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J W Sanger

Publications and source records attributed to J W Sanger.

At least 55 records · Page 3Linked to original sources
PubMed1992

Costameres are sites of force transmission to the substratum in adult rat cardiomyocytes.

Costameres, the vinculin-rich, sub-membranous transverse ribs found in many skeletal and cardiac muscle cells (Pardo, J. V., J. D. Siciliano, and S. W. Craig. 1983. Proc. Natl. Acad. Sci. USA. 80:363-367.) are thought to anchor the Z-lines of the myofibrils to the sarcolemma. In addition, it has been postulated that costameres provide mechanical linkage between the cells' internal contractile machinery and the extracellular matrix, but direct evidence for this supposition has been lacking. By combining the flexible silicone rubber substratum technique (Harris, A. K., P. Wild, and D. Stopak. 1980. Science (Wash. DC). 208:177-179.) with the microinjection of fluorescently labeled vinculin and alpha-actinin, we have been able to correlate the distribution of costameres in adult rat cardiac myocytes with the pattern of forces these cells exert on the flexible substratum. In addition, we used interference reflection microscopy to identify areas of the cells which are in close contact to the underlying substratum. Our results indicate that, in older cell cultures, costameres can transmit forces to the extracellular environment. We base this conclusion on the following observations: (a) adult rat heart cells, cultured on the silicone rubber substratum for 8 or more days, produce pleat-like wrinkles during contraction, which diminish or disappear during relaxation; (b) the pleat-like wrinkles form between adjacent alpha-actinin-positive Z-lines; (c) the presence of pleat-like wrinkles is always associated with a periodic, "costameric" distribution of vinculin in the areas where the pleats form; and (d) a banded or periodic pattern of dark gray or close contacts (as determined by interference reflection microscopy) has been observed in many cells which have been in culture for eight or more days, and these close contacts contain vinculin. A surprising finding is that vinculin can be found in a costameric pattern in cells which are contracting, but not producing pleat-like wrinkles in the substratum. This suggests that additional proteins or posttranslational modifications of known costamere proteins are necessary to form a continuous linkage between the myofibrils and the extracellular matrix. These results confirm the hypothesis that costameres mechanically link the myofibrils to the extracellular matrix. We put forth the hypothesis that costameres are composite structures, made up of many protein components; some of these components function primarily to anchor myofibrils to the sarcolemma, while others form transmembrane linkages to the extracellular matrix.

Actins↗
PubMed1992

Host cell actin assembly is necessary and likely to provide the propulsive force for intracellular movement of Listeria monocytogenes.

Listeria monocytogenes is able to escape from the phagolysosome and grow within the host cell cytoplasm. By 3 h after initiation of infection, actin filaments begin to concentrate at one end of the bacterium. Polarization of F-actin is associated with intracellular bacterial movement, long projections of actin filaments forming directly behind the moving bacteria. New actin monomers are added to the region of the projection in proximity to the bacterium. The rate of new actin filament growth correlates closely with the speed of bacterial migration. This actin structure is anchored within the cytoplasm, serving as a fixed platform for directional expansion of the actin filament network. The actin projection progressively lengthens as the bacterium migrates. Cytochalasin blocks both elongation of the projection and bacterial movement but does not result in complete depolymerization of the bacterially induced actin structure, residual actin and alpha-actinin persisting in proximity to one end of the bacterium. Bacteria initially migrate within the cortical cytoplasm but later move to the peripheral membrane, where they form filopodiumlike structures which pivot and undulate in the extracellular medium. In the filopodia, bacteria are occasionally seen to abruptly change direction, turn 180 degrees, and move back into the medullary region of the host cell. All filopodium movement ceases once the bacterium containing the F-actin projection returns to the cortical cytoplasm. These results indicate that host cell actin polymerization is necessary for intracellular migration of listeriae and suggest that directional actin assembly may in fact generate the propulsive force for bacterial and filopodial movement.

Actins↗
PubMed1990

Disruption of microfilament organization in living nonmuscle cells by microinjection of plasma vitamin D-binding protein or DNase I.

Plasma vitamin D-binding protein (DBP), which binds to monomeric actin, causes the breakdown of stress fibers when it is microinjected into nonmuscle cells. Disruption of the stress fiber network is also accompanied by shape changes in the cell that resemble those seen after cytochalasin treatment. When DBP was coinjected with fluorescently labeled alpha-actinin, no fluorescent stress fibers or attachment plaques were visible 30 min after injection. Twelve hours later the cells regained their flattened shape and their stress fibers. Fluorescently labeled DBP causes the same reversible changes in cell shape as the unlabeled protein. Upon injection, the labeled DBP diffuses throughout the cytoplasm, becoming localized by 12 hr in a punctate pattern, presumably due to lysozomal sequestration. Similar injections of DBP into skeletal myotubes and cardiac myocytes did not lead to shape changes or breakdown of nascent and/or fully formed myofibrils, even though DBP has a 2-fold higher binding affinity for muscle actin over that of the nonmuscle isoactins. Similar differential effects in nonmuscle cells were also observed after the microinjection of DNase I, another protein capable of binding monomer actin. The effects of these microinjected monomer actin-binding proteins imply that an accessible pool of monomer actin is needed to maintain stress fiber integrity in nonmuscle cells but not the integrity of the nascent or fully formed myofibrils in muscle cells.

Actin Cytoskeleton↗
PubMed1990

Angiopathic consequences of saturating the plasma scavenger system for actin.

Two plasma proteins, vitamin D-binding protein (actin monomer sequestrant) and gelsolin (actin polymer severing), have been found in association with actin in plasma from ill humans and during experimental injury. In vitro, these are the only plasma proteins that display a high affinity for actin. We infused increasing amounts of globular actin intravenously to rats to evaluate its disposition in plasma and tissues. Intravascular filament formation, microthrombi, and endothelial injury were observed, especially in the pulmonary circulation. These pathological changes were not observed when the globular actin in the infusate had been preincubated with the vitamin D-binding protein in vitro. Complexes of actin with both proteins were found in the plasma, suggesting a saturable, plasma actin-binding system in vivo. Our findings suggest that in vivo saturation of these proteins' actin-binding capacities may serve as a paradigm for pulmonary vascular disorders seen during widespread tissue trauma and cell lysis.

Actins↗
PubMed1990

Differential effects of myosin-antibody complexes on contractile rings and circumferential belts in epitheloid cells.

The role of myosin filaments during assembly and activity of microfilament rings was analyzed by microinjecting epitheloid cells (PtK2 and LLC-PK1 kidney cell lines) with specific anti-myosins. Six monoclonal antibodies directed against the light meromyosin (LMM) region of the myosin molecule were characterized with respect to epitope location, and their effects on actin-activated MgATPase as well as on assembly, structural integrity and stability of myosin filaments. All of these antibodies recognized LLC-PK1 myosin, but only three reacted with PtK2 myosin. The remaining three served as matching controls in experiments with this cell line. When injected in amounts sufficient to yield an excess of antibody over myosin, the reactive antibodies significantly delayed formation and constriction of the contractile ring in mitotic cells. These rings contained less myosin, but not less actin, than the controls. This indicates that the recruitment and alignment of actin in the cleavage furrow can occur independently of other components of the contractile ring. After completion of cytokinesis, the majority of the injected cells was unable to assemble a normal circumferential belt. This resulted in defective epitheloid sheets. Approximately one third of these cells showed grossly distorted cell shapes and an increase in locomotory activity. All these changes were fully reversible with time, suggesting that the effects of the antibodies were overcome by protein synthesis. The differential sensitivity seen between contractile rings and peripheral belts is discussed with respect to differences in their architecture, stability and proposed function.

Actin Cytoskeleton↗
PubMed1989

Aberrant postendocytotic fate of a 34-kDa molecular mass growth factor from human trophoblasts.

A 34-kDa growth factor expressed by trophoblasts and certain carcinomas binds to target fibroblastic cells through specific high-affinity receptors. Here we report studies on the cellular routing behavior of the receptor-bound 34-kDa protein. Internalization was visualized by using lissamine rhodamine-conjugated 34-kDa protein and was quantified by analyzing the acid dissociability of cell-bound radioiodinated protein after incubation at 37 degrees C. The protein was found to be rapidly internalized in a temperature-sensitive manner. However, in contrast with other protein ligands, the 34-kDa protein was not rapidly degraded. The extent of ligand degradation was small as quantified by gel filtration analysis. Studies on the receptor showed that there was an atypical up-regulation, i.e., increase in surface receptors in response to ligand binding at 37 degrees C. The up-regulation was partially blocked by cycloheximide, an inhibitor of protein biosynthesis, but not by known inhibitors of receptor recycling such as monensin, chloroquine, and methylamine, suggesting that enhanced receptor biosynthesis may be responsible for the process. These studies indicate that the cellular routing and receptor regulatory characteristics of the internalized 34-kDa growth factor are different from those of most growth factor ligands and imply the involvement of receptor up-regulation in signal transduction.

Cells, Cultured↗
PubMed1989

Visualization of intermediate filaments in living cells using fluorescently labeled desmin.

Fluorescently labeled desmin was incorporated into intermediate filaments when microinjected into living tissue culture cells. The desmin, purified from chicken gizzard smooth muscle and labeled with the fluorescent dye iodoacetamido rhodamine, was capable of forming a network of 10-nm filaments in solution. The labeled protein associated specifically with the native vimentin filaments in permeabilized, unfixed interphase and mitotic PtK2 cells. The labeled desmin was microinjected into living, cultured embryonic skeletal myotubes, where it became incorporated in straight fibers aligned along the long axis of the myotubes. Upon exposure to nocodazole, microinjected myotubes exhibited wavy, fluorescent filament bundles around the muscle nuclei. In PtK2 cells, an epithelial cell line, injected desmin formed a filamentous network, which colocalized with the native vimentin intermediate filaments but not with the cytokeratin networks and microtubular arrays. Exposure of the injected cells to nocadazole or acrylamide caused the desmin network to collapse and form a perinuclear cap that was indistinguishable from vimentin caps in the same cells. During mitosis, labeled desmin filaments were excluded from the spindle area, forming a cage around it. The filaments were partitioned into two groups either during anaphase or at the completion of cytokinesis. In the former case, the perispindle desmin filaments appeared to be stretched into two parts by the elongating spindle. In the latter case, a continuous bundle of filaments extended along the length of the spindle and appeared to be pinched in two by the contracting cleavage furrow. In these cells, desmin filaments were present in the midbody where they gradually were removed as the desmin filament network became redistributed throughout the cytoplasm of the spreading daughter cells.

Animals↗
PubMed1989

Dynamics of the endoplasmic reticulum in living non-muscle and muscle cells.

The dynamic changes of the endoplasmic reticulum (ER) in interphase and mitotic cells was detected by the vital fluorescent dye 3,3'-dihexyloxacarbocyanine iodide. Two types of arrays characterize the continuous ER system in the non-muscle PtK2 cell: 1) a lacy network of irregular polygons and 2) long strands of ER that are found aligned along stress fibers. In cross-striated myotubes there was a periodic localization of fluorescence over each I-band corresponding to the positions of the terminal cisternae of the sarcoplasmic reticulum (SR). In contrast to the arrangement in muscle cells, the alignment of the long strands of ER alon stress fibers showed no strict periodicity that could be correlated with the sarcomeric units of the stress fibers. The ER and SR arrays seen in living cells were also detected in fixed cells stained with antibodies directed against proteins of the endoplasmic reticulum and sarcoplasmic reticulum, respectively. Observations of vitally stained PtK2 cells at 1 to 2 minute intervals using low light level video cameras and image processing techniques enabled us to see the polygonal ER units form and undergo changes in their shapes. During cell division, the ER, rhodamine 123-stained mitochondria, and phagocytosed fluorescent beads were excluded from the mitotic spindle while soluble proteins were not. No obvious concentration or alignment of membranes could be found associated with the contractile proteins in the cleavage furrow. After completion of cell division there was a redeployment of the ER network in each daughter cell.

Animals↗
PubMed1989

Analysis of cell division using fluorescently labeled actin and myosin in living PtK2 cells.

Actin and the light chains of myosin were labeled with fluorescent dyes and injected into interphase PtK2 cells in order to study the changes in distribution of actin and myosin that occurred when the injected cells subsequently entered mitosis and divided. The first changes occurred when stress fibers in prophase cells began to disassemble. During this process, which began in the center of the cell, individual fibers shortened, and in a few fibers, adjacent bands of fluorescent myosin could be seen to move closer together. In most cells, stress fiber disassembly was complete by metaphase, resulting in a diffuse distribution of the fluorescent proteins throughout the cytoplasm with the greatest concentration present in the mitotic spindle. The first evidence of actin and myosin concentration in a cleavage ring occurred at late anaphase, just before furrowing could be detected. Initially, the intensity of fluorescence and the width of the fluorescent ring increased as the ring constricted. In cells with asymmetrically positioned mitotic spindles, both protein concentration and furrowing were first evident in the cortical regions closest to the equator of the mitotic spindle. As cytokinesis progressed in such asymmetrically dividing cells, fluorescent actin and myosin appeared at the opposite side of the cell just before furrowing activity could be seen there. At the end of cytokinesis, myosin and actin were concentrated beneath the membrane of the midbody and subsequently became organized in two rings at either end of the midbody.

Actins↗
PubMed1989

Talin dynamics in living microinjected nonmuscle cells.

To investigate the role of talin in the anchoring of actin-containing stress fibers to the cell membrane of nonmuscle cells, a fluorescent analog of the adhesion plaque protein talin was developed, characterized, and microinjected into living cells. Purified chicken gizzard talin was covalently labeled with the fluorescent dye lissamine rhodamine B sulfonyl chloride. The fluorescently labeled protein was then chromatographed on Sephadex G-25 and DEAE-cellulose in order to remove free dye and denatured protein. The fluorescent talin was able to bind purified vinculin and was localized in adhesion plaques, membrane ruffles, microspikes, and polygonal networks in acetone-permeabilized nonmuscle cells. In cells that were double-stained with fluorescent talin and an affinity-purified anti-talin antibody, a one-to-one correspondence of adhesion plaque staining was seen. Living epithelial cells (PtK2) were microinjected during interphase with fluorescent talin. Computer-enhanced video microscopy was used to document adhesion plaque dynamics such as 1) changes in plaque shape, 2) alterations in plaque positions, and 3) the appearance, growth, and dissolution of plaques. In cells that were followed during mitosis, the adhesion plaques disappeared during cell rounding and then subsequently reappeared upon spreading of the two daughter cells. Treatment of microinjected cells with DMSO in order to disassemble stress fibers resulted in an altered localization of the fluorescent talin. Upon recovery of the cell from the drug, the talin was visualized in its characteristic submembraneous position. These results are the first to document the role and distribution of talin in dynamic processes occurring in living microinjected nonmuscle cells.

Animals↗
PubMed1988

Somitogenesis in the mouse embryo.

This report describes the initiation of somitogenesis in the mouse embryo. Correlations are made with fibronectin distribution around the unsegmented mesoderm and the distribution of cytoskeletal elements within the cells as they undergo morphogenetic movements. The same temporal and topological changes in fibronectin, laminin, and cytoskeletal elements are seen in mouse somitogenesis as in the chick embryo. A notable exception is that the epithelial stage of somitogenesis in the mouse does not form a closed vesicle as it does in the chick. In the mouse the mesial portion of the forming somite does not become epithelial before the migration of sclerotomal cells.

Actins↗
PubMed1988

Incorporation of fluorescently labeled actin and tropomyosin into muscle cells.

The two major proteins in the I-bands of skeletal muscle, actin and tropomyosin, were each labeled with fluorescent dyes and microinjected into cultured cardiac myocytes and skeletal muscle myotubes. Actin was incorporated along the entire length of the I-band in both types of muscle cells. In the myotubes, the incorporation was uniform, whereas in cardiac myocytes twice as much actin was incorporated in the Z-bands as in any other area of the I-band. Labeled tropomyosin that had been prepared from skeletal or smooth muscle was incorporated in a doublet in the I-band with an absence of incorporation in the Z-band. Tropomyosin prepared from brain was incorporated in a similar pattern in the I-bands of cardiac myocytes but was not incorporated in myotubes. These results in living muscle cells contrast with the patterns obtained when labeled actin and tropomyosin are added to isolated myofibrils. Labeled tropomyosins do not bind to any region of the isolated myofibrils, and labeled actin binds to A-bands. Thus, only living skeletal and cardiac muscle cells incorporate exogenous actin and tropomyosin in patterns expected from their known myofibrillar localization. These experiments demonstrate that in contrast to the isolated myofibrils, myofibrils in living cells are dynamic structures that are able to exchange actin and tropomyosin molecules for corresponding labeled molecules. The known overlap of actin filaments in cardiac Z-bands but not in skeletal muscle Z-bands accounts for the different patterns of actin incorporation in these cells. The ability of cardiac myocytes and non-muscle cells but not skeletal myotubes to incorporate brain tropomyosin may reflect differences in the relative actin-binding affinities of non-muscle tropomyosin and the respective native tropomyosins. The implications of these results for myofibrillogenesis are presented.

Actins↗
PubMed1987

Stress fiber and cleavage furrow formation in living cells microinjected with fluorescently labeled alpha-actinin.

alpha-Actinins, isolated from muscle and nonmuscle sources and labeled with various fluorescent dyes, were microinjected into living PtK2 cells during interphase to observe the reformation of stress fibers following cell division. Fluorescently labeled ovalbumin and bovine serum albumin were also injected as control proteins. alpha-Actinin was incorporated into stress fibers within 5 minutes after injection and remained present in the fibers for up to 11 days. The pattern of incorporation was the same regardless of whether the alpha-actinin was isolated from muscle or nonmuscle tissues or whether it was labeled with fluorescein, Lucifer Yellow, or rhodamine dyes. In contrast, neither labeled ovalbumin nor bovine serum albumin were incorporated into stress fibers. When the injected cells entered prophase, all stress fibers disassembled, resulting in a distribution of the fluorescent alpha-actinin throughout the cytoplasm. During cytokinesis, the fluorescent alpha-actinin was concentrated in the broad area between the separated chromosomes and along the edge of the cell in the cleavage area. Within 10 minutes after the completion of cleavage, the first fluorescent stress fibers reformed parallel to the spreading edges of the daughter cells and in close association with the midbody with a concomitant loss of alpha-actinin in the former cleavage furrow. Additional fibers formed adjacent to these first stress fibers. In some cases, new stress fibers formed between two existing stress fibers and some stress fibers moved up to 4 micron apart from one another in the course of 2 hours. Thus, fluorescent alpha-actinin, injected into living cells, undergoes the same cyclical changes in distribution as endogenous alpha-actinin during the cell cycle: from stress fibers to cleavage furrow and back to stress fibers.

Actinin↗
PubMed1987

Stress fiber reformation after ATP depletion.

Fluorescently labeled heavy meromyosin, alpha-actinin, and vinculin were used to localize actin, alpha-actinin, and vinculin, respectively, in permeabilized and living cells during the process of stress fiber reassembly, which occurred when cells were removed from ATP-depleting medium (20 mM sodium azide and 10 mM 2-deoxyglucose). In 80% of the cells recovering from ATP depletion, small, scattered plaques containing actin, alpha-actinin, and vinculin were replaced by long, thin, periodic fibers within 5 minutes of removal of the inhibitors. These nascent stress fibers grew broader as recovery progressed, until they attained the thickness of stress fibers in control cells. In the other 20% of the cells, the scattered plaques aggregated within 5 minutes of reversal, and almost all the actin, alpha-actinin, and vinculin in the cells became localized in one perinuclear aggregate, with a diameter of approximately 15-25 micron. As recovery progressed, all aggregates resembled rings, with diameters that increased at about 0.5 micron/minute and grew to as large as 70 micron in some giant cells. As the size of the rings increased, fibers radiated outward from them and sometimes spanned the diameter of the rings. The shape of the cells did not change during this time. By 1 hour after reversal, the rings were no longer present and all cells had networks of stress fibers. Indirect immunofluorescence techniques used to localize tubulin and vimentin indicated that microtubules and intermediate filaments were not constituents of the rings, and the rings were not closely apposed to the substrate, judging from reflection contrast optics. The rapid rearrangement of attachment plaques into a perinuclear aggregate that spreads radially in the cytoplasm occurs at the same speed as fibroblast and chromosomal movement, but is unlike other types of intracytoplasmic motility.

Actinin↗
PubMed1987

Binding and distribution of fluorescently labeled filamin in permeabilized and living cells.

This study reports the first development of a fluorescently labeled filamin. Smooth muscle filamin was labeled with fluorescent dyes in order to study its interaction with stress fibers and myofibrils, both in living cells and in permeabilized cells. The labeled filamin bound to the Z bands of isolated cross-striated myofibrils and to the Z bands and intercalated discs in both permeabilized embryonic cardiac myocytes and in frozen sections of adult rat ventricle. In permeabilized embryonic chick myotubes, filamin bound to early myotubes but was absent at later stages. In living embryonic chick myotubes, the fluorescently labeled filamin was incorporated into the Z bands of myofibrils during early and late stages of development but was absent during an intermediate stage. In living cardiac myocytes, filamin-IAR was incorporated into nascent as well as fully formed sarcomeres throughout development. In permeabilized nonmuscle cells, labeled filamin bound to attachment plaques and foci of polygonal networks and to the dense bodies in stress fibers. The periodic bands of filamin in stress fibers had a longer spacing in fibroblasts than in epithelial cells. When injected into living cells, filamin was readily incorporated into stress fibers in a striated pattern. The fluorescent filamin bands were broader in injected cells, however, than they were in permeabilized cells. We have interpreted these results from living and permeabilized cells to mean that native filamin is distributed along the full length of the actin filaments in the stress fibers, with a higher concentration present in the dense bodies. A sarcomeric model is presented indicating the position of filamin with respect to other proteins in the stress fiber.

Actinin↗
PubMed1987

Visualization of myosin in living cells.

Myosin light chains labeled with rhodamine are incorporated into myosin-containing structures when microinjected into live muscle and nonmuscle cells. A mixture of myosin light chains was prepared from chicken skeletal muscle, labeled with the fluorescent dye iodoacetamido rhodamine, and separated into individual labeled light chains, LC-1, LC-2, and LC-3. In isolated rabbit and insect myofibrils, the fluorescent light chains bound in a doublet pattern in the A bands with no binding in the cross-bridge-free region in the center of the A bands. When injected into living embryonic chick myotubes and cardiac myocytes, the fluorescent light chains were also incorporated along the complete length of the A band with the exception of the pseudo-H zone. In young myotubes (3-4 d old), myosin was localized in aperiodic as well as periodic fibers. The doublet A band pattern first appeared in 5-d-old myotubes, which also exhibited the first signs of contractility. In 6-d and older myotubes, A bands became increasingly more aligned, their edges sharper, and the separation between them (I bands) wider. In nonmuscle cells, the microinjected fluorescent light chains were incorporated in a striated pattern in stress fibers and were absent from foci and attachment plaques. When the stress fibers of live injected cells were disrupted with DMSO, fluorescently labeled myosin light chains were present in the cytoplasm but did not enter the nucleus. Removal of the DMSO led to the reformation of banded, fluorescent stress fibers within 45 min. In dividing cells, myosin light chains were concentrated in the cleavage furrow and became reincorporated in stress fibers after cytokinesis. Thus, injected nonmuscle cells can disassemble and reassemble contractile fibers using hybrid myosin molecules that contain muscle light chains and nonmuscle heavy chains. Our experiments demonstrate that fluorescently labeled myosin light chains from muscle can be readily incorporated into muscle and nonmuscle myosins and then used to follow the dynamics of myosin distribution in living cells.

Actinin↗
PubMed1987

Differential response of stress fibers and myofibrils to gelsolin.

The actin-severing activity of human platelet gelsolin was analyzed on embryonic skeletal and cardiac myofibrils, and on stress fibers in non-muscle cells. These subcellular structures, although in all three cell types composed of contractile proteins arranged in sarcomeric units, were found to respond differently to gelsolin. The myofibrils in permeabilized myotubes or cardiac cells, as well as in living, microinjected muscle cells proved resistant to a wide concentration range of gelsolin. The same was found for the "mini-sarcomeres" which are seen in developing muscle cells. In contrast, stress fibers in microinjected fibroblasts or epithelial cells, as well as in permeabilized cells, were broken down rapidly by the platelet gelsolin. We conclude from these results that the mini-sarcomeres in embryonic myotubes and cardiac myocytes are not identical with stress fibers.

Actin Cytoskeleton↗