Biomedical subjects

Cornelis J Weijer

Publications and source records attributed to Cornelis J Weijer.

12 recordsLinked to original sources

PubMed2005

Paxillin is required for cell-substrate adhesion, cell sorting and slug migration during Dictyostelium development.

Paxillin is a key regulatory component of focal adhesion sites, implicated in controlling cell-substrate interactions and cell movement. We analyse the function of a Dictyostelium discoideum paxillin homologue, PaxB, which contains four highly conserved LD and four LIM domains, but lacks two characteristic tyrosine residues, that form the core of vertebrate SH2-binding domains. PaxB is expressed during growth and all stages of development, but expression peaks during slug formation. Using a paxB-gfp knockin strain we show the existence of focal adhesions and characterise their dynamics. During multicellular development PaxB is not only found in focal adhesions at the cell-substrate interface, but also in the tips of filopodial structures predominantly located at the trailing ends of cells. paxB- strains are less adhesive to the substrate, they can aggregate but multicellular development from the mound stage onwards is severely impeded. paxB- strains are defective in proper cell type proportioning, cell sorting, slug migration and form-defective fruiting bodies. Mutation of a conserved JNK phosphorylation site, implicated in the control of cell migration, does not have any major effects on cell sorting, slug migration or morphogenesis in Dictyostelium. PaxB does not appear to function redundantly with its closest relative Lim2 (paxA), which when deleted also results in a mound arrest phenotype. However, analysis of paxA- and paxB- single and double null mutants suggest that PaxB may act upstream of Lim2.

Amino Acid Sequence↗

PubMed2005

Analysis of tissue flow patterns during primitive streak formation in the chick embryo.

We have investigated the patterns of tissue flow underlying the formation of the primitive streak in the chick embryo. Analysis of time-lapse sequences of brightfield images to extract the tissue velocity field and of fluorescence images of small groups of DiI-labelled cells have shown that epiblast cells move in two large-scale counter-rotating streams, which merge at the site of streak formation. Despite the large-scale tissue flows, individual cells appear to move little relative to their neighbours. As the streak forms, it elongates in both the anterior and posterior directions. Inhibition of actin polymerisation via local application of the inhibitor latrunculin A immediately terminates anterior extension of the streak tip, but does not prevent posterior elongation. Inhibition of actin polymerisation at the base of the streak completely inhibits streak formation, implying that continuous movement of cells into the base of the forming streak is crucial for extension. Analysis of cycling cells in the early embryo shows that cell-cycle progression in the epiblast is quite uniform before the primitive streak forms then decreases in the central epiblast and incipient streak and increases at the boundary between the area pellucida and area opaca during elongation. The cell-cycle inhibitor aphidicolin, at concentrations that completely block cell-cycle progression, permits initial streak formation but arrests development during extension. Our analysis suggests that cell division maintains the cell-flow pattern that supplies the streak with cells from the lateral epiblast, which is critical for epiblast expansion in peripheral areas, but that division does not drive streak formation or the observed tissue flow.

Actins↗

PubMed2005

Signaling to cytoskeletal dynamics during chemotaxis.

We review insights in signaling pathways controlling cell polarization and cytoskeletal organization during chemotactic movement in Dictyostelium amoebae and neutrophils. We compare and contrast these insights with our current understanding of pathways controlling chemotactic movements in more-complex multicellular developmental contexts.

Actins↗

PubMed2004

In vivo analysis of 3-phosphoinositide dynamics during Dictyostelium phagocytosis and chemotaxis.

Phagocytosis and chemotaxis are receptor-mediated processes that require extensive rearrangements of the actin cytoskeleton, and are controlled by lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. We used a panel of pleckstrin homology (PH) domains with distinct binding specificities for PtdIns(3,4,5)P3 and PtdIns(3,4)P2 to study the spatiotemporal dynamics of these phosphoinositides in vivo. During phagocytosis and macropinocytosis PtdIns(3,4,5)P3 levels transiently increased at sites of engulfment, followed by a rapid PtdIns(3,4)P2 production round the phagosome/macropinosome upon its internalisation, suggesting that PtdIns(3,4,5)P3 is degraded to PtdIns(3,4)P2. PTEN null mutants, which are defective in phagocytosis, showed normal rates of PtdIns(3,4,5)P3 degradation, but unexpectedly an accelerated PtdIns(3,4)P2 degradation. During chemotaxis to cAMP only PtdIns(3,4,5)P3 was formed in the plasma membrane, and no PtdIns(3,4)P2 was detectable, showing that all PtdIns(3,4,5)P3 was degraded by PTEN to PtdIns(4,5)P2. Furthermore, we showed that different PtdIns(3,4,5)P3 binding PH domains gave distinct spatial and temporal readouts of the same underlying PtdIns(3,4,5)P3 signal, enabling distinct biological responses to one signal.

Animals↗

PubMed2004

Dictyostelium morphogenesis.

During starvation-induced Dictyostelium development, up to several hundred thousand amoeboid cells aggregate, differentiate and form a fruiting body. The chemotactic movement of the cells is guided by the rising phase of the outward propagating cAMP waves and results in directed periodic movement towards the aggregation centre. In the mound and slug stages of development, cAMP waves continue to play a major role in the coordination of cell movement, cell-type-specific gene expression and morphogenesis; however, in these stages where cells are tightly packed, cell-cell adhesion/contact-dependent signalling mechanisms also play important roles in these processes.

Animals↗

PubMed2003

The cyclase-associated protein CAP as regulator of cell polarity and cAMP signaling in Dictyostelium.

Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.

Actins↗

PubMed2003

Modelling of Dictyostelium discoideum slug migration.

The development of most multicellular organisms involves differential movement of cells resulting in the formation of tissues. The principles governing these movements are poorly understood. One exception is the formation of the slug in the social amoebae Dictyostelium discoideum. The slug forms by the chemotactic aggregation of up to 10(5) starving cells, it is motile and migrates in response to light and temperature gradients to the surface of the soil to form a fruiting body consisting of a stalk supporting a spore head. Slug migration and behaviour result from coordinated chemotactic movement of the individual cells in the slug. Waves of a chemoattractant, most likely cAMP, are periodically initiated in the tip of the slug and propagate towards the back of the slug resulting in periodic forward movement of individual cells as well as the whole slug. Here we develop a model to investigate how wave propagation and cell movement interacts to result in migration and shape changes of the slug. The slug tissue is modelled as an incompressible liquid, in which waves of chemoattractant are generated in an excitable manner. The liquid is "active", i.e. it is able to generate body forces in response to the gradients of the chemoattractant. These forces lead to the formation of flows (representing chemotactically moving cells) and result in slug movement and shape changes. The model provides a theoretical framework for the understanding of the interactions between cell-cell signalling and cell movement, which govern slug behaviour and tissue morphogenesis.

Animals↗

PubMed2003

Visualizing signals moving in cells.

Cells display a highly complex spatiotemporal organization, required to exert a wide variety of different functions, for example, detection, processing, and propagation of nerve impulses by neurons; contraction and relaxation by muscle cells; movement by leukocytes; and adsorption and secretion of nutrients and metabolites by epithelial cells lining the gut. Successful execution of these complex processes requires highly dynamic information transfer between different regions and compartments within cells. Through the development of fluorescent sensors for intracellular signaling molecules coupled with improved microscopic imaging techniques, it has now become possible to investigate signal propagation in cells with high spatial and temporal resolution.

Animals↗

PubMed2003

Chemotactic cell movement during development.

Chemotaxis is an important mechanism controlling cell migration over either short or long distances during different developmental processes. Small rapid diffusing chemo-attractants are detected through serpentine, G protein coupled receptors through graded activation of receptors along the length of the cell. Internal amplification results in polarisation of the actin and myosin cytoskeletal dynamics along the gradient and directed movement. The dynamics of these processes can now be studied in individual cells in developing organisms. Slow diffusing chemo-attractants such as growth factors, providing short-range guidance information, often signal through tyrosine kinase receptors. Detection of these signals may involve the active extension of very long cellular process up growth factor gradients, followed by translocation of the cell in the direction of the gradient.

Animals↗

PubMed2002

Visualizing PI3 kinase-mediated cell-cell signaling during Dictyostelium development.

BACKGROUND: Starving amoebae of Dictyostelium discoideum communicate by relaying extracellular cAMP signals, which direct chemotactic movement, resulting in the aggregation of thousands of cells into multicellular aggregates. Both cAMP relay and chemotaxis require the activation of PI3 kinase signaling. The spatiotemporal dynamics of PI3 kinase signaling can be followed in individual cells via the cAMP-induced membrane recruitment of a GFP-tagged PH domain-containing protein, CRAC, which is required for the activation of adenylylcyclase. RESULTS: We show that polarized periodic CRAC-GFP translocation occurs during the aggregation and mound stages of development in response to periodic cAMP signals. The duration of CRAC translocation to the membrane is determined by the duration of the rising phase of the cAMP signal. The system shows rapid adaptation and responds to the rate of change of the extracellular cAMP concentration. When the cells are in close contact, it takes 10 s for the signal to propagate from one cell to the next. In slugs, all cells show a permanent polarized PI3 kinase signaling in their leading edge, which is dependent on cell-cell contact. CONCLUSIONS: Measuring the redistribution of GFP-tagged CRAC has enabled us to study the dynamics of PI3 kinase-mediated cell-cell communication at the individual cell level in the multicellular stages of Dictyostelium development. This approach should also be useful to study the interactions between cell-cell signaling, cell polarization, and movement in the development of other organisms.

Animals↗

PubMed2002

Simultaneous quantification of cell motility and protein-membrane-association using active contours.

We present a new method for the quantification of dynamic changes in fluorescence intensities at the cell membrane of moving cells. It is based on an active contour method for cell-edge detection, which allows tracking of changes in cell shape and position. Fluorescence intensities at specific cortical subregions can be followed in space and time and correlated with cell motility. The translocation of two GFP tagged proteins (CRAC and GRP1) from the cytosol to the membrane in response to stimulation with the chemoattractant cAMP during chemotaxis of Dictyostelium cells and studies of the spatio-temporal dynamics of this process exemplify the method: We show that the translocation can be correlated with motility parameters and that quantitative differences in the rate of association and dissociation from the membrane can be observed for the two PH domain containing proteins. The analysis of periodic CRAC translocation to the leading edge of a cell responding to natural cAMP waves in a mound demonstrates the power of this approach. It is not only capable of tracking the outline of cells within aggregates in front of a noisy background, but furthermore allows the construction of spatio-temporal polar plots, capturing the dynamics of the protein distribution at the cell membrane within the cells' moving co-ordinate system. Compilation of data by means of normalised polar plots is suggested as a future tool, which promises the so-far impossible practicability of extensive statistical studies and automated comparison of complex spatio-temporal protein distribution patterns.

Algorithms↗

PubMed2002

Cell movement patterns during gastrulation in the chick are controlled by positive and negative chemotaxis mediated by FGF4 and FGF8.

During gastrulation in amniotes, epiblast cells ingress through the primitive streak and migrate away to form endodermal, mesodermal, and extraembryonic structures. Here we analyze the detailed movement trajectories of cells emerging at different anterior-posterior positions from the primitive streak, using in vivo imaging of the movement of GFP-tagged streak cells. Cells emerging at different anterior-posterior positions from the streak show characteristic cell migration patterns, in response to guidance signals from neighboring tissues. Streak cells are attracted by sources of FGF4 and repelled by sources of FGF8. The observed movement patterns of anterior streak cells can be explained by an FGF8-mediated chemorepulsion of cells away from the streak followed by chemoattraction toward an FGF4 signal produced by the forming notochord.

Animals↗