scholarly journals Stress fibers in cells in situ: immunofluorescence visualization with antiactin, antimyosin, and anti-alpha-actinin.

1982 ◽  
Vol 93 (3) ◽  
pp. 804-811 ◽  
Author(s):  
H R Byers ◽  
K Fujiwara
Keyword(s):  
Cultured Cells ◽  
Cellular Adhesion ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Goldfish Carassius Auratus ◽  
Culture Cells ◽  
Central Regions ◽  
In Cells

Stress fiber-like patterns are visualized by indirect immunofluorescence in scleroblasts (fibroblasts) in situ on the scale of the common goldfish, Carassius auratus, using an affinity-purified antiactin, antimyosin, and anti-alpha-actinin. These fibers demonstrate the classical convergent and parallel patterns exhibited by stress fibers in tissue culture cells. Because the dimensions, the composition, and the pattern of distribution of these cytoplasmic fibers correspond well with those of stress fibers in cultured cells, we will call these fibers stress fibers also. The staining patterns with anti-alpha-actinin and antimyosin along the stress fibers often reveal a periodicity of 1-2 microM, identical to that found in cells in vitro. The majority of scleroblasts do not exhibit stress fiber staining and they are specifically located in the central regions of the scale. Stress fibers are present in scleroblasts residing on or near the edges or radical ridges of the scale. They are consistently orientated perpendicular to these structures; however, unlike microtubules, stress fibers show no co-alignment with collagen fibers of the scale. The finding that stress fibers are located in regions of the scale more subject to shearing forces may indicate their role in increased cellular adhesion to the substratum.

Organization and Function of Stress Fibers in Cells in Vitro and in Situ

1984 ◽  
pp. 83-137 ◽  
Author(s):  
H. Randolph Byers ◽  
Glenn E. White ◽  
Keigi Fujiwara
Keyword(s):  
Stress Fibers ◽  
And Function ◽  
In Cells

Physico-Chemical Studies of Isolated Chromatin Compared with in situ Chromatin after Partial Hepatectomy in the Rat

1979 ◽  
Vol 34 (5-6) ◽  
pp. 442-448 ◽  
Author(s):  
Paul Miller ◽  
Walfried A. Linden ◽  
Claudio Nicolini
Keyword(s):  
Partial Hepatectomy ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Cultured Cells ◽  
Fluorescence Emission ◽  
Chromatin Organization ◽  
Chromatin Conformation ◽  
Functional Changes

Abstract Chromatin was isolated from rat liver cells at 0, 3, 5, 11, 18 and 24 h following partial hepatectomy. Consistent with findings in cultured cells stimulated to proliferate, there was an increase in chromatin molar ellipticity measured at 276 nm, and a decrease in thermal stability 3 to 8 h after surgery. These events occured prior to the onset of DNA synthesis. These early changes be­ tween non-proliferating (G0) and proliferating (G1) cells, as well as later chromatin conformational changes observed at S and G2 phases, mimic changes in template activity.Results with sheared and unsheared chromatin (both with in vitro and in vivo systems) prove that structural and functional changes can be caused by even the slightest shearing during chromatin preparation, suggesting the loss of native chromatin organization. To eliminate this problem, experiments were also conducted using chromatin in situ. A flow cytometer (FCM) was used to study unfixed liver cell suspensions stained with ethidium bromide (EB). Fluorescence was mea­ sured in the green spectral range after addition of increasing amounts of EB. Experimental evidence is provided that the same alteration in chromatin conformation can be best detected using low molar ratios of EB per unit DNA due to greater fluorescence emission in G1 respect to G0 cells.These correlated studies demonstrate that the same changes controlling chromatin organization in situ are detected also in the tertiary-quaternary structure of “isolated” chromatin. These changes in chromatin conformation are macromolecular events related to cell proliferation both at the G0 -G1 and G1 -S transitions.

scholarly journals Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes.

1989 ◽  
Vol 108 (6) ◽  
pp. 2355-2367 ◽  
Author(s):  
Z X Lin ◽  
S Holtzer ◽  
T Schultheiss ◽  
J Murray ◽  
T Masaki ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Stress Fiber ◽  
Protein Isoforms ◽  
Stress Fibers ◽  
Intercalated Discs ◽  
Adhesion Plaques ◽  
Alpha Actin ◽  
Muscle Myosin

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown.

scholarly journals Gene expression analysis by non-radioactive RNA-RNA in situ hybridization techniques

2004 ◽  
Vol 23 (2) ◽  
pp. 127-133 ◽  
Author(s):  
Danijela Drakulic ◽  
Milena Stevanovic ◽  
Gordana Nikcevic
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Cultured Cells ◽  
Specific Gene ◽  
Rna In Situ Hybridization ◽  
Sox Gene ◽  
Labeled Rna ◽  
Set Up

RNA-RNA in situ hybridization is a reliable method for studying tissue and cell specific gene expression, which enables visualization of labeled antisense RNA probe hybridized to specific mRNA. In this study we employed non-radioactive RNA-RNA in situ hybridization using biotin- or digoxigenin-labeled RNA probes in order to detect SOX gene expression in carcinoma cell lines. By this approach we confirmed results obtained by Northern blot analysis, where the presence of SOX2 mRNA in NT2/D1 and SOX14 mRNA in HepG2 cells has been established. Our aim was to set up RNA-RNA in situ hybridization method in in vitro cultured cells in order to perform further analyses of SOX gene expression on various normal and cancer tissues.

Macromolecular specializations that mediate lateral interactions between microfilaments and the membrane at focal contacts

1992 ◽  
Vol 50 (1) ◽  
pp. 724-725
Author(s):  
Steven J. Samuelsson ◽  
Paul W. Luther ◽  
David W. Pumplin ◽  
Robert J. Bloch
Keyword(s):  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Focal Contact ◽  
Lateral Interactions ◽  
Matrix Assembly ◽  
Cytoplasmic Surface ◽  
Focal Contacts ◽  
Specific Proteins

Focal contacts are membrane specializations of cultured cells where stress fibers terminate and where the cell is most closely applied to the substrate. The organization of this cytoskeletal-membrane-extracellular matrix assembly has been well characterized. Immunofluorescence microscopy has shown that two focal contact-specific proteins, vinculin and talin, colocalize with microfilaments for several microns before the stress fiber terminates. This result raises the question of whether microfilament-membrane interactions are limited to the ends of microfilaments, or if lateral interactions also occur. We addressed this question by examining the cytoplasmic surface of isolated focal contacts in detail.

scholarly journals Tetramethylpyrazine blocks TFAM degradation and up-regulates mitochondrial DNA copy number by interacting with TFAM

2017 ◽  
Vol 37 (3) ◽  
Author(s):  
Linhua Lan ◽  
Miaomiao Guo ◽  
Yong Ai ◽  
Fuhong Chen ◽  
Ya Zhang ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Lon Protease ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Dna Copy Number ◽  
Antitumour Agent ◽  
Small Molecule Compound ◽  
Mtdna Replication ◽  
Mitochondrial Transcription Factor A ◽  
In Cells

The natural small molecule compound: 2,3,5,6-tetramethylpyrazine (TMP), is a major component of the Chinese medicine Chuanxiong, which has wide clinical applications in dilating blood vessels, inhibiting platelet aggregation and treating thrombosis. Recent work suggests that TMP is also an antitumour agent. Despite its chemotherapeutic potential, the mechanism(s) underlying TMP action are unknown. Herein, we demonstrate that TMP binds to mitochondrial transcription factor A (TFAM) and blocks its degradation by the mitochondrial Lon protease. TFAM is a key regulator of mtDNA replication, transcription and transmission. Our previous work showed that when TFAM is not bound to DNA, it is rapidly degraded by the ATP-dependent Lon protease, which is essential for mitochondrial proteostasis. In cultured cells, TMP specifically blocks Lon-mediated degradation of TFAM, leading to TFAM accumulation and subsequent up-regulation of mtDNA content in cells with substantially low levels of mtDNA. In vitro protease assays show that TMP does not directly inhibit mitochondrial Lon, rather interacts with TFAM and blocks degradation. Pull-down assays show that biotinylated TMP interacts with TFAM. These findings suggest a novel mechanism whereby TMP stabilizes TFAM and confers resistance to Lon-mediated degradation, thereby promoting mtDNA up-regulation in cells with low mtDNA content.

Augmentation by epidermal growth factor of basal and stimulated progesterone production by human luteinized granulosa cells

1989 ◽  
Vol 121 (2) ◽  
pp. 397-402 ◽  
Author(s):  
M. C. Richardson ◽  
S. C. Gadd ◽  
G. M. Masson
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cells ◽  
Cultured Cells ◽  
Vitro Fertilization ◽  
Human Granulosa Cells ◽  
Culture Cells ◽  
Oocyte Collection ◽  
Epidermal Growth

ABSTRACT Human granulosa cells were prepared from follicular aspirates obtained during oocyte collection for in-vitro fertilization. Following several days in culture, cells were washed and then progesterone output was measured in 2-h incubations. After culture for 3 days, incubated cells responded well to human chorionic gonadotrophin (hCG) and prostaglandin (PG) E2 with similar levels of maximum response. Exposure of cultured cells to epidermal growth factor (EGF) for 2 days (days 3–5) led to substantial increases both in basal production and in responses to hCG and PGE2 during subsequent incubations. These effects of EGF were not accompanied by measurable increases in DNA levels in cultures over this time. Results may point to a possible paracrine role for EGF-like factors modulating the activity of cells forming the early corpus luteum. Journal of Endocrinology (1989) 121, 397–402

scholarly journals Tetracycline-regulated expression enables purification and functional analysis of recombinant connexin channels from mammalian cells

2004 ◽  
Vol 383 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Irina V. KOREEN ◽  
Wafaa A. ELSAYED ◽  
Yu J. LIU ◽  
Andrew L. HARRIS
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Expression System ◽  
Physiological Processes ◽  
Messenger Molecules ◽  
One Step ◽  
Cellular Control

Intercellular coupling mediated by gap junction channels composed of connexin protein underlies numerous physiological processes, such as cellular differentiation, tissue synchronization and metabolic homoeostasis. The distinct molecular permeability of junctional channels composed of different connexin isoforms allows cellular control of coupling via regulation of isoform expression. However, the permeability properties of most connexin isoforms have not been well characterized due to the difficulty of manipulating and measuring the diffusible concentrations of cytoplasmic messenger molecules and metabolites, and to a lack of control over channel isoform composition, in vivo. Here we present a method to express and purify active connexin hemichannels of a single isoform or a consistent ratio of two isoforms from cultured cells using the Tet-On inducible expression system and one-step anti-haemagglutinin immunoaffinity purification. The procedure yields 10–20 μg of pure connexin protein from 2.5×108 HeLa cells. The purified channels are shown to be useful for in vitro permeability analysis using well established techniques. This method has substantial advantages over existing methods for heterologous connexin expression, such as the ease of co-expression of two isoforms at a constant ratio, consistently high expression levels over many passages, and the ability to study channel properties in situ as well as in purified form. Furthermore, the generic cloning site of the new pBI-GT vector and the commercial availability of anti-haemagglutinin (clone HA-7)–agarose make this affinity tagging and purification procedure easily applicable to other proteins.

scholarly journals Microvascular pericytes contain muscle and nonmuscle actins.

1985 ◽  
Vol 101 (1) ◽  
pp. 43-52 ◽  
Author(s):  
I M Herman ◽  
P A D'Amore
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Stress Fibers ◽  
Fiber Bundles

We have affinity-fractionated rabbit antiactin immunoglobulins (IgG) into classes that bind preferentially to either muscle or nonmuscle actins. The pools of muscle- and nonmuscle-specific actin antibodies were used in conjunction with fluorescence microscopy to characterize the actin in vascular pericytes, endothelial cells (EC), and smooth muscle cells (SMC) in vitro and in situ. Nonmuscle-specific antiactin IgG stained the stress fibers of cultured EC and pericytes but did not stain the stress fibers of cultured SMC, although the cortical cytoplasm associated with the plasma membrane of SMC did react with nonmuscle-specific antiactin. Whereas the muscle-specific antiactin IgG failed to stain EC stress fibers and only faintly stained their cortical cytoplasm, these antibodies reacted strongly with the fiber bundles of cultured SMC and pericytes. Similar results were obtained in situ. The muscle-specific antiactin reacted strongly with the vascular SMC of arteries and arterioles as well as with the perivascular cells (pericytes) associated with capillaries and post-capillary venules. The non-muscle-specific antiactin stained the endothelium and the pericytes but did not react with SMC. These findings indicate that pericytes in culture and in situ possess both muscle and nonmuscle isoactins and support the hypothesis that the pericyte may represent the capillary and venular correlate of the SMC.

scholarly journals Interaction of BAG1 and Hsp70 Mediates Neuroprotectivity and Increases Chaperone Activity

2005 ◽  
Vol 25 (9) ◽  
pp. 3715-3725 ◽  
Author(s):  
Jan Liman ◽  
Sundar Ganesan ◽  
Christoph P. Dohm ◽  
Stan Krajewski ◽  
John C. Reed ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Heat Shock Protein 70 ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Full Length ◽  
Wild Type ◽  
Binding Characteristics ◽  
In Cells

ABSTRACT It was recently shown that Bcl-2-associated athanogene 1 (BAG1) is a potent neuroprotectant as well as a marker of neuronal differentiation. Since there appears to exist an equilibrium within the cell between BAG1 binding to heat shock protein 70 (Hsp70) and BAG1 binding to Raf-1 kinase, we hypothesized that changing BAG1 binding characteristics might significantly alter BAG1 function. To this end, we compared rat CSM14.1 cells and human SHSY-5Y cells stably overexpressing full-length BAG1 or a deletion mutant (BAGΔC) no longer capable of binding to Hsp70. Using a novel yellow fluorescent protein-based foldase biosensor, we demonstrated an upregulation of chaperone in situ activity in cells overexpressing full-length BAG1 but not in cells overexpressing BAGΔC compared to wild-type cells. Interestingly, in contrast to the nuclear and cytosolic localizations of full-length BAG1, BAGΔC was expressed exclusively in the cytosol. Furthermore, cells expressing BAGΔC were no longer protected against cell death. However, they still showed accelerated neuronal differentiation. Together, these results suggest that BAG1-induced activation of Hsp70 is important for neuroprotectivity, while BAG1-dependent modulation of neuronal differentiation in vitro is not.

Sign in / Sign up

Export Citation Format

Share Document