scholarly journals A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. II. Effects on cell movement, organization of microtubules, and intermediate filaments, and arrangement of Golgi elements.

1983 ◽  
Vol 97 (5) ◽  
pp. 1476-1490 ◽  
Author(s):  
J Wehland ◽  
M C Willingham
Keyword(s):  
Monoclonal Antibody ◽  
Intermediate Filaments ◽  
Golgi Complex ◽  
Cold Treatment ◽  
Cell Movement ◽  
Living Cells ◽  
Alpha Tubulin ◽  
Cell Biol ◽  
Saltatory Movement ◽  
High Concentrations

A rat monoclonal antibody against yeast alpha-tubulin (clone YL 1/2; Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) that reacts specifically with the tyrosylated form of alpha-tubulin and readily binds to tubulin in microtubules when injected into cultured cells (see Wehland, J., M. C. Willingham, and I. V. Sandoval, 1983, J. Cell Biol., 97:1467-1475) was used to study microtubule organization and function in living cells. Depending on the concentration of YL 1/2 that was injected the following striking effects were observed: (a) When injected at a low concentration (2 mg IgG/ml in the injection solution), where microtubules were decorated without changing their distribution, intracellular movement of cell organelles (saltatory movement) and cell translocation were not affected. Intermediate concentrations (6 mg IgG/ml) that induced bundling but no perinuclear aggregation of microtubules abolished saltatory movement and cell translocation, and high concentrations (greater than 12 mg IgG/ml) that induced perinuclear aggregation of microtubules showed the same effect. (b) YL 1/2, when injected at intermediate and high concentrations, arrested cells in mitosis. Such cells showed no normal spindle structures. (c) Injection of an intermediate concentration of YL 1/2 that stopped saltatory movement caused little or no aggregation of intermediate filaments and no dispersion of the Golgi complex. After injection of high concentrations, resulting in perinuclear aggregation of microtubules, intermediate filaments formed perinuclear bundles and the Golgi complex became dispersed analogous to results obtained after treatment of cells with colcemid. (d) When rhodamine-conjugated YL 1/2 was injected at concentrations that stopped saltatory movement and arrested cells in mitosis, microtubule structures could be visualized and followed for several hours in living cells by video image intensification microscopy. They showed little or no change in distribution and organization during observation, even though these microtubule structures appeared not to be stabilized by injected YL 1/2 since they were readily depolymerized by colcemid or cold treatment and repolymerized upon drug removal or rewarming to 37 degrees C, respectively. These results are discussed in terms of the participation of microtubules in cellular activities such as cell movement and cytoplasmic organization and in terms of the specificity of YL 1/2 for the tyrosylated form of alpha-tubulin.

Morphogenetic machines revealed: Microscopy of living cells in the embryo of the frog, Xenopus laevis

1993 ◽  
Vol 51 ◽  
pp. 172-173
Author(s):  
Ray Keller
Keyword(s):  
Image Processing ◽  
Fluorescent Dyes ◽  
Cell Movement ◽  
Living Cells ◽  
Ease Of Use ◽  
Low Light ◽  
Amphibian Embryo ◽  
Large Populations ◽  
Light Fluorescence ◽  
Video Image Processing

The amphibian embryo offers advantages of size, availability, and ease of use with both microsurgical and molecular methods in the analysis of fundamental developmental and cell biological problems. However, conventional wisdom holds that the opacity of this embryo limits the use of methods in optical microscopy to resolve the cell motility underlying the major shape-generating processes in early development.These difficulties have been circumvented by refining and adapting several methods. First, methods of explanting and culturing tissues were developed that expose the deep, nonepithelial cells, as well as the superficial epithelial cells, to the view of the microscope. Second, low angle epi-illumination with video image processing and recording was used to follow patterns of cell movement in large populations of cells. Lastly, cells were labeled with vital, fluorescent dyes, and their behavior recorded, using low-light, fluorescence microscopy and image processing. Using these methods, the details of the cellular protrusive activity that drives the powerful convergence (narrowing)

HVEM Analysis of Microfilament Patterns in The Margins of Tissue Cells

1980 ◽  
Vol 38 ◽  
pp. 808-811
Author(s):  
Carol Allen
Keyword(s):  
Cell Movement ◽  
Cell Spreading ◽  
Living Cells ◽  
Tissue Cell ◽  
Large Sample Size ◽  
Cell Periphery ◽  
Whole Cell ◽  
Critical Point Drying ◽  
Lamellar Region ◽  
Tissue Cells

When provided with a suitable solid substrate, tissue cells undergo a rapid conversion from the spherical form expressed in suspension culture to a characteristic flattened morphology. As a result of this conversion, called cell spreading, the cell nucleus and organelles come to occupy a central region of “deep cytoplasm” which slopes steeply into a peripheral “lamellar” region less than 1 pm thick at its outer edge and generally free of cell organelles. Cell spreading is accomplished by a continuous outward repositioning of the lamellar margins. Cell translocation on the substrate results when the activity of the lamellae on one side of the cell become dominant. When this occurs, the cell is “polarized” and moves in the direction of the “leading lamellae”. Careful analysis of tissue cell locomotion by time-lapse microphotography (1) has shown that the deformational movements of the leading lamellae occur in a repeating cycle of advance and retreat in the direction of cell movement and that the rate of such deformations are positively correlated with the speed of cell movement. In the present study, the physical basis for these movements of the cell margin has been examined by comparative light microscopy of living cells with whole-mount electron microscopy of fixed cells. Ultrastructural observations were made on tissue cells grown on Formvar-coated grids, fixed with glutaraldehyde, further processed by critical-point drying, and then photographed in the High Voltage Electron Microscope. This processing and imaging system maintains the 3-dimensional organization of the whole cell, the relationship of the cell to the substrate, and affords a large sample size which facilitates quantitative analysis. Comparative analysis of film records of living cells with the whole-cell micrographs revealed that specific patterns of microfilament organization consistently accompany recognizable stages of lamellar formation and movement. The margins of spreading cells and the leading lamellae of locomoting cells showed a similar pattern of MF repositionings (Figs. 1-4). These results will be discussed in terms of a working model for the mechanics of lamellar motility which includes the following major features: (a) lamellar protrusion results when an intracellular force is exerted at a locally weak area of the cell periphery; (b) the association of cortical MFs with one another determines the local resistance to this force; (c) where MF-to-MF association is weak, the cell periphery expands and some cortical MFs are dragged passively forward; (d) contact of the expanded area with the substrate then triggers the lateral association and reorientation of these cortical MFs into MF bundles parallel to the direction of the expansion; and (e) an active interaction between these MF bundles associated with the cortex of the expanded lamellae and the cortical MFs which remained in the sub-lamellar region then pulls the latter MFs forward toward the expanded area. Thus, the advance of the cell periphery on the substrate occurs in two stages: a passive phase in which some cortical MFs are dragged outward by the force acting to expand the cell periphery, and an active phase in which additional cortical MFs are pulled forward by interaction with the first set. Subsequent interactions between peripheral microfilament bundles and filaments in the deeper cytoplasm could then transmit the advance gained by lamellar expansion to the bulk of the cytoplasm.

Visualization of intermediate filaments in living cells using fluorescently labeled desmin

1989 ◽  
Vol 12 (3) ◽  
pp. 127-138 ◽  
Author(s):  
B. Mittal ◽  
J. M. Sanger ◽  
J. W. Sanger
Keyword(s):  
Intermediate Filaments ◽  
Living Cells

A centrosomal antigen localized on intermediate filaments and mitotic spindle poles

1990 ◽  
Vol 97 (2) ◽  
pp. 259-271
Author(s):  
B. Buendia ◽  
C. Antony ◽  
F. Verde ◽  
M. Bornens ◽  
E. Karsenti
Keyword(s):  
Monoclonal Antibody ◽  
Intermediate Filaments ◽  
Mitotic Spindle ◽  
Mdck Cells ◽  
Large Fraction ◽  
Human Lymphocytes ◽  
Spindle Poles ◽  
Pericentriolar Material ◽  
Xenopus Egg

A monoclonal antibody (CTR2611) raised against centrosomes isolated from human lymphocytes (KE37) stains the pericentriolar material and intermediate filaments in the same cells. In MDCK cells, where most of the microtubules do not originate from the pericentriolar region during interphase, the antigen is distributed along intermediate filaments. At the onset of mitosis, a large fraction of the CTR2611 antigen associates with the minus-end domain of the microtubules of the mitotic spindle but not with the pericentriolar region itself. Treatment of mitotic MDCK cells with taxol leads to the assembly of many microtubule asters in the cytoplasm at the expense of the mitotic spindle. The CTR2611 antigen is present in the center of each of these asters. Similar asters can also be produced in vitro by adding taxol to concentrated Xenopus egg mitotic cytoplasm. Again, the antigen is found close to the center of the asters. These results suggest that CTR2611 antigen is associated with a material involved in microtubule nucleation or microtubule minus-end stabilization. The monoclonal antibody recognizes a 74 × 10(3) Mr polypeptide and other polypeptides at 120 × 10(3) Mr and 170 × 10(3) Mr. The 74 × 10(3) Mr polypeptide is found in all species examined so far, suggesting that it contains a highly conserved epitope.

Phenotypic consequences of tubulin overproduction in Saccharomyces cerevisiae: differences between alpha-tubulin and beta-tubulin

1990 ◽  
Vol 10 (10) ◽  
pp. 5295-5304
Author(s):  
B Weinstein ◽  
F Solomon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Microtubule Assembly ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Data Support ◽  
Cell Biol

Overexpression of alpha- and beta-tubulin genes in Saccharomyces cerevisiae, separately or together, leads to accumulation of large excesses of each of the polypeptides and arrest of cell division. However, other consequences of overexpression of these genes differ in several ways. As shown previously (D. Burke, P. Gasdaska, and L. Hartwell, Mol. Cell. Biol. 9:1049-1059, 1989), overexpression of beta-tubulin leads, at early times, to loss of microtubule structures and loss of viability. Eventually, the excess beta-tubulin forms abnormal structures. We show here that, in contrast, overexpression of alpha-tubulin led to none of these phenotypes and in fact could suppress each of the phenotypes associated with beta-tubulin accumulation. Truncated forms of beta-tubulin that were not competent to carry out microtubule functions also failed to elicit the beta-tubulin-specific phenotypes when overexpressed. The data support the hypothesis that beta-tubulin in excess over alpha-tubulin is uniquely toxic, perhaps because it interferes with normal microtubule assembly.

Tau confers drug stability but not cold stability to microtubules in living cells

1994 ◽  
Vol 107 (1) ◽  
pp. 135-143 ◽  
Author(s):  
P.W. Baas ◽  
T.P. Pienkowski ◽  
K.A. Cimbalnik ◽  
K. Toyama ◽  
S. Bakalis ◽  
...  
Keyword(s):  
Sympathetic Neurons ◽  
Drug Stability ◽  
Living Cells ◽  
Microtubule Depolymerization ◽  
Cell Biol ◽  
Cold Stability ◽  
The Stability ◽  
Cultured Sympathetic Neurons ◽  
Very High

We previously defined two classes of microtubule polymer in the axons of cultured sympathetic neurons that differ in their sensitivity to nocodazole by roughly 35-fold (Baas and Black (1990) J. Cell Biol. 111, 495–509). Here we demonstrate that virtually all of the microtubule polymer in these axons, including the drug-labile polymer, is stable to cold. What factors account for the unique stability properties of axonal microtubules? In the present study, we have focused on the role of tau, a microtubule-associated protein that is highly enriched in the axon, in determining the stability of microtubules to nocodazole and/or cold in living cells. We used a baculovirus vector to express very high levels of tau in insect ovarian Sf9 cells. The cells respond by extending processes that contain dense bundles of microtubules (Knops et al. (1991) J. Cell Biol. 114, 725–734). Cells induced to express tau were treated with either cold or 2 micrograms/ml nocodazole for times ranging from 5 minutes to 6 hours. The results with each treatment were very different from one another. Virtually all of the polymer was depolymerized within the first 30 minutes in cold, while little or no microtubule depolymerization was detected even after 6 hours in nocodazole. Based on these results, we conclude that tau is almost certainly a factor in conferring drug stability to axonal microtubules, but that factors other than or in addition to tau are required to confer cold stability.

The membrane skeleton of acetylcholine receptor domains in rat myotubes contains antiparallel homodimers of beta-spectrin in filaments quantitatively resembling those of erythrocytes

1995 ◽  
Vol 108 (9) ◽  
pp. 3145-3154 ◽  
Author(s):  
D.W. Pumplin
Keyword(s):  
Monoclonal Antibody ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Cytoplasmic Membrane ◽  
Membrane Surface ◽  
Membrane Skeleton ◽  
Immunogold Labeling ◽  
Platinum Coating ◽  
High Concentrations ◽  
Receptor Clusters

I used immunogold labeling and quick-freeze, deep-etch, rotary replication to characterize the membrane skeleton at regions with high concentrations of acetylcholine receptor domains in receptor clusters of cultured rat muscle cells. This membrane skeleton consists of a network of filaments closely applied to the cytoplasmic membrane surface. The filaments are specifically decorated by immunogold labeling with a monoclonal antibody, VIIF7, that recognizes an isoform of beta-spectrin colocalizing with acetylcholine receptors. The filaments are 32 +/- 11 nm in length and three to four filaments (average 3.1-3.3) join at each intersection to form the network. These parameters are nearly identical to those reported previously for the membrane skeleton of erythrocytes. Depending on the amount of platinum coating, filament diameters range from 9 to 11 nm in diameter, and are 1.4 nm larger on average than spectrin filaments of erythrocytes replicated at the same time. Filaments are decorated with gold particles close to one end, consistent with the location of the epitope recognized by the monoclonal antibody. Computer modeling shows that all filament intersections in the membrane skeletal network are equally capable of being labeled by the monoclonal antibody. This pattern of labeling is consistent with a network containing antiparallel homodimers of beta-spectrin.

scholarly journals Thrombospondin stimulates motility of human neutrophils.

1990 ◽  
Vol 111 (6) ◽  
pp. 3077-3086 ◽  
Author(s):  
P J Mansfield ◽  
L A Boxer ◽  
S J Suchard
Keyword(s):  
Monoclonal Antibody ◽  
Cell Types ◽  
Human Neutrophils ◽  
Heparin Binding ◽  
Directed Movement ◽  
Surface Receptors ◽  
Chemotactic Peptides ◽  
Heparin Binding Domain ◽  
High Concentrations ◽  
Priming Activity

Polymorphonuclear leukocytes (PMNs) migrate to sites of inflammation or injury in response to chemoattractants released at those sites. The presence of extracellular matrix (ECM) proteins at these sites may influence PMN accumulation at blood vessel walls and enhance their ability to move through tissue. Thrombospondin (TSP), a 450-kD ECM protein whose major proteolytic fragments are a COOH-terminal 140-kD fragment and an NH2-terminal heparin-binding domain (HBD), is secreted by platelets, endothelial cells, and smooth muscle cells. TSP binds specifically to PMN surface receptors and has been shown, in other cell types, to promote directed movement. TSP in solution at low concentrations (30-50 nM) "primed" PMNs for f-Met-Leu-Phe (fMLP)-mediated chemotaxis, increasing the response two- to fourfold. A monoclonal antibody against the HBD of TSP totally abolished this priming effect suggesting that the priming activity resides in the HBD of TSP. Purified HBD retains the priming activity of TSP thereby corroborating the antibody data. TSP alone, in solution at high concentrations (0.5-3.0 microM), stimulated chemotaxis of PMNs and required both the HBD and the 140-kD fragment of TSP. In contrast to TSP in solution, TSP bound to nitrocellulose filters in the range of 20-70 pmol stimulated random locomotion of PMNs. The number of PMNs migrating in response to bound TSP was approximately two orders of magnitude greater than the number of cells that exhibited chemotaxis in response to soluble TSP or fMLP. Monoclonal antibody C6.7, which recognizes an epitope near the carboxyl terminus of TSP, blocked migration stimulated by bound TSP, suggesting that the activity resides in this domain. Using proteolytic fragments, we demonstrated that bound 140-kD fragment, but not HBD, promoted migration of PMNs. Therefore, TSP released at injury sites, alone or in synergy with chemotactic peptides like fMLP, could play a role in directing PMN movement.

scholarly journals Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers

2020 ◽  
Vol 1867 (8) ◽  
pp. 118726 ◽  
Author(s):  
Mariano Smoler ◽  
Giovanna Coceano ◽  
Ilaria Testa ◽  
Luciana Bruno ◽  
Valeria Levi
Keyword(s):  
Intermediate Filaments ◽  
Living Cells ◽  
Apparent Stiffness
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