the use of optimized, controlled mechanical forces. Cell sorting and selective cellular recovery from heterogenous samples have also gained traction in biosensors, microfluidic systems, and other diagnostic devices. Together, these recent developments in tissue disaggregation and targeted cellular retrieval have contributed to the development of increasingly streamlined sample preparation workflows for single-cell analysis technologies, which minimize equipment requirements, enable lower processing times and costs, and pave the way for high-throughput, automated technologies. In this chapter, we survey recent developments and emerging trends in this field.

scholarly journals Extracellular Domains I and II of cell-surface glycoprotein CD44 mediate its trans-homophilic dimerization and tumor cluster aggregation

2020 ◽  
Vol 295 (9) ◽  
pp. 2640-2649 ◽  
Author(s):  
Madoka Kawaguchi ◽  
Nurmaa Dashzeveg ◽  
Yue Cao ◽  
Yuzhi Jia ◽  
Xia Liu ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Single Cells ◽  
Cluster Formation ◽  
Cell Aggregation ◽  
Biochemical Properties ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Extracellular Domains ◽  
Pak2 Activation

CD44 molecule (CD44) is a well-known surface glycoprotein on tumor-initiating cells or cancer stem cells. However, its utility as a therapeutic target for managing metastases remains to be fully evaluated. We previously demonstrated that CD44 mediates homophilic interactions for circulating tumor cell (CTC) cluster formation, which enhances cancer stemness and metastatic potential in association with an unfavorable prognosis. Furthermore, CD44 self-interactions activate the P21-activated kinase 2 (PAK2) signaling pathway. Here, we further examined the biochemical properties of CD44 in homotypic tumor cell aggregation. The standard CD44 form (CD44s) mainly assembled as intercellular homodimers (trans-dimers) in tumor clusters rather than intracellular dimers (cis-dimers) present in single cells. Machine learning–based computational modeling combined with experimental mutagenesis tests revealed that the extracellular Domains I and II of CD44 are essential for its trans-dimerization and predicted high-score residues to be required for dimerization. Substitutions of 10 these residues in Domain I (Ser-45, Glu-48, Phe-74, Cys-77, Arg-78, Tyr-79, Ile-88, Arg-90, Asn-94, and Cys-97) or 5 residues in Domain II (Ile-106, Tyr-155, Val-156, Gln-157, and Lys-158) abolished CD44 dimerization and reduced tumor cell aggregation in vitro. Importantly, the substitutions in Domain II dramatically inhibited lung colonization in mice. The CD44 dimer-disrupting substitutions decreased downstream PAK2 activation without affecting the interaction between CD44 and PAK2, suggesting that PAK2 activation in tumor cell clusters is CD44 trans-dimer–dependent. These results shed critical light on the biochemical mechanisms of CD44-mediated tumor cell cluster formation and may help inform the development of therapeutic strategies to prevent tumor cluster formation and block cluster-mediated metastases.

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