Biomedical subjects

C J Weijer

Publications and source records attributed to C J Weijer.

At least 19 recordsLinked to original sources

PubMed2005

The regulation of cell migration by PTEN.

In vertebrates, the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) regulates many cellular processes through its PtdIns(3,4,5)P3 lipid phosphatase activity, antagonizing PI3K (phosphoinositide 3-kinase) signalling. Given the important role of PI3Ks in the regulation of directed cell migration and the role of PTEN as an inhibitor of migration, it is somewhat surprising that data now indicate that PTEN is able to regulate cell migration independent of its lipid phosphatase activity. Here, we discuss the role of PTEN in the regulation of cell migration.

Animals↗

PubMed2001

A Dictyostelium nuclear phosphatidylinositol phosphate kinase required for developmental gene expression.

The generation of diacylglycerol (DAG) in response to receptor stimulation is a well-documented signalling mechanism that leads to activation of protein kinase C (PKC). Putative alternative effectors contain sequences that interact with DAGs, but the mechanisms of signal transduction are unknown. We have identified a Dictyostelium gene encoding a novel protein which contains a domain with high identity to the DAG-binding domain of PKC. It does not encode a PKC homologue as the conservation does not extend outside this region. We confirm that the proposed DAG-binding domain is sufficient to mediate interaction of a fusion protein with vesicles containing DAG. The protein also shows significant homology to mammalian phosphatidylinositol phosphate (PIP) kinases and we show that this domain has PIP kinase activity. The protein, PIPkinA, is enriched in the nucleus and abrogation of gene function by homologous recombination inhibits early developmental gene expression, blocking development at an early stage. Thus, we have identified a PIP kinase from Dictyostelium which is required for development, is a candidate effector for DAG and has the potential to synthesize nuclear PIP(2).

Animals↗

PubMed2001

Propagating chemoattractant waves coordinate periodic cell movement in Dictyostelium slugs.

Migration and behaviour of Dictyostelium slugs results from coordinated movement of its constituent cells. It has been proposed that cell movement is controlled by propagating waves of cAMP as during aggregation and in the mound. We report the existence of optical density waves in slugs; they are initiated in the tip and propagate backwards. The waves reflect periodic cell movement and are mediated by cAMP, as injection of cAMP or cAMP phosphodiesterase disrupts wave propagation and results in effects on cell movement and, therefore, slug migration. Inhibiting the function of the cAMP receptor cAR1 blocks wave propagation, showing that the signal is mediated by cAR1. Wave initiation is strictly dependent on the tip; in decapitated slugs no new waves are initiated and slug movement stops until a new tip regenerates. Isolated tips continue to migrate while producing waves. We conclude from these observations that the tip acts as a pacemaker for cAMP waves that coordinate cell movement in slugs.

Animals↗

PubMed2001

Inducible nuclear translocation of a STAT protein in Dictyostelium prespore cells: implications for morphogenesis and cell-type regulation.

Dd-STATa, the Dictyostelium STAT (signal transducer and activator of transcription) protein, is selectively localised in the nuclei of a small subset of prestalk cells located in the slug tip. Injection of cAMP into the extracellular spaces in the rear of the slug induces rapid nuclear translocation of a Dd-GFP:STATa fusion protein in prespore cells surrounding the site of injection. This suggests that cAMP signals that emanate from the tip direct the localised nuclear accumulation of Dd-STATa. It also shows that prespore cells are competent to respond to cAMP, by Dd-STATa activation, and it implies that cAMP signalling is in some way limiting in the rear of the slug. Co-injection of a specific inhibitor of the cAR1 serpentine cAMP receptor almost completely prevents the cAMP-induced nuclear translocation, showing that most or all of the cAMP signal is transduced by cAR1. Dd-GFP:STATa also rapidly translocates into the nuclei of cells adjoining the front and back cut edges when a slug is bisected. Less severe mechanical disturbances, such as pricking the rear of a slug with an unfilled micropipette, also cause a more limited nuclear translocation of Dd-GFP:STATa. We propose that these signalling events form part of a repair mechanism that is activated when the migrating slug suffers mechanical damage.

Animals↗

PubMed2001

cAMP receptor affinity controls wave dynamics, geometry and morphogenesis in Dictyostelium.

Serpentine G-protein-coupled cAMP receptors are key components in the detection and relay of the extracellular cAMP waves that control chemotactic cell movement during Dictyostelium development. During development the cells sequentially express four closely related cAMP receptors of decreasing affinity. In this study, we investigated the effect of cAMP receptor type and affinity on the dynamics of cell-cell signalling in vivo, by measuring the dynamics of wave initiation and propagation in a variety of cAMP receptor mutants. We found that receptor affinity controls the frequency of wave initiation, but it does not determine wave propagation velocity, thus resulting in dramatic changes in wave geometry. In the limiting case, the affinity of the receptor is so low that waves can still be initiated but no stable centres form - thus, the cells cannot aggregate. In mounds, expression of low affinity receptors results in slow concentric waves instead of the normally observed multi-armed spiral waves. Under these conditions there is no rotational cell movement and the hemispherical mounds cannot transform into slugs. These results highlight the importance of receptor number and affinity in the proper control of cell-cell signalling dynamics required for the successful completion of development.

Animals↗

PubMed2000

The control of chemotactic cell movement during Dictyostelium morphogenesis.

Differential cell movement is an important mechanism in the development and morphogenesis of many organisms. In many cases there are indications that chemotaxis is a key mechanism controlling differential cell movement. This can be particularly well studied in the starvation-induced multicellular development of the social amoeba Dictyostelium discoideum. Upon starvation, up to 10(5) individual amoebae aggregate to form a fruiting body The cells aggregate by chemotaxis in response to propagating waves of cAMP, initiated by an aggregation centre. During their chemotactic aggregation the cells start to differentiate into prestalk and prespore cells, precursors to the stalk and spores that form the fruiting body. These cells enter the aggregate in a random order but then sort out to form a simple axial pattern in the slug. Our experiments strongly suggest that the multicellular aggregates (mounds) and slugs are also organized by propagating cAMP waves and, furthermore, that cell-type-specific differences in signalling and chemotaxis result in cell sorting, slug formation and movement.

Animals↗

PubMed2000

A temperature-sensitive adenylyl cyclase mutant of Dictyostelium.

Dictyostelium development starts with the chemotactic aggregation of up to 10(6) amoebae in response to propagating cAMP waves. cAMP is produced by the aggregation stage adenylyl cyclase (ACA) and cells lacking ACA (aca null) cannot aggregate. Temperature-sensitive mutants of ACA were selected from a population of aca null cells transformed with a library of ACA genes, a major segment of which had been amplified by error-prone PCR. One mutant (tsaca2) that can complement the aggregation null phenotype of aca null cells at 22 degrees C but not at 28 degrees C was characterized in detail. The basal catalytic activity of the enzyme in this mutant was rapidly and reversibly inactivated at 28 degrees C. Using this mutant strain we show that cell movement in aggregates and mounds is organized by propagating waves of cAMP. Synergy experiments between wild-type and tsaca2 cells, shifted to the restrictive temperature at various stages of development, showed that ACA plays an important role in the control of cell sorting and tip formation.

Adenylyl Cyclases↗

PubMed1999

Morphogenetic cell movement in Dictyostelium.

Dictyostelium morphogenesis starts with the chemotactic aggregation of starving individual cells. The cells move in response to propagating waves of the chemoattractant cyclic AMP initiated by cells in the aggregation centre. During aggregation the cells begin to differentiate into several types with different signalling and chemotactic properties. These cell types sort out from each other to form an axial pattern in the slug. There is now good evidence that periodic chemotactic signals not only control aggregation, but also later stages of morphogenesis. These signals take the form of target patterns, spirals, multi-armed spirals and scroll waves. I will discuss their role in the control of cell movement during mound and slug formation and in the formation of the fruiting body.

Animals↗

PubMed1999

Modeling chemotactic cell sorting during Dictyostelium discoideum mound formation.

Coordinated cell movement is a major mechanism of the multicellular development of most organisms. The multicellular morphogenesis of the slime mould Dictyostelium discoideum, from single cells into a multicellular fruiting body, results from differential chemotactic cell movement. During aggregation cells differentiate into prestalk and prespore cells that will form the stalk and spores in the fruiting body. These cell types arise in a salt and pepper pattern after what the prestalk cells chemotactically sort out to form a tip. The tip functions as an organizer because it directs the further development. It has been difficult to get a satisfactory formal description of the movement behavior of cells in tissues. Based on our experiments, we consider the aggregate as a drop of a viscous fluid and show that this consideration is very well suited to mathematically describe the motion of cells in the tissue. We show that the transformation of a hemispherical mound into an elongated slug can result from the coordinated chemotactic cell movement in response to scroll waves of the chemoattractant cAMP. The model calculations furthermore show that cell sorting can result from differences in chemotactic cell movement and cAMP relay kinetics between the two cell types. During this process, the faster moving and stronger signaling cells collect on the top of the mound to form a tip. The mound then extends into an elongated slug just as observed in experiments. The model is able to describe cell movement patterns in the complex multicellular morphogenesis of Dictyostelium rather well and we expect that this approach may be useful in the modeling of tissue transformations in other systems.

Animals↗

PubMed1998

Induction of optical density waves and chemotactic cell movement in Dictyostelium discoideum by microinjection of cAMP pulses.

The development of most multicellular organisms involves coordinated cell movement. The early aggregation of Dictyostelium cells has been shown to be mediated by chemotactic movement to propagating waves of cAMP. We have proposed that propagating waves of a chemoattractant, most likely cAMP, also control the movement of cells in mounds and slugs. We have now used periodic pressure injection of pulses of cAMP in the extracellular space of aggregation streams, mounds, and slugs to investigate whether these signals can be relayed and control cell movement, using quantitative digital time-lapse microscopy. Our major findings are (1) short (0.1 s) pulses of cAMP (10(7) molecules) were able to elicit optical density (OD) waves in fields of aggregating amoebae. They propagate from the micropipet outward and interact with endogenous OD waves. (2) Periodic injection of cAMP pulses into aggregation streams blocked the pulses coming from the center and led to the rapid accumulation of cells downstream of the pipet around the pipet. (3) Injection of pulses of cAMP into mounds elicited OD waves, which propagated from the pipet outward and interacted with the endogenous waves, indicating that the same propagator carries them. (4) Periodic microinjection of cAMP in the prespore zone of slugs led to accumulation of anterior-like cells around the micropipet followed by tip formation. Furthermore, the cAMP signal could control the spacing of the endogenous sorting pattern. These results strongly support the hypothesis that the optical density waves observed during early development up to the mound stage represent propagating cAMP waves. They suggest furthermore that cAMP is the morphogen that controls cell movements in slugs.

Animals↗

PubMed1998

Propagating waves control Dictyostelium discoideum morphogenesis.

The morphogenesis of Dictyostelium results from the coordinated movement of starving cells to form a multicellular aggregate (mound) which transforms into a motile slug and finally a fruiting body. Cells differentiate in the mound and sort out to form an organised pattern in the slug and fruiting body. During aggregation, cell movement is controlled by propagating waves of the chemo-attractant cAMP. We show that mounds are also organised by propagating waves. Their geometry changes from target or single armed spirals during aggregation to multi-armed spiral waves in the mound. Some mounds develop transiently into rings in which multiple propagating wave fronts can still be seen. We model cell sorting in the mound stage assuming cell type specific differences in cell movement speed and excitability. This sorting feeds back on the wave geometry to generate twisted scroll waves in the slug. Slime mould morphogenesis can be understood in terms of wave propagation directing chemotactic cell movement.

Animals↗

PubMed1997

Null mutations of the Dictyostelium cyclic nucleotide phosphodiesterase gene block chemotactic cell movement in developing aggregates.

Extracellular cAMP is a critical messenger in the multicellular development of the cellular slime mold Dictyostelium discoideum. The levels of cAMP are controlled by a cyclic nucleotide phosphodiesterase (PDE) that is secreted by the cells. The PDE gene (pdsA) is controlled by three promoters that permit expression during vegetative growth, during aggregation, and in prestalk cells of the older structures. Targeted disruption of the gene aborts development, and complementation with a modified pdsA restores development. Two distinct promoters must be used for full complementation, and an inhibitory domain of the PDE must be removed. We took advantage of newly isolated PDE-null cells and the natural chimerism of the organism to ask whether the absence of PDE affected individual cell behavior. PDE-null cells aggregated with isogenic wild-type cells in chimeric mixtures, but could not move in a coordinated manner in mounds. The wild-type cells move inward toward the center of the mound, leaving many of the PDE-null cells at the periphery of the aggregate. During the later stages of development, PDE-null cells in the chimera segregate to regions which correspond to the prestalk region and the rear of the slug. Participation in the prespore/spore population returns with the restoration of a modified pdsA to the null cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

Analysis of cell movement and signalling during ring formation in an activated G alpha1 mutant of Dictyostelium discoideum that is defective in prestalk zone formation.

Mound formation in the cellular slime mould Dictyostelium results from the chemotactic aggregation of competent cells. Periodic cAMP signals propagate as multiarmed spiral waves and coordinate the movement of the cells. In the late aggregate stage the cells differentiate into prespore and several prestalk cell types. Prestalk cells sort out chemotactically to form the tip, which then controls all further development. The tip organises cell movement via a scroll wave that converts to planar waves in the prespore zone leading to rotational cell movement in the tip and periodic forward movement in the prespore zone. Expression of an activated G alpha1 protein under its own promoter leads to a severely altered morphogenesis from the mound stage onwards. Instead of forming a tipped mound, the cells form a ring-shaped structure without tip. Wave propagation pattern and dynamics during aggregation and mound formation in the mutant are indistinguishable from the parental strain AX3. However, at the time of tip formation the spiral waves that organise the late aggregate do not evolve in a scroll-organising centre in the tip but transform into a circularly closed (twisted) scroll ring wave. This leads to the formation of a doughnut-shaped aggregate. During further development, the doughnut increases in diameter and the twisted scroll wave converts into a train of planar waves, resulting in periodic rotational cell movement. Although biochemical consequences resulting from this mutation are still unclear, it must affect prestalk cell differentiation. The mutant produces the normal proportion of prespore cells but is unable to form functional prestalk cells, i.e., prestalk cells with an ability to sort out from the prespore cells and form a prestalk zone. Failure of sorting leads to an altered signal geometry, ring-shaped scroll waves, that then directs ring formation. This mutant demonstrates the importance of prestalk cell sorting for the stabilisation of the scroll wave that organises the tip.

Animals↗

PubMed1996

Spatial pattern formation during aggregation of the slime mould Dictyostelium discoideum.

Stream formation and spiral wave behaviour during the aggregation of Dictyostelium discoideum (Dd) are studied in a model based on the Martiel-Goldbeter equations for cAMP relay, combined with chemotactic motion of Dd cells. The results show that stream formation occurs if the turnover rate of intracellular cAMP is increased. This increase in the turnover rate of cAMP[in] leads to a dependence of the speed of the cAMP wave on the cell density. We propose that this dependence of wave speed on cell density is the underlying mechanism for stream formation. Besides stream formation, increasing the turnover rate of cAMP[in] also results in a spiral wave period that decreases during aggregation, a phenomenon that is commonly observed in situ. Furthermore, the dependence of wave speed on cell density is measured empirically. The speed of the cAMP wave is found to decrease as the wave travels from high to low cell density. This indicates that in situ, wave speed does depend on cell density.

Animals↗

PubMed1996

Analysis of optical density wave propagation and cell movement during mound formation in Dictyostelium discoideum.

Aggregation fields of Dictyostelium amoebae are organized by propagating concentric or spiral waves of cAMP. These waves coordinate cell movement directed toward the aggregation center. We now systematically investigated dark-field wave propagation and chemotactic cell movement during late aggregation and mound formation. The period and the signal propagation velocity decreased continuously during aggregation leading to a 15-fold decrease of the chemical wavelength. By analyzing the behavior of single GFP-labeled cells in aggregates and mounds we measured cell movement velocity, changes in cell shape, periodicity of cell movement, and cell trajectories. In early mounds of strain AX-3 dark-field waves propagated frequently as multiarmed (high-frequency) spirals. During the high-frequency waves observed in the early mound stage, cell movement speed is low and cell movement rather undirected. During tip formation the wave period decreased again and the cells started to rotate in the mound at unusually high average speeds of 40 microns/min. The rotation was almost monotonic with no clear periodicity. Since at this time the majority of the cells had already differentiated into prespore cells, this implies that prespore cells moved faster than aggregation stage cells. At 12 hr of development cell movement velocity dropped again and became highly periodic. These measurements show that the relay system is characterized by a specific temporal evolution, which is closely correlated with cellular differentiation. The remarkable changes in cell movement speed and period indicate a qualitative change in signal and movement parameters which might well be caused by the observed switch from high- to low-affinity cAMP receptors during mound formation. This switch might be required to copy with the increase in cell density and most likely plays a crucial role in the process of cell sorting.

Animals↗

PubMed1996

Periodic phenomena in Proteus mirabilis swarm colony development.

Proteus mirabilis colonies exhibit striking geometric regularity. Basic microbiological methods and imaging techniques were used to measure periodic macroscopic events in swarm colony morphogenesis. We distinguished three initial phases (lag phase, first swarming phase, and first consolidation phase) followed by repeating cycles of subsequent swarming plus consolidation phases. Each Proteus swarm colony terrace corresponds to one swarming-plus-consolidation cycle. The duration of the lag phase was dependent upon inoculation density in a way that indicated the operation of both cooperative and inhibitory multicellular effects. On our standard medium, the second and subsequent swarm phases displayed structure in the form of internal waves visible with reflected and dark-field illumination. These internal waves resulted from organization of the migrating bacteria into successively thicker cohorts of swarmer cells. Bacterial growth and motility were independently modified by altering the composition of the growth medium. By varying the glucose concentration in the substrate, it was possible to alter biomass production without greatly affecting the kinetics of colony surface area expansion. By varying the agar concentration in the substrate, initial bacterial biomass production was unaffected but colony expansion dynamics were significantly altered. Higher agar concentrations led to slower, shorter swarm phases and longer consolidation phases. Thus, colony growth was restricted by higher agar concentrations but the overall timing of the swarming-plus-consolidation cycles remained constant. None of a variety of factors which had significant effects on colony expansion altered terracing frequencies at 32 degrees C, but the length of the swarming-plus-consolidation cycle was affected by temperature and medium enrichment. Some clinical isolates displayed significant differences in terracing frequencies at 32 degrees C. Our results defined a number of readily quantifiable parameters in swarm colony development. The data showed no connection between nutrient (glucose) depletion and the onset of different phases in swarm colony morphogenesis. Several observations point to the operation of density-dependent thresholds in controlling the transitions between distinct phases.

Agar↗

PubMed1996

Analysis of cell movement during the culmination phase of Dictyostelium development.

Co-ordinated cell movement of tens of thousands of cells and periodic signals characterise the multicellular development of the cellular slime mould Dictyostelium discoideum. We investigated cell movement by analysing time-lapse video recordings made during the slug stage and the culmination phase of Dictyostelium development. Slugs viewed from the side showed an even, straight forward movement with the tip slightly raised in the air. Slugs that had migrated for a prolonged period of time either culminated or showed a behaviour best described as abortive culmination. Culmination is initiated by a local aggregation of anterior-like cells at the base of the slug at the prestalk-prespore boundary, where they form a stationary mass of cells. Prespore cells continue to move forward over this stationary pile and, as a result, are lifted into the air. The stationary group of anterior-like cells thereby end up to the back of the slug. At this point the slug either falls back on the agar surface or continues culmination. If the slug continues to migrate these cells regain motility, move forward to the prespore-prestalk boundary and form a new pile again. In the case of culmination the neutral red stained cells in the pile move to the back of the slug and form a second signalling centre beside the tip. Both centres are characterised by vigorous rotational cell movement. The cells belonging to the basal centre will form the basal disc and the lower cup in the fruiting body. The upper cup will be formed by the prestalk cells rotating most vigorously at the prestalk-prespore boundary. The remaining neutral red stained anterior-like cells in the prespore zone sort either to the upper or lower organising centre in the fruiting body.

Animals↗

PubMed1996

The role of the cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress, ensure cell size, cell shape and motility, and contribute to phagocytosis and development.

We generated Dictyostelium double mutants lacking the two F-actin crosslinking proteins alpha-actinin and gelation factor by inactivating the corresponding genes via homologous recombination. Here we investigated the consequences of these deficiencies both at the single cell level and at the multicellular stage. We found that loss of both proteins severely affected growth of the mutant cells in shaking suspension, and led to a reduction of cell size from 12 microns in wild-type cells to 9 microns in mutant cells. Moreover the cells did not exhibit the typical polarized morphology of aggregating Dictyostelium cells but had a more rounded cell shape, and also exhibited an increased sensitivity towards osmotic shock and a reduced rate of phagocytosis. Development was heavily impaired and never resulted in the formation of fruiting bodies. Expression of developmentally regulated genes and the final developmental stages that were reached varied, however, with the substrata on which the cells were deposited. On phosphate buffered agar plates the cells were able to form tight aggregates and mounds and to express prespore and prestalk cell specific genes. Under these conditions the cells could perform chemotactic signalling and cell behavior was normal at the onset of multicellular development as revealed by time-lapse video microscopy. Double mutant cells were motile but speed was reduced by approximately 30% as compared to wild type. These changes were reversed by expressing the gelation factor in the mutant cells. We conclude that the actin assemblies that are formed and/or stabilized by both F-actin crosslinking proteins have a protective function during osmotic stress and are essential for proper cell shape and motility.

Actinin↗