scholarly journals Relation between cell activity and the distribution of cytoplasmic actin and myosin.

1981 ◽  
Vol 90 (1) ◽  
pp. 84-91 ◽  
Author(s):  
I M Herman ◽  
N J Crisona ◽  
T D Pollard
Keyword(s):  
Hela Cells ◽  
Experimental Approach ◽  
Cultured Cells ◽  
Cell Activity ◽  
Time Lapse ◽  
Entire Surface ◽  
Cytoplasmic Actin ◽  
Filament Bundles ◽  
Slow Moving ◽  
Diffuse Distribution

We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.

Desmosomes: interconnected calcium-dependent structures of remarkable stability with significant integral membrane protein turnover

2002 ◽  
Vol 115 (8) ◽  
pp. 1717-1732 ◽  
Author(s):  
Reinhard Windoffer ◽  
Monika Borchert-Stuhlträger ◽  
Rudolf E. Leube
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Structural Integrity ◽  
Time Lapse ◽  
Fluorescence Recovery ◽  
Adhesion Sites ◽  
Time Space ◽  
Molecular Stability ◽  
Filament Bundles ◽  
Cytokeratin Filaments

Desmosomes are prominent cell adhesion structures that are major stabilizing elements, together with the attached cytoskeletal intermediate filament network, of the cytokeratin type in epithelial tissues. To examine desmosome dynamics in tightly coupled cells and in situations of decreased adhesion, fluorescent desmosomal cadherin desmocollin 2a (Dsc2a) chimeras were stably expressed in human hepatocellular carcinoma-derived PLC cells (clone PDc-13) and in Madin-Darby canine kidney cells (clone MDc-2) for the continuous monitoring of desmosomes in living cells. The hybrid polypeptides integrated specifically and without disturbance into normal-appearing desmosomes that occurred in association with typical cytokeratin filament bundles. Tracking of labeled adhesion sites throughout the cell cycle by time-lapse fluorescence microscopy revealed that they were immobile and that they maintained their structural integrity for long periods of time. Time-space diagrams further showed that desmosomal positioning was tightly controlled, even during pronounced cell shape changes, although the desmosomal arrays extended and contracted, suggesting that they were interconnected by a flexible system with intrinsic elasticity. Double-fluorescence microscopy detecting Dsc2a chimeras together with fluorescent cytokeratin 18 chimeras revealed the association and synchronous movement of labeled desmosomes and fluorescent cytokeratin filaments. Only a minor destabilization of desmosomes was observed during mitosis, demonstrated by increased diffuse plasma membrane fluorescence and the fusion of desmosomes into larger structures. Desmosomes did not disappear completely at any time in any cell, and residual cytokeratin filaments remained in association with adhesion sites throughout cell division. On the other hand, a rapid loss of desmosomes was observed upon calcium depletion, with irreversible uptake of some desmosomal particles. Simultaneously, diffusely distributed desmosomal cadherins were detected in the plasma membrane that retained the competence to nucleate the reformation of desmosomes after the cells were returned to a standard calcium-containing medium. To examine the molecular stability of desmosomes, exchange rates of fluorescent chimeras were determined by fluorescence recovery after photobleaching, thereby identifying considerable Dsc2a turnover with different rates of fluorescence recovery for PDc-13 cells (36±17% recovery after 30 minutes) and MDc-2 cells (60±20% recovery after 30 minutes). Taken together, our observations suggest that desmosomes are pliable structures capable of fine adjustment to functional demands despite their overall structural stability and relative immobility.

scholarly journals Spectrin redistributes to the cytosol and is phosphorylated during mitosis in cultured cells.

1992 ◽  
Vol 119 (6) ◽  
pp. 1559-1572 ◽  
Author(s):  
V M Fowler ◽  
E J Adam
Keyword(s):  
Cell Body ◽  
Hela Cells ◽  
Plasma Membranes ◽  
Cultured Cells ◽  
Beta Subunit ◽  
Actin Binding ◽  
Culture Dish ◽  
Cell Rounding ◽  
Interphase Cells ◽  
Mitotic Cells

Dramatic changes in morphology and extensive reorganization of membrane-associated actin filaments take place during mitosis in cultured cells, including rounding up; appearance of numerous actin filament-containing microvilli and filopodia on the cell surface; and disassembly of intercellular and cell-substratum adhesions. We have examined the distribution and solubility of the membrane-associated actin-binding protein, spectrin, during interphase and mitosis in cultured CHO and HeLa cells. Immunofluorescence staining of substrate-attached, well-spread interphase CHO cells reveals that spectrin is predominantly associated with both the dorsal and ventral plasma membranes and is also concentrated at the lateral margins of cells at regions of cell-cell contacts. In mitotic cells, staining for spectrin is predominantly in the cytoplasm with only faint staining at the plasma membrane on the cell body, and no discernible staining on the membranes of the microvilli and filopodia (retraction fibers) which protrude from the cell body. Biochemical analysis of spectrin solubility in Triton X-100 extracts indicates that only 10-15% of the spectrin is soluble in interphase CHO or HeLa cells growing attached to tissue culture plastic. In contrast, 60% of the spectrin is soluble in mitotic CHO and HeLa cells isolated by mechanical "shake-off" from nocodazole-arrested synchronized cultures, which represents a four- to sixfold increase in the proportion of soluble spectrin. This increase in soluble spectrin may be partly due to cell rounding and detachment during mitosis, since the amount of soluble spectrin in CHO or HeLa interphase cells detached from the culture dish by trypsin-EDTA or by growth in spinner culture is 30-38%. Furthermore, mitotic cells isolated from synchronized spinner cultures of HeLa S3 cells have only 2.5 times as much soluble spectrin (60%) as do synchronous interphase cells from these spinner cultures (25%). The beta subunit of spectrin is phosphorylated exclusively on serine residues both in interphase and mitosis. Comparison of steady-state phosphorylation levels of spectrin in mitotic and interphase cells demonstrates that solubilization of spectrin in mitosis is correlated with a modest increase in the level of phosphorylation of the spectrin beta subunit in CHO and HeLa cells (a 40% and 70% increase, respectively). Two-dimensional phosphopeptide mapping of CHO cell spectrin indicates that this is due to mitosis-specific phosphorylation of beta-spectrin at several new sites. This is independent of cell rounding and dissociation from other cells and the substratum, since no changes in spectrin phosphorylation take place when cells are detached from culture dishes with trypsin-EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Fluctuation in radioresponse of HeLa cells during the cell cycle evaluated based on micronucleus frequency

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hiroaki Shimono ◽  
Atsushi Kaida ◽  
Hisao Homma ◽  
Hitomi Nojima ◽  
Yusuke Onozato ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Time Lapse ◽  
S Phase ◽  
G1 Phase ◽  
M Phase ◽  
G2 Phase ◽  
The Difference ◽  
Time Lapse Imaging ◽  
First Time

AbstractIn this study, we examined the fluctuation in radioresponse of HeLa cells during the cell cycle. For this purpose, we used HeLa cells expressing two types of fluorescent ubiquitination-based cell cycle indicators (Fucci), HeLa-Fucci (CA)2 and HeLa-Fucci (SA), and combined this approach with the micronucleus (MN) assay to assess radioresponse. The Fucci system distinguishes cell cycle phases based on the colour of fluorescence and cell morphology under live conditions. Time-lapse imaging allowed us to further identify sub-positions within the G1 and S phases at the time of irradiation by two independent means, and to quantitate the number of MNs by following each cell through M phase until the next G1 phase. Notably, we found that radioresponse was low in late G1 phase, but rapidly increased in early S phase. It then decreased until late S phase and increased in G2 phase. For the first time, we demonstrated the unique fluctuation of radioresponse by the MN assay during the cell cycle in HeLa cells. We discuss the difference between previous clonogenic experiments using M phase-synchronised cell populations and ours, as well as the clinical implications of the present findings.

scholarly journals Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia.

1979 ◽  
Vol 83 (2) ◽  
pp. 320-337 ◽  
Author(s):  
E Wang ◽  
R K Cross ◽  
P W Choppin
Keyword(s):  
Protein Synthesis ◽  
Cell Fusion ◽  
Structural Integrity ◽  
Immunofluorescence Microscopy ◽  
Time Lapse ◽  
Nuclear Migration ◽  
High Concentration ◽  
Nuclear Movement ◽  
Filament Bundles ◽  
10 Nm Filaments

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501-520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.

Computer-assisted analysis of time-lapse cinemicrographs of cultured cells

1978 ◽  
Vol 11 (4) ◽  
pp. 363-379 ◽  
Author(s):  
A.P. Tolmach ◽  
A.R. Mitz ◽  
S.L. Von Rump ◽  
M.L. Pepper ◽  
L.J. Tolmach
Keyword(s):  
Cultured Cells ◽  
Time Lapse ◽  
Computer Assisted ◽  
Assisted Analysis

Fibroblast Growth Factors Regulate Stem Cell Functioning In Vitro and in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1705-1705
Author(s):  
Joyce S.G Yeoh ◽  
Ronald van Os ◽  
Ellen Weersing ◽  
Bert Dontje ◽  
Edo Vellenga ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
Cultured Cells ◽  
Cell Activity ◽  
Fibroblast Growth Factors ◽  
Fibroblast Growth ◽  
Stem Cell Activity

Abstract Fibroblast Growth Factors (FGF) are a large family of signaling molecules widely involved in tissue development, maintenance and repair. Little is known about the role of FGF/FGF-receptor signaling in the regulation of adult hematopoietic stem cells (HSC). In this study, we assessed the potential of exogenously added FGF-1/2, or retrovirally overexpressed FGF-1 to preserve HSC function in vitro and in vivo. First, we demonstrate that in vitro culture of unfractionated mouse bone marrow cells, in serum-free medium, supplemented with FGF-1 or FGF-2 or FGF-1 + 2 resulted in the robust generation of long-term repopulating (LTR) HSCs. Cultures were maintained for 12 weeks and during that time in vivo competitive reconstitution assays were performed. Stem cell activity was detectable at 3, 5, and 8 weeks after initiation of culture, but lost after 12 weeks. However, whereas 3 and 5 week cultured cells provided radioprotection in non-competitive assays, animals transplanted with 8 or 12 week cultured cells succumbed due to bone marrow failure. So far, we have been unable to expand single, highly purified Lin−Sca-1+c-Kit+ using FGF-1 + 2. Consequently, we speculated that essential intermediate cell populations or signals are required for FGF-induced stem cell conservation. To test this we cultured highly purified CD45.1 Lin−Sca-1+c-Kit+ cells in a co-culture with CD45.2 unfractionated BM. Co-cultured cells were transplanted after 5 weeks in lethally irradiated recipients, and CD45.1 chimerism levels were assessed. High levels of CD45.1 chimerism confirmed that Lin−Sca-1+c-Kit+ cells require an accessory signal in addition to FGF to induced stem cell activity in vitro. We subsequently tested stem cell potential of cells cultured in FGF-1 + 2 for 5 weeks, with the addition of SCF + IL-11 + Flt3L for the last 2, 4 or 7 days. Cell numbers increased with increasing time of growth factor presence. However, only when growth factors were present for 2 days engraftment of cultured cells in a competitive repopulation assay was increased 3.5-fold. Finally, we show by immunohistochemistry that ~10% of freshly isolated Lin−Sca-1+c-Kit+ expresses high levels of FGF-1. Retroviral overexpression of FGF-1 in stem cells resulted in increased growth potential and sustained clonogenic activity in vitro. Upon transplantation of transduced stem cells, FGF-1 overexpression resulted in increased white blood cell counts 4 weeks post-transplant compared to control animals. Most notable was a marked granulocytosis in FGF-1 overexpressing recipients Our results reveal FGF as an important regulator of HSC signaling and homeostasis. Importantly, in the presence of FGF stem cells can be maintained in vitro for 2 months. These findings open novel avenues for in vitro manipulation of stem cells for future clinical therapies.

Ciclopirox Ethanolamine Is a Novel Modifier of Human Hematopoietic Stem Cell Ex Vivo Expansion

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 36-36
Author(s):  
Mehrnaz Safaee Talkhoncheh ◽  
Fredrik Ek ◽  
Aurelie Baudet ◽  
Christine Karlsson ◽  
Roger Olsson ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Cell Activity ◽  
Cell Number ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem Cell Activity ◽  
Cells Cultured

Abstract Despite extensive studies over the last decades, little is known about the mechanisms governing human hematopoietic stem cell (HSC) fate decisions. In particular, it has been challenging to define culture conditions in which HSCs can be expanded for clinical benefit. Application of small molecule screening to modulate stem cells has emerged as a useful tool for identification of new compounds with ability to expand hematopoietic stem and progenitor cells (HSPCs). Such screens have mainly relied on the expression of CD34 as predictor of stem cell activity in cultured cells. However, CD34 defines a broad repertoire of progenitor cells and does not define stem cell function. We found that the long-term repopulation potential of cultured human HSPCs is exclusively contained within a discrete cell population co-expressing CD34 and CD90, while the vast majority of progenitor cells are found in the CD34+CD90- population. Tracking the CD34+ CD90+ population is therefore a sensitive and specific tool to predict stem cell activity in cultured hematopoietic cells and provides a good basis for a screen aimed at discovering modifiers of stem cell expansion. To search broadly for novel and potential modifiers of ex vivo HSCs expansion we next developed and optimized a small molecule screen in human cord blood (CB) derived CD34+ cells. We screened >500 small molecules from 8 different annotated chemical libraries for the phenotypic expansion of CD34+ CD90+ cells following a 6-day culture in serum-free medium supplemented with stem cell factor (SCF), thrombopoietin (TPO) and fms-like tyrosine kinase 3 ligand (FL). The numbers of CD34+ CD90+ cells for each molecule, tested at two different concentrations, was compared to DMSO treated controls. Following the initial screen, several candidate hits were selected and subjected to a dose response validation experiment from which we selected four top candidate molecules. Two of these molecules were histone deacetylase (HDAC) inhibitors, which recently have been reported to facilitate expansion of CB derived HSCs. One of the top candidates, Ciclopirox ethanolamine (CE), had previously not been implicated in HSC expansion. Ciclopirox ethanolamine is known as an antifungal agent and iron chelator. It has further been shown to suppress cancer cell survival through inhibition of Wnt/beta catenin signaling. We found that CB cells cultured with CE had a 4-fold increase in CD34+90+ cell number compared to DMSO treated controls following 6 days of culture. Interestingly, the total cell count was not different, suggesting a specific increase in CD34+ CD90+ cell number rather than an overall higher proliferation rate. When plated in methylcellulose, CE cultured cells generated increased numbers of myeloid colonies. Moreover, CE treated cells gave rise to multilineage colonies (CFU-GEMM) that could not be detected from the control cultures. To further test the functional capacity of cells cultured with CE, we transplanted cultured equivalents of 30,000 CB CD34+ cells (cultured with or without CE) into sub lethally irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Human hematopoietic reconstitution in peripheral blood was determined 16 weeks later. Mice transplanted with CE cultured cells showed higher human CD45 engraftment 16 weeks post transplant compared to control cells (33.2±6.7% vs 14.6±5% p=0.04). The engrafted cells contributed to both myeloid and lymphoid lineages. This shows that Ciclopirox ethanolamine enhances the long-term engraftment capacity of ex vivo cultured HSCs and suggests that it should be considered in stem cell expansion protocols, either alone or in combination with other molecules. We are currently addressing the basis for the increased stem cell activity mediated by Ciclopirox ethanolamine using parameters for differentiation, cell cycling and apoptosis. In addition, we are comparing Ciclopirox ethanolamine with other recently defined modifiers of HSC expansion. Disclosures No relevant conflicts of interest to declare.

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