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Biomedical subjects

G A Dunn

Publications and source records attributed to G A Dunn.

At least 19 recordsLinked to original sources
PubMed2002

Phenotype modulation in non-adherent and adherent sublines of Walker carcinosarcoma cells: the role of cell-substratum contacts and microtubules in controlling cell shape, locomotion and cytoskeletal structure.

We characterised two sublines of Walker carcinosarcoma cells generated by epigenetic changes. Subline 1 cells were mostly polarised and made no or only non-adhesive cell-substratum contacts. Subline 2 cells were spread, adhesive and mainly non-polar. Subline 1 cells migrate in a non-adhesive mode which is very efficient but operates only in a 3D environment, whereas subline 2 cells migrate in an adhesive mode, which is less efficient but works on 2D and 3D substrata. Nocodazole had little or no effect on shape, polarity and locomotion of subline 1 cells. In glass-adherent subline 2 cells, 10(-6)M nocodazole increased the proportion of polarised cells migrating in an adhesive mode and decreased adhesion to the substratum, whereas 10(-5)M nocodazole further reduced the contacts and the cells reverted to a non-adhesive mode of locomotion. When non-polar subline 2 cells were detached mechanically or by nocodazole, they became polarised and morphologically indistinguishable from non-adherent subline 1 cells. On more adhesive plastic substrata, subline 2 cells produced heterogeneous responses to nocodazole including loss of polarity. The phenotypes of Walker carcinosarcoma sublines have similarities with a broad range of cell types ranging from leucocytes to fibroblast-like cells, suggesting that these phenotypic differences can be controlled by the adhesive and contractile state rather than the cell type. Adhesion modulates contractility (isometric or isotonic contraction) and vice versa and this determines morphology (shape, F-actin, myosin and alpha-actinin), locomotion and responses to microtubule-disassembly. The model may be applied to analyse the mechanisms controlling the phenotype of cells in general.

Actinin↗
PubMed2002

Alignment of myoblasts on ultrafine gratings inhibits fusion in vitro.

During development, skeletal muscle precursor cells fuse to form multi-nucleated myotubes. However, it is unclear how this fusion is regulated such that linear myotubes are produced. In a previous study, we found that linear arrays of myoblasts cultured on micropatterns of laminin fused to form linear myotubes of a constant diameter, independent of the width of the laminin track. This suggested that a mechanism exists to prevent myoblasts from fusing laterally [Exp. Cell Res. 230 (1997) 275]. In this study, we have investigated this further by culturing myoblasts on ultrafine grooved surfaces previously shown to align fibroblasts and epithelial cells. We found that all the individual myoblasts were highly aligned along the groove axis, and time-lapse recordings showed that motility was mostly restricted to a direction parallel to the grooves. In contrast to the previous study, however, there was a strong tendency for early differentiating cells to form aggregates either at an angle of approximately 45 degrees or perpendicular to the groove axis. Nevertheless, we rarely saw myotubes formed at those angles, supporting our earlier idea that the ability of cells to fuse laterally is prohibited. Our data strongly suggest that myoblasts are most likely to fuse in an end-to-end configuration, and it is this that enables them to form linear, rather than irregular myotubes.

Animals↗
PubMed2002

Fluorescence localization after photobleaching (FLAP): a new method for studying protein dynamics in living cells.

FLAP is a new method for localized photo-labelling and subsequent tracking of specific molecules within living cells. It is simple in principle, easy to implement and has a wide potential application. The molecule to be located carries two fluorophores: one to be photobleached and the other to act as a reference label. Unlike the related methods of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), the use of a reference fluorophore permits the distribution of the photo-labelled molecules themselves to be tracked by simple image differencing. In effect, FLAP is therefore comparable with methods of photoactivation. Its chief advantage over the method of caged fluorescent probes is that it can be used to track chimaeric fluorescent proteins directly expressed by the cells. Although methods are being developed to track fluorescent proteins by direct photoactivation, these still have serious drawbacks. In order to demonstrate FLAP, we have used nuclear microinjection of cDNA fusion constructs of beta-actin with yellow (YFP) and cyan (CFP) fluorescent proteins to follow both the fast relocation dynamics of monomeric (globular) G-actin and the much slower dynamics of filamentous F-actin simultaneously in living cells.

Actins↗
PubMed2001

Extracellular control of cell size.

Both cell growth (cell mass increase) and progression through the cell division cycle are required for sustained cell proliferation. Proliferating cells in culture tend to double in mass before each division, but it is not known how growth and division rates are co-ordinated to ensure that cell size is maintained. The prevailing view is that coordination is achieved because cell growth is rate-limiting for cell-cycle progression. Here, we challenge this view. We have investigated the relationship between cell growth and cell-cycle progression in purified rat Schwann cells, using two extracellular signal proteins that are known to influence these cells. We find that glial growth factor (GGF) can stimulate cell-cycle progression without promoting cell growth. We have used this restricted action of GGF to show that, for cultured Schwann cells, cell growth rate alone does not determine the rate of cell-cycle progression and that cell size at division is variable and depends on the concentrations of extracellular signal proteins that stimulate cell-cycle progression, cell growth, or both.

Animals↗
PubMed2001

Specific changes to the mechanism of cell locomotion induced by overexpression of beta-actin.

Overexpression of beta-actin is known to alter cell morphology, though its effect on cell motility has not been documented previously. Here we show that overexpressing beta-actin in myoblasts has striking effects on motility, increasing cell speed to almost double that of control cells. This occurs by increasing the areas of protrusion and retraction and is accompanied by raised levels of beta-actin in the newly protruded regions. These regions of the cell margin, however, show decreased levels of polymerised actin, indicating that protrusion can outpace the rate of actin polymerisation in these cells. Moreover, the expression of beta*-actin (a G244D mutant, which shows defective polymerisation in vitro) is equally effective at increasing speed and protrusion. Concomitant changes in actin binding proteins show no evidence of a consistent mechanism for increasing the rate of actin polymerisation in these actin overexpressing cells. The increase in motility is confined to poorly spread cells in both cases and the excess motility can be abolished by blocking myosin function with butanedione monoxime (BDM). Our observations on normal myoblasts are consistent with the view that they protrude by the assembly and cross linking of actin filaments. In contrast, the additional motility shown by cells overexpressing beta-actin appears not to result from an increase in the rate of actin polymerisation but to depend on myosin function. This suggests that the additional protrusion arises from a different mechanism. We discuss the possibility that it is related to retraction-induced protrusion in fibroblasts. In this phenomenon, a wave of increased protrusion follows a sudden collapse in cell spreading. This view could explain why it is only the additional motility that depends on spreading, and has implications for understanding the differences in locomotion that distinguish tissue cells from highly invasive cell types such as leucocytes and malignant cells.

Actin Depolymerizing Factors↗
PubMed1999

TGFbeta1 induces a cell-cycle-dependent increase in motility of epithelial cells.

We have previously shown that addition of type 1 transforming growth factor-beta (TGFbeta1) to an exponentially growing population of mink lung CCl64 cells increases their average intermitotic time from 14.4 to 20.3 hours, predominantly by extending G1 from 7.5 to 13.5 hours. Here we have used the DRIMAPS system (digitally recorded interference microscopy with automatic phase-shifting) for obtaining data on cellular mass distribution, cell motility and morphology. We found no significant change in the cells' rate of mass increase following TGFbeta1 treatment, which implies that the treated cells attained a higher mass during their extended cell cycle and this was confirmed by direct measurement of cell size. However, the cells showed a dramatic motile response to treatment: TGFbeta1-treated cells had a significantly higher time-averaged speed of 36.2 microm hour-1 compared to 14.5 microm hour-1 for the control cells. The time course of the response was gradual, reaching a maximum mean speed of 52.6 microm hour-1 after 15 hours exposure. We found that the gradual onset of the response was probably not due to a slow accumulation of a secondary factor but because cells were dividing throughout the experiment and most of the response to TGFbeta1 occurred only after the first cell division in its presence. Thus, taking only those cells that had not yet divided, the time-averaged speed of treated cells (26.1 micrometer hour-1) was only moderately higher than that of untreated cells (14.9 micrometer hour-1) whereas, for those cells that had divided, the difference in speed between treated cells (45.1 micrometer hour-1) and untreated cells (14.1 microm hour-1) was much greater. Increased speed was a consequence of enhanced protrusion and retraction of the cell margin coupled with an increase in cell polarity. TGFbeta1 also increased the mean spreading of the cells, measured as area-to-mass ratio, from 3.2 to 4.4 micrometer2 pg-1, and the intracellular mass distribution became more asymmetric. The observations indicate that a G2 signal may be necessary to reach maximal motility in the presence of TGFbeta1.

Animals↗
PubMed1999

Using molecular genetics as a tool in understanding crawling cell locomotion in myoblasts.

We have used digitally recorded interference microscopy with automatic phase shifting (DRIMAPS) to investigate the crawling locomotion of normal and mutant mouse myoblasts. Contraction forces that give rise to cell body movement, tail retraction and cell adhesion to the substrate in myoblasts and other locomoting tissue cells arise from the interactions of actin and non-muscle myosin II. The activity of non-muscle myosin II is regulated differently from that of skeletal myosin. Using DRIMAPS, we found that crawling locomotion was altered in myoblasts that heterologously expressed human beta-cardiac myosin heavy chain (MHC); the cells moved more slowly and had reduced rates of protrusion and retraction. Immunolocalization demonstrated that MHC and non-muscle myosin II were not co-localized, suggesting that MHC does not compete directly with myosin II, but interferes with cell locomotion by binding inappropriately to actin filaments and possibly cross-linking them. Myosin I may be involved in protrusion of the lamellipodia. However, using DRIMAPS, we found that crawling locomotion was unaltered in myoblasts that heterologously expressed a truncated myosin I which lacked the membrane-binding tail domain. This suggests that, if endogenous myosin I is important for cell locomotion, this mutant was unable to interfere with its action. We conclude that the effects on locomotion of expressing foreign or mutant proteins of the cytoskeleton in vertebrate cells can be subtle and can be swamped by the intrinsic variability of the cells. Their characterization requires automated methods of acquiring data, such as DRIMAPS, and careful statistical analysis in order to take account of other sources of variation.

Actins↗
PubMed1998

Disruption of the talin gene compromises focal adhesion assembly in undifferentiated but not differentiated embryonic stem cells.

We have used gene disruption to isolate two talin (-/-) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of beta1 integrin, although levels of alpha5 and alphaV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (-/-) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (-/-) ES cells were able to assemble talin-containing focal adhesions. Both talin (-/-) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (-/-) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the beta1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for beta1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.

Actins↗
PubMed1998

Chemotaxis of macrophages is abolished in the Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare disease characterized by microthrombocytopenia, eczema and immune deficiency. In this study a direct-viewing chemotaxis chamber was used to analyse chemotactic responses of WAS neutrophils and macrophages in stable linear concentration gradients. In five patients with classic WAS, chemotaxis of macrophages but not of neutrophils was found to be abolished, whereas the speed of random motility of both cell types was found to be indistinguishable from control cells. This supports the existence of an essential functional link, previously suggested by biochemical studies, between Cdc42, WAS protein (WASp) and the actin cytoskeleton in primary human macrophages. Moreover, these data suggest that Cdc42-WASp-mediated filopodial extension is a requirement for chemotaxis but not for chemokinesis in these cells. Abnormal directional cell motility of macrophages and related antigen-presenting cells may play a significant part in the immune deficiency and eczema of WAS.

Chemotaxis↗
PubMed1997

Deficiency of p67phox, p47phox or gp91phox in chronic granulomatous disease does not impair leucocyte chemotaxis or motility.

Chronic granulomatous disease (CGD) is a syndrome characterized by failure of the NADPH oxidase of phagocytes that generates superoxide, which is central to the microbicidal process. Cytosolic components of this oxidase system include the proteins p67phox and p47phox, deficiencies of which cause the autosomal recessive form of CGD, whereas the X-linked form of the disease is characterized by a deficiency in the plasma membrane component gp91phox. Components of the oxidase system have been reported to be associated with the cytoskeleton and neutrophils from CGD patients have been reported to have a defective chemotactic response in Boyden chambers. Using a chamber that permits the direct observation of cell behaviour in a linear gradient of a chemoattractant, we have analysed the chemotactic response of neutrophils from a patient lacking p67phox; from another lacking p47phox and from a third lacking gp91phox. The results of measuring the speeds and directions of locomotion of the cells show that their speeds are undiminished relative to cells from healthy control subjects and that their directions of migration are at least as strongly biased in the direction of the gradient as those of the control cells. We conclude that these definitive aspects of the chemotactic response are not abnormal in either the autosomal recessive or the X-linked forms of CGD and that they are therefore not factors in the predisposition to infection that characterizes the syndrome.

Adult↗
PubMed1997

Rapid, microtubule-dependent fluctuations of the cell margin.

Using data automatically acquired by microinterferometry from large numbers of chick fibroblasts, we have detected rapid microfluctuations in the rates of protrusion and retraction of the cell margin which were strongly suppressed by colcemid, nocodazole and taxol. Fluctuations in the rate of retraction of the margin were about twice as powerful as fluctuations in the rate of protrusion. High-frequency fluctuations were also apparent in the cell track and in measures of cell spreading, shape and speed. These rapid fluctuations were also all suppressed by colcemid and nocodazole, sometimes by doses insufficient to disrupt the majority of microtubules. Taxol on the other hand did not suppress fluctuations in direction of the cell track nor fluctuations in the spreading of the cells but it was very effective at suppressing variations in protrusion and retraction and in cell speed and shape. We discovered that much slower, larger-scale variations in protrusion, retraction, spreading, shape and speed resulted from the accumulation of these rapid, microtubule-dependent fluctuations of the cell margin. These large-scale variations in cell behaviour were also suppressed by the same drug treatments that were effective in suppressing the corresponding high-frequency fluctuations. We speculate that a function of microtubules is to enhance the fibroblast's responses to its environment by causing microfluctuations of the cell's margin which give rise to large-scale variations in cell behaviour.

Animals↗
PubMed1995

Are growth factors chemotactic agents?

We report the first direct observation of chemotaxis in slowly moving malignant cells. Two sarcoma cell lines of different metastatic potential were used. In a direct-viewing chemotaxis chamber with two concentric wells, the pooled trajectories of highly malignant rat T15 cells were strongly biased toward the outer well which contained platelet-derived and insulin-like growth factors. In individual experiments, however, the trajectories of the T15 cells showed a significant directional bias which, depending on the cell distribution, sometimes deviated by as much as 170 degrees from the gradient direction. Cells of a less malignant rat line, K2, did not respond to the gradient but a strong K2 response appeared if T15 cells were placed in the outer well along with the growth factors. We conclude that stimulated T15 cells release a chemoattractant, for both T15 and K2 cells, which overrides any chemoattractive effect of the growth factors. These results call into question whether growth factors are ever directly chemotactic in this system and demonstrate the need for direct observation in determining whether any substance is a direct chemoattractant.

Animals↗
PubMed1995

Dynamics of fibroblast spreading.

A new technique of microinterferometry permits cellular growth and motile dynamics to be studied simultaneously in living cells. In isolated chick heart fibroblasts, we have found that the non-aqueous mass of each cell tends to increase steadily, with minor fluctuations, throughout the cell cycle. The spread area of each cell also tends to increase during interphase but fluctuates between wide limits. These limits are dependent on the cell's mass and the upper limit is particularly sharp and directly proportional to mass. From a dynamical point of view, the spread area of a cell is determined by the balance between the rates of two antagonistic processes: protrusion of cellular material into new territory and retraction of material from previously occupied territory. The spatial asymmetry of these processes determines the translocation of the cell. We have found with the chick fibroblasts that the rates of the two processes are generally closely matched to each other and appear to be dependent on the cell's area of spreading. Both continue incessantly in well spread cells, even when there is no net translocation of the cell, and the lower limit of each activity is directly proportional to spread area. The two processes show different behaviour, however, during changes in the spread area of the cell. Both increases and decreases in area appear to be brought about by changes in the rate of retraction, the rate of protrusion remaining relatively constant. A simple stochastic model based on a limited supply of adhesion molecules can simulate all our observations including the mass-limited spreading, the strong correlation between protrusion and retraction and the retraction-dominated changes in area. We conclude that the spread area of the cell is actively regulated, possibly by a simple automatic mechanism that adjusts the area of spreading in relation to the mass of the cell and controls the rate of protrusion to compensate rapidly for spontaneous fluctuations in retraction.

Animals↗
PubMed1995

Origins of the parasitophorous vacuole membrane of the malaria parasite: surface area of the parasitized red cell.

There is conflicting evidence on whether the parasitophorous vacuole membrane, in which the malaria parasite becomes encapsulated when it enters the red cell, represents a part of the host cell membrane or is derived, at least in part, from the parasite. We have measured the surface area of populations of red cells before and after invasion by up to four merozoites of the malaria parasite, Plasmodium falciparum. The dimensions of the merozoite are such that, if it enveloped itself entirely in host cell membrane during entry, the loss of surface area would amount to some 4 square microns 2 or 3% of the total for each parasite internalized. Our measurements show that within the 99% level of confidence any loss of surface area is less than 1 square micron 2 per parasite internalized. Area measurements on red cells that have been allowed to lose known proportions of their membrane by metabolically induced vesiculation reveal, moreover, that diminutions in surface area in the range of interest are readily detectable. Our observations on recently invaded (young ring-stage) parasites appear to exclude any significant change in surface area of the host cell following invasion. This implies that, if indeed there is internalization of host cell membrane lipid on invasion, as the best evidence shows, it is compensated by parasite-derived lipid, and conversely the parasitophorous vacuole membrane probably contains a contribution of parasite-derived material, presumably that seen to be discharged by the apical organelles, the rhoptries, at the time of invasion.

Animals↗
PubMed1993

Phase-shifting interference microscopy applied to the analysis of cell behaviour.

The theory of phase-shifting interferometry is not new but it is only recently, with the advent of solid-state detector arrays and fast image processors, that it has become a practical imaging technique. In conjunction with transmission interference microscopy, phase-shifting presents a new way of introducing contrast into the images of transparent microscopic objects such as cultured cells. An earlier paper from our laboratory has emphasised the advantages of transmission interference microscopy over phase contrast or differential interference contrast microscopy for the computerised analysis of cell behaviour. Phase-shifting greatly improves the accuracy, long-term stability and range of application of this technique but it has not previously, to our knowledge, been combined with transmission interference microscopy for the study of cultured cells. The resulting image is especially well suited to quantitative analysis by computer since it is a direct representation of the distribution of non-aqueous cellular material in the specimen. The image is not degraded by uneven illumination; by heterogeneous sensitivity of the detector array; or by differential absorption of light in the optics or specimen. Our main purpose in developing the method is to obtain sequences of images of the motile behaviour of cells in culture for analysis by computer. This type of analysis is potentially a powerful tool for studying the motile responses of cells and the operation and control of their locomotory machinery. Not only can the method be used for studying cell translocation and the dynamics of intracellular movement of non-aqueous material, but it is now possible to study in detail the time course of growth in individual cultured cells.

Animals↗
PubMed1993

Long-term chemotaxis of neutrophils in stable gradients: preliminary evidence of periodic behavior.

We observed and recorded the behavior of human neutrophils in stable linear gradients of the chemotactic attractant f-Met-Leu-Phe in a recently developed, direct-viewing, chemotaxis chamber. The cells were kept cold for the first hour after setting up the chemotaxis chamber in order to establish a stable gradient before allowing the cells to spread and begin to move by warming them up to 37 degrees C. We found that the cells showed very significant chemotaxis at different times throughout the 2-hour period of observation but, unexpectedly, the activity occurred in periodic or quasi-periodic cycles during which the cells entered phases of undirected motility lasting for several minutes. We discuss whether the periodicity is a population or single cell-based phenomenon and briefly speculate on its possible analogy to the well-known periodic chemotaxis of slime mould amoebas.

Chemotaxis, Leukocyte↗