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R M Warn

Publications and source records attributed to R M Warn.

At least 37 records · Page 2Linked to original sources
PubMed1991

Microtubules rich in post-translationally modified alpha-tubulin form distinct arrays in frog lens epithelial cells.

Isolated frog lens epithelia were stained with antibodies against tyrosinated, detyrosinated or acetylated alpha-tubulin and observed by several means including a scanning confocal microscope. The most prominent feature of Rana pipiens lens cells was a primary cilium close to the apical surface of the cells above the centrosome. This structure was associated with microtubules rich in modified alpha-tubulin. The cilium was less pronounced but still discernible in the cells of another species R. ridibunda. In both species, the modified (acetylated or detyrosinated) microtubules formed arrays spatially distinct from the unmodified (tyrosinated) microtubules. The modified microtubules formed a basket of microtubules with a curly distribution around the nucleus while the tyrosinated array consisted predominantly of rather straighter microtubules running from the apical centrosome to the cell periphery, down the lateral sides of the cells and across the basal surface adjacent to the lens capsule and basement membrane. It is concluded that the organization of modified microtubules previously described for several types of cultured cells may represent a remnant of the three-dimensional perinuclear array of such microtubules described here for the cells of an intact epithelium.

Animals↗
PubMed1991

Diamide induces reversible changes in morphology, cytoskeleton and cell-cell coupling in lens epithelial cells.

The isolated frog lens epithelium can be maintained with its cell shape, cytoskeletal organization and membrane electrophysiological characteristics intact for more than 24 hr. Perifusion with the permeant oxidant diamide (1 mM) led to drastic, but reversible, changes in all the above parameters. After a 20 min exposure to diamide, the regular polygonal arrangement of the epithelial cells become increasingly disrupted as the cells reorganized and a 'rosette' pattern formed. The cells at the edges of the rosette pulled apart from one another while those in the centre maintained a relatively normal appearance. Blebs formed on the apical surface of all of the cells on prolonged exposure and the internal structure was also found to be severely disrupted. The cytoplasm became granular, vacuolated and the nucleus had a banded, non-homogeneous appearance. Phalloidin staining of F-actin microfilaments revealed that there was a general disruption of organization, with actin losing its association with the membrane. The microtubule array, organized around the centrosome, was also severely disrupted although microtubules were still discernible in most cells. During exposure to diamide the membrane potential depolarized and both electrical and dye coupling, which are normally extremely efficient in these cells, were disturbed. If the epithelium was exposed to 1 mM diamide for more than 45 min then all of the above changes were irreversible and cell death followed. If exposure was restricted to less than 30 min, then all of the above changes occurred and, in fact, progressed for over 1 hr; but if the epithelium was perifused for a further 20 hr in control medium, then most of the changes were reversible.

Actin Cytoskeleton↗
PubMed1991

Scatter factor affects major changes in the cytoskeletal organization of epithelial cells.

The effects of scatter factor on the cytoskeleton of MDCK and PtK2 cells are described. During the first 6 h after the addition of scatter factor, MDCK cells were found to increase their projected areas twofold, as well as the number and size of their F-actin stress fibers. In contrast PtK2 cells showed no change in their projected areas or in their stress fiber content. However, when both MDCK and PtK2 cells began to separate and scatter after approximately 6 h, the size and number of stress fibers was found to decrease considerably. Unscattered PtK2 cells and cells treated with scatter factor which had yet to scatter showed focal contacts present over the whole ventral surface, as judged by staining for both vinculin and talin. After treated cells separated, both vinculin and talin staining were mainly present in focal contacts on the ventral surfaces of the cell bodies and the distal ends of the processes. However, the cell processes showed few focal contacts along their lengths. The distribution of microtubules and vimentin and keratin intermediate filaments also did not change significantly until scattering had occurred. After cell separation, the processes were always packed with microtubules which were often, but not always, rich in detyrosinated alpha-tubulin and often, but not always, packed with intermediate filaments. All these changes in cytoskeletal organization are consistent with the adoption of a much more motile phenotype. The changes found are compared with those brought about by transformation.

Actins↗
PubMed1990

Distribution of microtubules containing post-translationally modified alpha-tubulin during Drosophila embryogenesis.

The distribution of microtubules (MTs) enriched in detyrosinated alpha-tubulin (Glu-tubulin) was studied in Drosophila embryos by immunofluorescence microscopy by using a monoclonal antibody (ID5) which was raised against a 14-residue synthetic peptide spanning the carboxyterminal sequence of Glu-tubulin (Wehland and Weber: J. Cell Sci. 88:185-203, 1987). While all MT arrays contained tyrosinated alpha-tubulin (Tyr-tubulin), MTs rich in Glu-tubulin were not found during early stages of development even by using an image intensification camera. Elevated levels of microtubular Glu-tubulin were first detected after CNS condensation in neurone processes. In addition, sperm tails, which remained remarkably stable inside the embryo until late stages of development, were decorated by ID5. This was in marked contrast to the distribution of microtubule arrays containing acetylated alpha-tubulin, which could already be detected during the cellular blastoderm stage. Additional experiments with taxol suggested that the absence of MTs rich in Glu-tubulin during early stages of development was not due to the rapid turnover rate of MTs, which would be too fast for alpha-tubulin to be detyrosinated. The possible significance of the differential detyrosination and acetylation of microtubules during development is discussed.

Acetylation↗
PubMed1990

F-actin organization during the cellularization of the Drosophila embryo as revealed with a confocal laser scanning microscope.

The changes in F-actin organization during the cellularization of the Drosophila embryo have been studied with a confocal laser scanning microscope using fluorescein-phalloidin as a specific stain. Particular study has been made of the changes in the organization of the F-actin network associated with the leading edges of the growing membranes. The role of this actin network in the cellularization process is considered. Other actin-containing structures have also been examined, including the cortical actin layer and a conspicuous region of F-actin aggregates, present beneath the level of the forming cell membranes.

Actins↗
PubMed1989

Incorporation of tubulin from an evolutionarily diverse source, Physarum polycephalum, into the microtubules of a mammalian cell.

Physarum myxamoebal tubulin was injected into PtK2 cells to determine whether tubulin from this eukaryotic microbe could act as a reporter for microtubule growth and dynamics in a mammalian cell. The distribution of Physarum tubulin was determined by the use of a monoclonal antibody specific for Physarum tubulin and unable to detect mammalian tubulin. Physarum tubulin was incorporated into the microtubules of both interphase arrays and the mitotic spindle. Measurements of microtubule turnover kinetics were found to be similar to those of other studies in which chemically modified brain tubulin has been used. Results using this heterologous system demonstrate that tubulin from an evolutionarily diverse organism can be used as a marker for microtubule growth in mammalian cells. Furthermore, the Physarum tubulin was able to endow the injected cells with novel properties. Resistance to colchicine-induced microtubule disassembly, a characteristic of Physarum tubulin, was conferred on the injected PtK2 cells. Use of this heterologous reporter tubulin system has also revealed features of variation in microtubule dynamics both within individual cells and between cells.

Animals↗
PubMed1989

Microtubules rich in modified alpha-tubulin characterize the tail processes of motile fibroblasts.

The organisation of microtubules rich in post-translationally modified alpha-tubulin has been investigated in a fibroblast cell line (NIH-3T3-T15) that can be reversibly transformed. An immunofluorescence microscopy study of the static non-transformed cells has revealed a central distribution of wavy microtubules showing post-translational modifications. When transformed there is a marked increase in cell motility and the appearance of long thin cytoplasmic 'tails'. These tails have been found to contain conspicuous bundles of post-translationally modified microtubules that run down the length of the processes and terminate close to the plasmalemma. Both detyrosinated and acetylated alpha-tubulin are present as major species in these modified microtubules. Such a pattern of modified microtubules is only occasionally seen in the untransformed NIH-3T3-T15 cells. We have also found them to be present in other transformed fibroblast lines. The presence of bundles of microtubules rich in modified alpha-tubulin in the cell tails is correlated with a marked reduction in the numbers of F-actin stress fibres. The possible role of these modified stable microtubules in cell motility is discussed.

Cell Line↗
PubMed1988

Membrane and junctional properties of the isolated frog lens epithelium.

The isolated frog lens epithelium can be maintained intact in both appearance and electrical properties for more than 24 hours. The mean resting membrane potential was -80 mV and the cells were depolarized by both high potassium and low calcium Ringer's solution in a manner very similar to that of the whole lens. The epithelial cells were found to be well coupled using both electrical and dye-injection techniques. Electrical coupling was measured using separate current-injection and voltage-measuring electrodes and the relationship between the induced voltage and distance from the current-passing electrode could be well fitted by a Bessel Function solution to the cable equation. The values obtained from the fit for the membrane and internal resistances were 1.95 omega m2 and 25 omega m, respectively. Exposure to octanol (500 microM) or low external Ca2+ (less than 1 microM) failed to disrupt significantly the intercellular flow of current. There was evidence to suggest that raised intracellular calcium does, however, uncouple the cells. Dye coupling was investigated by microinjecting Lucifer Yellow CH into single epithelial cells. Diffusion into surrounding cells was rapid and, in control medium, occurred in a radially symmetrical manner. In contrast to the electrical coupling data, dye transfer appeared to be blocked by exposure to 500 microM octanol and was severely restricted on perfusing with low external calcium. Differences between the electrical and dye-coupling experiments indicate either that there are two types of junction within the cell and only the larger type, permeable to Lucifer Yellow, is capable of being uncoupled or that there is only one large type of junction which can be partially closed by uncoupling agents.

Animals↗
PubMed1988

Effects of elevated intracellular magnesium on cytoskeletal integrity.

Increasing the intracellular magnesium concentration of PtK2 cells by 1 mM or more resulted in the disassembly of the interphase microtubule array over a period of 5 min after microinjection. This effect was found to be both transient and fully reversible, with the microtubule arrays reforming after further incubation. These effects were studied using immunofluorescence microscopy of fixed cells, and also in living cells using rhodamine-tubulin or rhodamine-conjugated anti-tubulin antibodies and image intensification and enhancement techniques. Simultaneously and accompanying the disassembly of the microtubule arrays the F-actin stress fibres also disappeared, usually leaving the peripheral and perinuclear F-actin microfilaments intact. In contrast, increasing intracellular magnesium appeared to have no effect on the vimentin-containing intermediate filaments of PtK2 cells. These effects on the cytoskeleton were specific to magnesium and could not be mimicked by either microinjection of injection buffer of equivalent ionic strength or sham injection. Raising the intracellular free calcium to the same extent resulted in the disassembly of the microtubule network, but appeared to have no effect on the F-actin stress fibres.

Actin Cytoskeleton↗
PubMed1987

An investigation of microtubule organization and functions in living Drosophila embryos by injection of a fluorescently labeled antibody against tyrosinated alpha-tubulin.

Rhodamine-labeled monoclonal antibodies, which react with tyrosinated alpha-tubulin (clone YL 1/2; Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) and label microtubules in vivo (Wehland, J., M. C. Willingham, and I. Sandoval, 1983, J. Cell Biol., 97:1467-1475) were microinjected into syncytial stage Drosophila embryos. At 1 mg/ml antibody concentration, the microtubule arrays of the surface caps became labeled by YL 1/2 but normal development was found to continue. The results are compared with the data from fixed material particularly with regard to interphase microtubules, centrosome separation, and spindle and midbody formation. At 5 mg/ml antibody concentration the microtubules took up larger quantities of antibodies and clumped around the nuclei. Nuclei with clumped microtubules lost their position in the surface layer and moved into the interior. As a result, the F-actin cap meshwork associated with such nuclei either failed to form or subsided. It is concluded that microtubule activity is required to maintain the nuclei in the surface layer and organize the F-actin meshwork of the caps.

Actin Cytoskeleton↗
PubMed1986

Microtubule arrays present during the syncytial and cellular blastoderm stages of the early Drosophila embryo.

The organization of microtubules within the surface caps of Drosophila embryos is described for the mitotic cycles of the syncytial blastoderm stage (particularly cycle 10), and for the subsequent cellularization process. Tubulin was labelled with the well characterized monoclonal antibody YL 1/2 (Kilmartin et al., J cell biol 93 (1982) 576). Each surface cap was found to contain an array of microtubules running around the nucleus. The microtubules originated at prominent centrosomes located close to the apical surface of each cap nucleus. During mitosis the spindle microtubules stained strongly for tubulin. A novel finding was that the spindle microtubules of the interzone region appeared to reduce their connections with the centrosomes at the end of anaphase. The spindle remnant remained in position during telophase but then became smaller in size, disappearing by interphase. At this phase of the cell cycle duplication of the aster centrosomes occurred. The cellular blastoderm stage was marked by a change in the main axis of microtubule orientation. The centrosomes of each cap separated somewhat and formed initiation centres for the development of a well developed basket of microtubules around each nucleus, but now perpendicular to the surface. The microtubule baskets were seen to extend in parallel with nuclear elongation, but not in concert with growth of the cell membranes, which extended some way beneath the bases of the nuclei.

Anaphase↗
PubMed1986

The cytoskeleton of the early Drosophila embryo.

The organization and roles of the cytoskeleton are described for a complex developing system (the early Drosophila embryo) at a time when the basic embryonic plan is mapped out. This type of embryo shows a separation of mitosis from cytokinesis during the early stages of development. Most cells are only formed when a syncytium of approximately 6000 nuclei are present. The functions of the cytoskeleton are considered for the process of nuclear migration (pre-blastoderm), which distributes the nuclei throughout the embryo and brings most of them close to the surface. They are also described for the subsequent mitoses of the syncytial blastoderm where the cortex and its well-developed cytoskeleton is reorganized into cell-like surface protrusions known as 'caps' or 'buds'. A comparison is made of the very different cytoskeletal organization present during the cleavages that form the two cell types of early development (pole cell and blastoderm cell), together with information from mutations that affect various aspects of these cleavages via factors laid down during oogenesis.

Animals↗
PubMed1985

F-actin rings are associated with the ring canals of the Drosophila egg chamber.

Staining of Drosophila egg chambers with rhodaminyl-lysine-phallotoxin (RLP), a specific stain for F-actin, has demonstrated the presence of dense F-actin rings associated with the inner surfaces of the ring canals. They were first observed in the distal part of the germarium where rings of four different size classes were found, differing in diameter by up to twofold. The ring sizes are considered to correspond to the ring canals formed at each of four successive incomplete cleavages. During the growth of the egg chamber the actin rings were found to increase in diameter from less than 1 micron to approx. 10 micron. Concomitantly a secondary outer ring of more diffuse material is built up in association with the cell membranes. A well developed array of microfilament bundles was also associated with the nurse cell plasmalemma. In stages where the transfer of the bulk of the nurse cell cytoplasm into the oocyte was occurring the rings came closer together in a central area. In late stage chambers the F-actin rings and the microfilament bundles appeared to be incorporated into large irregular masses of actin, which subsequently disappeared as the mature oocyte formed. The F-actin rings are suggested to act as mechanical strengthening elements for the canal plasmalemma, whilst cytoplasmic transport occurs through the ring canals.

Actins↗
PubMed1985

Three distinct distributions of F-actin occur during the divisions of polar surface caps to produce pole cells in Drosophila embryos.

The F-actin distribution was studied during pole cell formation in Drosophila embryos using the phalloidin derivative rhodaminyl-lysine-phallotoxin. Nuclei were also stained with 4'-6 diamidine-2-phenylindole dihydrochloride to correlate the pattern seen with the nuclear cycle. The precursors of the pole cells, the polar surface caps, were found to have an F-actin-rich cortex distinct from that of the rest of the embryo surface and an interior cytoplasm that was less intensely stained but brighter than the cytoplasm deeper in the embryo. They were found to divide once without forming true cells and then a second time when cells formed as a result of a meridional and a basal cleavage. Three distinct distributions of the cortical F-actin have been identified during these cleavages. It is concluded that the first division, which cleaves the polar caps but does not separate them from the embryo, involves very different processes from those that lead to the formation of the pole cells. A contractile-ring type of F-actin organization may not be present during the first cleavage but is suggested to occur during the second.

Actins↗