Embryogenesis from protoplasts of haploid European larch

1992 ◽  
Vol 22 (3) ◽  
pp. 397-402 ◽  
Author(s):  
Patrick Von Aderkas
Keyword(s):  
Cell Walls ◽  
Gradient Centrifugation ◽  
Time Lapse ◽  
Colony Development ◽  
European Larch ◽  
Digestion Mixture ◽  
Embryogenic Line ◽  
Dividing Cells ◽  
Half Strength ◽  
Haploid Megagametophyte

Haploid megagametophyte-derived lines of European larch (Larixdecidua Mill.) displaying different degrees of embryogenicity had their protoplasts isolated and cultured. Embryoids were produced from two lines. Colonies of dividing cells but no embryoids were produced from the third, least embryogenic, line. The isolation procedure consisted of preplasmolysis in 0.44 M mannitol for 1 h, followed by enzyme digestion of the cell walls using cellulase and Macerozyme. The released protoplasts were separated from the digestion mixture by screening through cheesecloth and Nitex meshes and by differential gradient centrifugation. The protoplasts were plated in modified half-strength LM medium solidified with agarose. At the end of 1 week the agarose was suspended in a regeneration medium that had lower osmolarity. Osmotic levels were progressively lowered over a period lasting 3 weeks. An alternative method for osmotic reduction was used in which protoplasts were transferred directly from regeneration to maintenance medium. In either method, regeneration of protoplasts occurred in agarose. Regenerated embryoids underwent limited development in suspension, so maturation was completed by plating them onto semisolid half-strength LM medium, and they were raised in the dark for another month. Development from single protoplasts is described, and time-lapse illustrations of colony development from multinucleate protoplasts (coenocytes) are also included.

scholarly journals Cytokinesis and Midzone Microtubule Organization inCaenorhabditis elegans Require the Kinesin-like Protein ZEN-4

1998 ◽  
Vol 9 (8) ◽  
pp. 2037-2049 ◽  
Author(s):  
William B. Raich ◽  
Adrienne N. Moran ◽  
Joel H. Rothman ◽  
Jeff Hardin
Keyword(s):  
Cell Division ◽  
Null Allele ◽  
Time Lapse ◽  
Equatorial Region ◽  
Microtubule Organization ◽  
Dividing Cells ◽  
Spindle Midzone ◽  
Cross Links ◽  
Complete Cell

Members of the MKLP1 subfamily of kinesin motor proteins localize to the equatorial region of the spindle midzone and are capable of bundling antiparallel microtubules in vitro. Despite these intriguing characteristics, it is unclear what role these kinesins play in dividing cells, particularly within the context of a developing embryo. Here, we report the identification of a null allele ofzen-4, an MKLP1 homologue in the nematodeCaenorhabditis elegans, and demonstrate that ZEN-4 is essential for cytokinesis. Embryos deprived of ZEN-4 form multinucleate single-celled embryos as they continue to cycle through mitosis but fail to complete cell division. Initiation of the cytokinetic furrow occurs at the normal time and place, but furrow propagation halts prematurely. Time-lapse recordings and microtubule staining reveal that the cytokinesis defect is preceded by the dissociation of the midzone microtubules. We show that ZEN-4 protein localizes to the spindle midzone during anaphase and persists at the midbody region throughout cytokinesis. We propose that ZEN-4 directly cross-links the midzone microtubules and suggest that these microtubules are required for the completion of cytokinesis.

scholarly journals Changes in the Min Oscillation Pattern before and after Cell Birth

2010 ◽  
Vol 192 (16) ◽  
pp. 4134-4142 ◽  
Author(s):  
Jennifer R. Juarez ◽  
William Margolin
Keyword(s):  
Cell Division ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
The Other ◽  
Cell Pole ◽  
Daughter Cells ◽  
Division Site ◽  
Before And After ◽  
Dividing Cells ◽  
Green Fluorescent

ABSTRACT The Min system regulates the positioning of the cell division site in many bacteria. In Escherichia coli, MinD migrates rapidly from one cell pole to the other. In conjunction with MinC, MinD helps to prevent unwanted FtsZ rings from assembling at the poles and to stabilize their positioning at midcell. Using time-lapse microscopy of growing and dividing cells expressing a gfp-minD fusion, we show that green fluorescent protein (GFP)-MinD often paused at midcell in addition to at the poles, and the frequency of midcell pausing increased as cells grew longer and cell division approached. At later stages of septum formation, GFP-MinD often paused specifically on only one side of the septum, followed by migration to the other side of the septum or to a cell pole. About the time of septum closure, this irregular pattern often switched to a transient double pole-to-pole oscillation in the daughter cells, which ultimately became a stable double oscillation. The splitting of a single MinD zone into two depends on the developing septum and is a potential mechanism to explain how MinD is distributed equitably to both daughter cells. Septal pausing of GFP-MinD did not require MinC, suggesting that MinC-FtsZ interactions do not drive MinD-septal interactions, and instead MinD recognizes a specific geometric, lipid, and/or protein target at the developing septum. Finally, we observed regular end-to-end oscillation over very short distances along the long axes of minicells, supporting the importance of geometry in MinD localization.

Regulation of Mitosis

1969 ◽  
Vol 5 (3) ◽  
pp. 745-755
Author(s):  
W. T. JACKSON
Keyword(s):  
Mitotic Spindle ◽  
Time Lapse ◽  
Polarization Microscopy ◽  
Animal Cells ◽  
Higher Plant ◽  
Competitive Antagonism ◽  
Dividing Cells ◽  
Spindle Microtubules ◽  
Giant Nuclei

Earlier studies on the effects of the herbicide isopropyl N-phenylcarbamate (IPC) on mitosis revealed blocked metaphases, multinucleate cells, giant nuclei and an increase in number of partly contracted chromosomes. It was assumed that IPC, like colchicine, was causing these effects by disruption of the spindle apparatus by destroying the spindle microtubules. The animal hormone melatonin causes an increase in birefringence of the mitotic spindle in animal cells, presumably by increasing the number of microtubules. We have studied the effects of IPC, melatonin, and combinations of the two on mitosis in dividing endosperm cells of the African blood lily (Haemanthus katherinae Baker) in vivo by phase-contrast and polarization microscopy. Both qualitative and quantitative data are presented. Interpretation of these results has been aided materially by a time-lapse cinemicrographic analysis of dividing cells subjected to 1 and 10 p.p.m. IPC (unpublished) and by an accompanying fine-structural analysis of untreated and IPC-treated cells. Mitosis was disrupted by 0.01-10 p.p.m. IPC, the severity of the effect depending on both concentration and stage of mitosis of the cell at the time of treatment. Concentrations of IPC that caused cessation of chromosome movement also caused loss of birefringence of the mitotic spindle. Melatonin increased birefringence of the mitotic spindle in these plant cells and partly nullified the adverse effects of IPC. The results of this study demonstrate that the herbicide IPC, under our conditions, causes disruption of mitosis and loss of birefringence of the spindle. And it has been established that an animal hormone is capable of increasing the birefringence, and presumably the number of microtubules, of the mitotic spindle in dividing endosperm cells of a higher plant. Although melatonin is capable of partly nullifying the effects of IPC, a competitive antagonism is not postulated.

Characterization of a protein-lipopolysaccharide complex released from cell walls of Pseudomonas aeruginosa by ethylenediaminetetraacetic acid

1969 ◽  
Vol 15 (7) ◽  
pp. 743-748 ◽  
Author(s):  
S. W. Rogers ◽  
H. E. Gilleland Jr. ◽  
R. G. Eagon
Keyword(s):  
Electron Microscopy ◽  
Pseudomonas Aeruginosa ◽  
Cell Walls ◽  
Ethylenediaminetetraacetic Acid ◽  
Gradient Centrifugation ◽  
Hollow Spheres ◽  
Gel Filtration ◽  
A Chain ◽  
Ultracentrifugal Analysis

Results from analytical ultracentrifugal analysis, Sephadex gel filtration, isopycnic density-gradient centrifugation, and polyacrylamide disc-gel electrophoresis revealed that ethylenediaminetetraacetic acid liberated a protein–lipopolysaccharide complex from cell walls of Pseudomonas aeruginosa with an estimated molecular weight of not less than 160 000 and probably about one million. Electron microscopy of this complex revealed spherules and rodlets. The diameter of the former was approximately 70 ± 10 Å while the dimensions of the latter were 70 ± 10 Å × 200 ± 50 Å. The rodlets appeared to be composed of three or more spherules arranged in a chain-like fashion. Electron microscopy of protein-free lipopolysaccharide revealed predominantly hollow spheres from 300 Å to 1500 Å in diameter, morphologically resembling membrane sacculi. It is proposed that the protein–lipopolysaccharide complex, but not the protein-free lipopolysaccharide, is representative of the in situ form of native endotoxin.

Cell wall associations of some antigenic proteins of slime-forming, encapsulated Streptococcus cremoris from the fermented milk product "viili"

1986 ◽  
Vol 32 (2) ◽  
pp. 176-178 ◽  
Author(s):  
Raili Forsén ◽  
Teuvo Hentunen ◽  
Kaua Valkonen ◽  
Sirpa Kontusaari
Keyword(s):  
Cell Wall ◽  
Molecular Mass ◽  
Cell Walls ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fermented Milk ◽  
Milk Product ◽  
Streptococcus Cremoris ◽  
Protein Components

Cell walls were isolated from mechanically disrupted cells of the slime-forming, encapsulated Streptococcus cremoris strains T5 and MLS96 by using sucrose gradient centrifugation as the last purification step. This cell wall isolation procedure was developed to obtain cell wall associated protein components. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis revealed several polypeptide bands; the 50 kiloDalton band was major in strain T5 cell walls and the 26 and 30 kiloDalton bands were major in strain MLS96 cell walls. Both strains contained five antigenic polypeptides with molecular radius (Mr) values of 40, 47, 50, 54, and 70 kiloDaltons as analysed by immunoblotting and autoradiography. The polypeptides of strain MLS96 with molecular mass of 40 and 70 kiloDaltons reacted most strongly with homologous anti-whole cell serum. In addition, antigenic polypeptides with molecular mass of 100 and 160 kiloDaltons were also detected in strain T5.

scholarly journals A Spindle Checkpoint Functions during Mitosis in the Early Caenorhabditis elegans Embryo

2005 ◽  
Vol 16 (3) ◽  
pp. 1056-1070 ◽  
Author(s):  
Sandra E. Encalada ◽  
John Willis ◽  
Rebecca Lyczak ◽  
Bruce Bowerman
Keyword(s):  
Caenorhabditis Elegans ◽  
Chromosome Segregation ◽  
Spindle Checkpoint ◽  
Metaphase Plate ◽  
Time Lapse ◽  
Cell Types ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Animal Cells ◽  
Dividing Cells

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.

scholarly journals Time-lapse microscopic observation of non-dividing cells in cultured human osteosarcoma MG-63 cell line

Cell Cycle ◽  
2017 ◽  
Vol 17 (2) ◽  
pp. 174-181 ◽  
Author(s):  
John Dosch ◽  
Elise Hadley ◽  
Cal Wiese ◽  
Marissa Soderberg ◽  
Tori Houwman ◽  
...  
Keyword(s):  
Cell Line ◽  
Microscopic Observation ◽  
Time Lapse ◽  
Human Osteosarcoma ◽  
Dividing Cells

scholarly journals Cell-Projection Pumping of Fibroblast Contents into Osteosarcoma SAOS-2 Cells Correlates with Increased SAOS-2 Proliferation and Migration, as well as Altered Morphology

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1875
Author(s):  
Swarna Mahadevan ◽  
James A Cornwell ◽  
Belal Chami ◽  
Elizabeth Kelly ◽  
Hans Zoellner
Keyword(s):  
Cancer Cell ◽  
Cell Tracking ◽  
Time Lapse ◽  
Cell Phenotype ◽  
Phenotypic Change ◽  
Histopathological Diagnosis ◽  
Daughter Cells ◽  
Dividing Cells ◽  
And Migration ◽  
Cell Profile

We earlier reported that cell-projection pumping transfers fibroblast contents to cancer cells and this alters the cancer cell phenotype. Here, we report on single-cell tracking of time lapse recordings from co-cultured fluorescent fibroblasts and SAOS-2 osteosarcoma cells, tracking 5201 cells across 7 experiments. The fluorescent lipophilic marker DiD was used to label fibroblast organelles and to trace the transfer of fibroblast cytoplasm into SAOS-2 cells. We related SAOS-2 phenotypic change to levels of fluorescence transfer from fibroblasts to SAOS-2 cells, as well as what we term ‘compensated fluorescence’, that numerically projects mother cell fluorescence post-mitosis into daughter cells. The comparison of absolute with compensated fluorescence allowed us to deduct if the phenotypic effects in mother SAOS-2 cells were inherited by their daughters. SAOS-2 receipt of fibroblast fluorescence correlated by Kendall’s tau with cell-profile area and without evidence of persistence in daughter cells (median tau = 0.51, p < 0.016); negatively and weakly with cell circularity and with evidence of persistence (median tau = −0.19, p < 0.05); and very weakly with cell migration velocity and without evidence of persistence (median tau = 0.01, p < 0.016). In addition, mitotic SAOS-2 cells had higher rates of prior fluorescence uptake (median = 64.9 units/day) than non-dividing cells (median = 35.6 units/day, p < 0.016) and there was no evidence of persistence post-mitosis. We conclude that there was an appreciable impact of cell-projection pumping on cancer cell phenotype relevant to cancer histopathological diagnosis, clinical spread and growth, with most effects being ‘reset’ by cancer cell mitosis.

Isolation, culture, and uses of plant protoplasts

1973 ◽  
Vol 51 (10) ◽  
pp. 1795-1799 ◽  
Author(s):  
Oluf L. Gamborg ◽  
R. A. Miller
Keyword(s):  
Genetic Information ◽  
Cell Walls ◽  
Cultured Cells ◽  
Culture Conditions ◽  
Hybrid Plant ◽  
Plant Production ◽  
Plant Protoplasts ◽  
Cell Hybridization ◽  
Hybrid Plants ◽  
Dividing Cells

Techniques are now available for isolating plant protoplasts in any quantity from leaves or from cultured cells by enzymatic removal of the cell walls. The culture conditions for regenerating dividing cells and complete plants has been established for a few species. Intact viruses enter plant protoplasts and multiply. Spontaneous fusion occurs in protoplasts of the same species. Mitosis frequently takes place synchronously in the nuclei of the multinucleated protoplasts. Induced fusion of widely different species such as wheat and soybean has been demonstrated. Hybrid plants of two tobacco species have been produced by cell fusion. Transformation has been achieved by feeding isolated DNA to plants. Isolated DNA supplied to protoplasts is absorbed. The progress in plant cell and protoplast research demonstrates the feasibility of transferring genetic information and makes hybrid plant production by somatic cell hybridization a realistic objective.

scholarly journals Antagonism between Bacteriostatic and Bactericidal Antibiotics Is Prevalent

2014 ◽  
Vol 58 (8) ◽  
pp. 4573-4582 ◽  
Author(s):  
Paolo S. Ocampo ◽  
Viktória Lázár ◽  
Balázs Papp ◽  
Markus Arnoldini ◽  
Pia Abel zur Wiesch ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Bacterial Infections ◽  
Time Lapse ◽  
Cellular Responses ◽  
Inhibition Of Growth ◽  
Drug Classes ◽  
Dividing Cells ◽  
Evolution Of Resistance ◽  
Time Kill ◽  
Antagonistic Interactions

ABSTRACTCombination therapy is rarely used to counter the evolution of resistance in bacterial infections. Expansion of the use of combination therapy requires knowledge of how drugs interact at inhibitory concentrations. More than 50 years ago, it was noted that, if bactericidal drugs are most potent with actively dividing cells, then the inhibition of growth induced by a bacteriostatic drug should result in an overall reduction of efficacy when the drug is used in combination with a bactericidal drug. Our goal here was to investigate this hypothesis systematically. We first constructed time-kill curves using five different antibiotics at clinically relevant concentrations, and we observed antagonism between bactericidal and bacteriostatic drugs. We extended our investigation by performing a screen of pairwise combinations of 21 different antibiotics at subinhibitory concentrations, and we found that strong antagonistic interactions were enriched significantly among combinations of bacteriostatic and bactericidal drugs. Finally, since our hypothesis relies on phenotypic effects produced by different drug classes, we recreated these experiments in a microfluidic device and performed time-lapse microscopy to directly observe and quantify the growth and division of individual cells with controlled antibiotic concentrations. While our single-cell observations supported the antagonism between bacteriostatic and bactericidal drugs, they revealed an unexpected variety of cellular responses to antagonistic drug combinations, suggesting that multiple mechanisms underlie the interactions.

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