ISSN:0886-1544    

DOI:10.1002/cm.970260102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractWe compared, on lysed polymorphonuclear leukocytes (PMNs), the spatial distributions of sites that nucleate actin polymerization with the spatial distribution of endogenous F‐actin. Sites nucleating polymerization of exogenous actin were detected by incubating lysed cells with rhodamine‐labeled G‐actin under polymerizing conditions. Endogenous F‐actin was stabilized and stained by lysis of cells into fluorescein‐labeled (FITC) phalloidin. We found the distributions of rhodamine and fluorescein intensities in a given cell, resting or stimulated with chemoattractant, to be similar. Thus, after lysis the number of sites able to nucleate actin polymerization is proportional to the local F‐actin concentration.Quantitative fluorescence microscopic analysis also demonstrated that (1) if cells were stimulated with chemoattractant shortly before lysis, the total fluorescence per cell of both fluorophors went up; (2) if peptide was diluted shortly before lysis, the endogenous F‐actin in the lamellae was dramatically reduced, but nucleation sites persisted, giving a high rhodamine to fluorescein ratio; and (3) there was a small increase in the ratio of rhodamine (exogenously grown actin) to fluorescein (endogenous F‐actin) in a region near the lamellar/endoplasm border, centripetal to regions of the highest concentration of endogenous F‐actin.The rhodamine signal appeared to be due to in situ actin polymerization probably nucleated by existing free barbed ends, since (1) the rhodamine signal increased linearly with time with no detectable lag if the actin concentration was above that of the critical concentration of the barbed end; (2) the rhodamine signal was dramatically reduced if lysates were incubated with gelsolin–actin complex (which stably caps barbed ends), then washed before the rhodamine G‐actin was added; and (3) the number of nucleation sites at the time of lysis is similar to the number of the barbed ends of actin filaments determined by the kinetics of depolymerization [Cano et al., 1991].The fact that the distribution of exogenous actin polymerization paralleled the endogenous F‐actin suggests that the number of free barbed ends per F‐actin is roughly constant. If all filament ends were free, or if a constant fraction of the filaments ends were free, these data would suggest that the mean filament length is roughly constant throughout the cel

ISSN:0886-1544    

DOI:10.1002/cm.970260103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe distribution and dynamics of the membranous organelles in two cell types were investigated during cell division. Live cells (either PtK2or LLC‐PK1) labeled with the vital dye 3,3′‐dihexyloxacarbocyanine iodide [DiOC6(3)] were observed via serial optical sectioning with the laser‐scanning confocal microscope. Z‐series of labeled, dividing cells were collected every 1–2 minutes throughout mitosis, beginning at prophase and extending to the spreading of the daughter cells. Membrane distribution began to change from the onset of prophase in both cell types. When the mitotic spindle formed in prometaphase, fine tubular membranes, similar to those extending out to the edges of interphase cells aligned along the kinetochore spindle fibers. The lacy polygonal network typical of interphase cells persisted beneath the spindle, and a membrane network was also associated with the dorsal layer of the cell. As PtK2cells reached metaphse, their spindles were nearly devoid of membrane staining, whereas the spindles of LLC‐PK1cells contained many tubular and small vesicular membranous structures. X–Z series of the LLC‐PK1metaphase spindle revealed a small cone of membranes that was separated from the rest of the cytoplasm by kinetochore MTs. In both cell types, as chromosome separation proceeded, the interzone remained nearly devoid of membranes until the onset of anaphase B. At this time the elongating interzonal microtubules were closely associated with the polygonal network of endoplasmic reticulum. Cytokinesis caused a compression, and then an exclusion of organelles from the midbody. Immunofluorescence staining with anti‐tubulin antibodies suggested that spindle membranes were associated with microtubules throughout mitosis. In addition, taxol induced a dense and extensive collection of small vesicles to collect at the spindle poles of both cell types. Nocodazole treatment induced a distinct loss of organization of the membranous components of the spindles. Together these results suggest that microtubules organize the membrane distribution in mitotic cells, and that this organization may vary in different cell types depending on the quantity of microtubules within the spindle. ©

ISSN:0886-1544    

DOI:10.1002/cm.970260104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractUltraviolet radiation in the near range (UVA) causes lens opacification and disrupts the actin cytoskeleton in rabbit and gray squirrel lenses. Changes were noted using transmission electron microscopy of tangential sections and rhodaminephalloidin fluorescence microscopy of epithelial whole mounts of irradiated and unirradiated lenses, and corresponded with gross cataract formation. Irradiated lenses lacked microfilament polygonal arrays at the inner surface of the apical plasma membrane (i.e., in the cell pole next to the lens fibers) in lens epithelia of both species; a condensed actin bundle was present instead. This bundle, and scattered small actin clumps in the cytoplasm, were identified by immunogold TEM, using a specific antibody and a secondary antibody conjugated with coloidal gold. Similar techniques showed breakdown of tubulin and vimentin, but after longer intervals than for the breakdown of actin. Generalized cytologic damage was also present in epithelial cells, but not in the underlying cortical lens fibers. Damage began to occur after 4 hr of irradiation and became more severe with increased exposure. Shielded controls remained clear, had normal cytology and polygonal arrays, and no clumping of actin filaments. © 1993 Wiley‐Liss, I

ISSN:0886-1544    

DOI:10.1002/cm.970260105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe fatty acid 12(S)‐HETE may be a new second messenger capable of activating PKC. In tumor cells 12(S)‐HETE stimulates cytoskeleton‐dependent cellular responses such as adhesion and spreading. Analysis of 12(S)‐HETE effects on B16a melanoma cell cytoskeleton revealed reversible rearrangement of microtubules, microfilaments, the actin‐binding proteins, vinculin, myosin heavy (MHC) and light chains (MLC), as well as bundling of vimentin intermediate filaments. The alterations in microfilaments and intermediate filaments occurred very rapidly, i.e., 5 min after exposure of tumor cells to 12(S)‐HETE. The 12(S)‐HETE‐induced cytoskeletal alterations were accompanied by centrifugal organelle‐translocation. Interestingly, MLC exhibited clear association with the cytoplasmic organelles. Biochemical analysis of the 12(S)‐HETE effect indicated a PKC‐mediated reversible hyperphosphorylation of MLC, vimentin, and a 130 kD cytoskeletal‐associated protein. Optimal effects were obtained after 5 min treatment with 12(S)‐HETE at 0.1 μM concentration. 12(S)‐HETE pretreatment induced tumor cell spreading on a fibronectin matrix which required the intactness of all three major cytoskeletal components. The spreading process was dependent upon the activity of PKC. Our data suggest that 12(S)‐HETE is a physiological stimulant of PKC. Further, it induces rearrangement of the cytoskeleton of tumor cells in interphase resulting in the stimulation of cytoskeleton‐dependent cell activity such as s

ISSN:0886-1544    

DOI:10.1002/cm.970260106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractBovine sperm, extracted with 0.1% Triton X‐100, frozen at −20°C for 48–120 hours, and thawed, disintegrated by microtubule sliding when 1 mM MgATP was added. Microtubules and outer dense fibers (ODFs) were usually extruded in groups or “bundles.” A total of 44.5% of the cells extruded two distinct bundles, one from each side of the connecting piece, exhibiting opposite curvatures. Only one bundle was observed in 46.2% of the cells, and 9.2% showed no signs of sliding. Transmission electron microscopy (T.E.M.) showed one group consisting of the 4,5‐6,7 elements, with the 9,1,2 elements on the other side of the axoneme making up the other bundle. T.E.M. revealed that when only one side of the axoneme had extruded elements, they werealwaysfrom the 4,5‐6,7 group. The remainder of the axoneme (8,9,1,2,3 and the central pair) was left relatively intact, suggesting a difference in the sliding response of the nine pairs of axonemal microtubules. These results indicate a predisposition for sliding between elements 7 and 8 over that between doublets 2 and 3, perhaps due to a disparity in activation thresholds. Also, both Ni2+and Cd2+appear to selectively block activation of 2–3 interdoublet sliding.Incubation with 0.25 mM Ni2+prior to adding MgATP modified the percentages of sliding patterns: 8.6% demonstrated two‐sided extrusion, 58.2% showed one‐sided, and 33.2% had no extruded bundles. Again, when half the axoneme was missing, it wasalwaysthe 4,5‐6,7 group. Ten micromolar Cd2+altered the sliding pattern similarly to Ni2+, with 28% two‐sided extrusion, 55.9% one‐sided extrusion and 16.1% with no extruded bundles.Either pretreatment regimen impeded extrusion of the 9,1,2 group in a high percentage of cells, compared to untreated cells. This specific inhibition of the 9,1,2 side by Ni2+or Cd2+is especially significant since Ni2+also inhibits spontaneous wave initiation in bull sperm (Lindemann et al.: Journal of Cell Biology 87:420–426, 1980), and both Ni2+and Cd2+reportedly block the flagellar Ca2+‐response in rat sperm (Lindemann and Goltz: Cell Motility and the Cytoskeleton 10:420–431, 1988; Lindemann et al.: Cell Motility and the Cytoskeleton 20:316–

ISSN:0886-1544    

DOI:10.1002/cm.970260107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractKeratins are a subgroup of cytoskeletal intermediate filament proteins found in most epithelial cells. Some reports have suggested that keratins may be found on the cell surface as well as their well‐accepted cytoskeletal location. A major part of the evidence in the interpretation of cell surface expression of keratins is cell surface radioiodination. Here we show that lactoperoxidase‐catalyzed iodination of colonic and breast tissue culture cells results in radiolabeling of the keratins when cells are manipulated. No labeling of keratins is detected when cells are labeled directly on the tissue culture dish. A similar result was obtained when intact cells were biotinylated using water‐soluble sulfo–NHS–biotin. Partitioning of the keratins to a soluble and an insoluble pool after “cell surface”125I‐labeling showed that both pools became iodinated. Indirect immunofluorescence showed that binding of a panel of anti‐keratin antibodies to intact epithelial cells occurs only on the cells that are more adherent, which are the cells that require longer manipulation to remove from the tissue culture dish. Taken together, our results indicate that the reported expression of cell surface keratins in some cells likely reflects intracellular keratins. In addition, the method of epithelial cell handling can dramatically alter the leakiness of cell surface iodination techniques. © 19

ISSN:0886-1544    

DOI:10.1002/cm.970260108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe importance of cell adhesion in a variety of physiological phenomena requires development of an understanding of the factors and molecular mechanisms underlying these behaviors. Cell adhesion is a multistep process involving primary receptor‐ligand interactions followed by secondary events that may lead to the formation of focal contacts. Due to the lack of well‐defined assays to study adhesion stabilization, little is known about this process, except that it may involve signaling events, receptor recruitment, and, as we have demonstrated, covalent peptide cross‐linking by cell membrane‐associated transglutaminase [Menter et al.: Cell Biophys. 18:123–143, 1992]. To study the stabilization process we have developed a dynamic assay employing a parallel plate flow chamber coupled with video microscopy and digital image processing. Our studies utilize wheat germ agglutinin‐selected human metastatic melanoma cell variants that exhibit differences in their experimental metastatic potential and expression of transglutaminase. Using this assay, quantifying cell‐substrate stabilization was found to be quick, reliable, reproducible, and useful in evaluating agents that block this process. © 1993 W

ISSN:0886-1544    

DOI:10.1002/cm.970260109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970260101

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY