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D Zicha

Publications and source records attributed to D Zicha.

At least 19 recordsLinked to original sources
PubMed2004

Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing.

Fluorescence resonance energy transfer (FRET) by acceptor photobleaching is a simple but effective tool for measurements of protein-protein interactions. Until recently, it has been restricted to qualitative or relative assessments owing to the spectral bleed-through contamination resulting from fluorescence overlap between the donor and the acceptor. In this paper, we report a quantitative algorithm that combines the spectral unmixing technique with FRET by acceptor photobleaching. By spectrally unmixing the emissions before and after photobleaching, it is possible to resolve the spectral bleed-through and retrieve the FRET efficiency/interaction distance quantitatively. Using a human keratinocyte cell line transfected with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged Cx26 connexins as an example, FRET information at homotypic gap junctions is measured and compared with well-established methods. Results indicate that the new approach is sensitive, flexible, instrument independent and solely FRET dependent. It can achieve FRET estimations similar to that from a sensitized emission FRET method. This approach has a great advantage in providing the relative concentrations of the donor and the acceptor; this is, for example, very important in the comparative study of cell populations with variable expression levels.

Algorithms↗
PubMed2003

Two dynamic morphotypes of sarcoma cells, asymmetric stellate and triangle with leading lamella, are related to malignancy.

A notion of the dynamic morphotype was developed as a conjunction between cell shape and migration. This enabled the investigation of the relationship between malignancy and patterns of dynamic morphology in neoplastic cells in vitro. Time-lapse cinemicroscopy was used to analyse the cell behaviour of three rat neoplastic cell lines (K2, T15, and A8), differing in metastatic potential, that were instrumental in revealing a coincidence between high migratory activity and appearance of the 3D structure of actin cables in high-malignant A8 cells (Pokorná et al., 1994). A set of criteria was established for visual classification of cell morphology. Matching the pattern of cell morphology with locomotory activity led to identification of four dynamic morphotypes. Cell speed was determined by tracking and the dynamic morphotypes assigned by the operator. All the three cell populations were studied for incidence of the dynamic morphotypes in culture media differing in pH: 6.6 simulating acid extracellular condition in tumours, physiological 7.4, and alkaline 8.2. The results showed that acid pH stimulated motile activity in the intermediate-malignant T15 and most malignant A8 cells. The T15 and A8 cells also manifested a prolonged continuation of fast locomotion in the early G1 phase and displayed a prevalence of two fast moving dynamic morphotypes: asymmetric stellate and triangle with leading lamella.

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

Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility.

Protein kinase C (PKC) alpha has been implicated in beta1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and beta1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. We provide the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo.

Amino Acid Substitution↗
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

A role for Cdc42 in macrophage chemotaxis.

Three members of the Rho family, Cdc42, Rac, and Rho are known to regulate the organization of actin-based cytoskeletal structures. In Bac1.2F5 macrophages, we have shown that Rho regulates cell contraction, whereas Rac and Cdc42 regulate the formation of lamellipodia and filopodia, respectively. We have now tested the roles of Cdc42, Rac, and Rho in colony stimulating factor-1 (CSF-1)-induced macrophage migration and chemotaxis using the Dunn chemotaxis chamber. Microinjection of constitutively activated RhoA, Rac1, or Cdc42 inhibited cell migration, presumably because the cells were unable to polarize significantly in response to CSF-1. Both Rho and Rac were required for CSF-1-induced migration, since migration speed was reduced to background levels in cells injected with C3 transferase, an inhibitor of Rho, or with the dominant-negative Rac mutant, N17Rac1. In contrast, cells injected with the dominant-negative Cdc42 mutant, N17Cdc42, were able to migrate but did not polarize in the direction of the gradient, and chemotaxis towards CSF-1 was abolished. We conclude that Rho and Rac are required for the process of cell migration, whereas Cdc42 is required for cells to respond to a gradient of CSF-1 but is not essential for cell locomotion.

Animals↗
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↗
PubMed1998

Effect of pH on proteinase secretion by transformed fibroblast populations.

Effect of pH on secretion of proteolytic enzymes in cell cultures of three clonal lines of transformed fibroblasts (K2, T15 and K4) was studied by using 14C-labelled denatured proteins as substrate. One line of malignant macrophages derived from mouse reticulum cell sarcoma (J774.1) was used for comparison. The relative motility index of all cell lines was derived by computer analysis of quantitative estimations of cell dispersion in single-cell-derived colonies. Cultivation at pH 6.5 decreased the growth rate in most experiments as compared with that at pH 7.4, and stimulated cell motility to a different extent. The population of mouse malignant macrophages produced several-fold higher extracellular proteolytic activity than the fibroblast lines. Secretion of proteinases by the malignant macrophages was significantly stimulated by the lower pH. Enzyme secretion by two of the three fibroblast derivatives was also stimulated by acidic pH but to a lesser extent than the secretion of the malignant macrophages. The assessment of motility done by measurement of dispersion of cells in colony proved a positive correlation between motility and proteinase secretion in J774.1 cells and one transformed fibroblast clone (T15) but not in the two other clonal lines.

Animals↗
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↗