Speed Of Cell Movement Research Articles

P12.14.B HIGHLY INVASIVE GLIOMA CELLS MOTILITY MEASUREMENTS ON A VARIETY OF EXTRACELLULAR MATRIX PROTEINS

Abstract BACKGROUND Glioblastoma multiforme (GBM) is a heterogeneous highly invasive malignant brain tumor. The extracellular matrix (ECM) produced by tumor microenvironment plays an important role in the regulation of cell motility and invasion into surrounding healthy tissue. In this research we determined the effect of various ECM proteins on the motility of primary tumor cells. MATERIAL AND METHODS Primary tumor cells were isolated from biopsy material obtained during GBM surgical removal. Cultivation of the piece of tumor (0.5 mm3) was carried out in the form of an 3D explant model at 37°C in 5% CO2-incubator. After several months, cells migrated from the tumor spontaneously formed spheroids. To assess the rate of cell migration, spheroids were seeded on 24-well plate coated with poly-L-lysine, fibrinogen, collagen type I, and Matrigel. After 3 days, the field of view in the well (n=9) was microphotographed every 15 min for 56 hours, after which the tracks of cells outside the spheroids were manually marked (n=50) on the obtained images. The average and maximum speeds were calculated, as well as the straightness of the track (the ratio of the Euclidean distance to the track length). The Mean square displacement (MSD) was plotted against time for each protein studied in comparison with random walk simulation calculated for the equal track parameters (number of steps, mean and sd) on correspondent protein. RESULTS Live-cell imaging of the patient-derived cells revealed the heterogeneous population of actively moving cells outside the spheroids. The following results of measuring cell motility were obtained (mean ± sd): (1) Mean speed on poly-L-lysine 4.3 ± 2.5 μm/h; on fibrinogen 11.1 ± 5.7 μm/h; on collagen type I 8.8 ± 5.1 μm/h; on Matrigel 9.7 ± 3.6 μm/h. (2) Max speed on poly-L-lysine 63.8 ± 30.1 μm/h; on fibrinogen 90.0 ± 38.9 μm/h; on collagen type I 96.1 ± 42.1 μm/h; on Matrigel 84.1 ± 34.1 μm/h. (3) Straightness on poly-L-lysine 0.24 ± 0.14 μm/h; on fibrinogen 0.18 ± 0.11 μm/h; on collagen type I 0.14 ± 0.09 μm/h; on Matrigel 0.20 ± 0.12 μm/h. MSD analysis showed the greatest contribution of random walk in the motility of cells on collagen type I, the smallest - on Matrigel. CONCLUSION Proteins of ECM affect not only the speed of cell movement, but also the tortuosity of their tracks. Further studies will clarify the mechanisms of GBM invasion depending on the composition of the ECM.

Investigation of electrical and viscoelastic parameters of erythrocytes in patients with autoimmune gastritis

Patients with autoimmune gastritis (AIG) often have anemia of various origins . Hematological disorders usually portend severe atrophy , and in many cases , are the only indicators of the disease . Aim of the study was to investigate the electrical and viscoelastic parameters of erythrocytes in patients with AIG for their possible use in diagnostics . Material and methods . 73 patients with AIG ( mean age 55.3 ± 12.54 years ) and 38 people of the control group were examined . Electrical and viscoelastic parameters of erythrocytes were studied by dielectrophoresis . Results . Statistically significant decrease in the average cell diameter , the proportion of discocytes cells and an increase in the proportion of spherocytes , deformed forms were found in the group of patients with AIG in combination with Helicobaсter рylori (H. pylori , H.p .) infection compared with healthy individuals . Patients with AIG had significantly lower levels of amplitude of deformation , membrane capacity , dipole moment , speed of cell movement to the electrodes , polarizability at high frequencies of the electric field ( 106 , 0.5×106 Hz ), relative polarizability , and , conversely , higher values of membrane conductivity , aggregation , destruction indexes , summarized viscosity , rigidity , than those in the comparison group . Between groups of patients with and without H. pylori infection , differences were found in indicators reflecting the surface charge of erythrocytes – the speed of movement to the electrodes (p = 0.019), the dipole moment (p < 0.001 ) and the state of the membranes – its capacity (p = 0.004). The diagnostic model , which includes three parameters of erythrocytes – the dipole moment , the speed of movement to the electrodes , the capacity of the cell membrane , provided high diagnostic accuracy of distinguishing AIG H.p . (+) and H.p . (–) – area under ROC curve AUC 0.925, sensitivity 92.4 %, specificity 89.7 %. Conclusions : Electrical and viscoelastic parameters of erythrocytes are promising in the diagnosis of AIG, including on the background of H. pylori infection .

Substrate stiffness modulates migration and local intercellular membrane motion in pulmonary endothelial cell monolayers.

The pulmonary artery endothelium forms a semipermeable barrier that limits macromolecular flux through intercellular junctions. This barrier is maintained by an intrinsic forward protrusion of the interacting membranes between adjacent cells. However, the dynamic interactions of these membranes have been incompletely quantified. Here, we present a novel technique to quantify the motion of the peripheral membrane of the cells, called paracellular morphological fluctuations (PMFs), and to assess the impact of substrate stiffness on PMFs. Substrate stiffness impacted large-length scale morphological changes such as cell size and motion. Cell size was larger on stiffer substrates, whereas the speed of cell movement was decreased on hydrogels with stiffness either larger or smaller than 1.25 kPa, consistent with cells approaching a jammed state. Pulmonary artery endothelial cells moved fastest on 1.25 kPa hydrogel, a stiffness consistent with a healthy pulmonary artery. Unlike these large-length scale morphological changes, the baseline of PMFs was largely insensitive to the substrate stiffness on which the cells were cultured. Activation of store-operated calcium channels using thapsigargin treatment triggered a transient increase in PMFs beyond the control treatment. However, in hypocalcemic conditions, such an increase in PMFs was absent on 1.25 kPa hydrogel but was present on 30 kPa hydrogel-a stiffness consistent with that of a hypertensive pulmonary artery. These findings indicate that 1) PMFs occur in cultured endothelial cell clusters, irrespective of the substrate stiffness; 2) PMFs increase in response to calcium influx through store-operated calcium entry channels; and 3) stiffer substrate promotes PMFs through a mechanism that does not require calcium influx.

Correlation and Differentiation Based Algorithms for Cell Mobility Quantification

Quantification of cell mobility in confluent cell cultures or on coverslips with high cell density is a challenge, because tracking of individual cells is not readily possible. Two procedures have been developed and tested to solve this problem, namely a differential (MoveDif) and a correlation (MoveCor) algorithm. Both compare successive image frames and calculate a difference parameter between them. Image preprocessing was required to neutralize brightness changes and inhomogeneous illumination. Additionally, structures and membranes in the images had to be enhanced and standardized by edge-detection filters. We showed that both algorithms had advantages in different experimental setups, depending of the complexity of the cellular movement. Besides the determination of global cell mobility, the membrane contractility and movement of single cells, small cell groups and intracellular structures could be quantified, if individual cells were separated and magnified. The discriminatory potency and sensitivity of both algorithms were tested by drug induced changes of cellular movement speeds and their robustness against brightness changes were analyzed too. To demonstrate the mobility effects of ketamine on dexamethasone-treated hippocampal neurons, we found that the correlation algorithm MoveCor could quantify the ketamine-dependent increase in cell movement speed after prior treatment with dexamethasone had reduced it distinctively. We also could measure how staurosporine slowed down the mobility of acidic intracellular organelles in highly magnified glial cells from chicken telencephalon. The differentiation algorithm MoveDif was employed to demonstrate that the contractility of pericyte membranes slowed down after induction of oxygen glucose deprivation. The limitations of both methods were given by their inability to produce absolute mobility values but these drawbacks can be solved by appropriate image preprocessing.

Stochastic model explains formation of cell arrays on H/O-diamond patterns.

Cell migration plays an important role in many biological systems. A relatively simple stochastic model is developed and used to describe cell behavior on chemically patterned substrates. The model is based on three parameters: the speed of cell movement (own and external), the probability of cell adhesion, and the probability of cell division on the substrate. The model is calibrated and validated by experimental data obtained on hydrogen- and oxygen-terminated patterns on diamond. Thereby, the simulations reveal that: (1) the difference in the cell movement speed on these surfaces (about 1.5×) is the key factor behind the formation of cell arrays on the patterns, (2) this difference is provided by the presence of fetal bovine serum (validated by experiments), and (3) the directional cell flow promotes the array formation. The model also predicts that the array formation requires mean distance of cell travel at least 10% of intended stripe width. The model is generally applicable for biosensors using diverse cells, materials, and structures.

Patterns of Proliferative Activity in the Colonic Crypt Determine Crypt Stability and Rates of Somatic Evolution

Epithelial cells in the colon are arranged in cylindrical structures called crypts in which cellular proliferation and migration are tightly regulated. We hypothesized that the proliferation patterns of cells may determine the stability of crypts as well as the rates of somatic evolution towards colorectal tumorigenesis. Here, we propose a linear process model of colonic epithelial cells that explicitly takes into account the proliferation kinetics of cells as a function of cell position within the crypt. Our results indicate that proliferation kinetics has significant influence on the speed of cell movement, kinetics of mutation propagation, and sensitivity of the system to selective effects of mutated cells. We found that, of all proliferation curves tested, those with mitotic activities concentrated near the stem cell, including the actual proliferation kinetics determined in in vivo labeling experiments, have a greater ability of delaying the rate of mutation accumulation in colonic stem cells compared to hypothetical proliferation curves with mitotic activities focused near the top of the crypt column. Our model can be used to investigate the dynamics of proliferation and mutation accumulation in spatially arranged tissues.

SDF and GABA interact to regulate axophilic migration of GnRH neurons

Stromal derived growth factor (SDF-1) and gamma-aminobutyric acid (GABA) are two extracellular cues that regulate the rate of neuronal migration during development and may act synergistically. The molecular mechanisms of this interaction are still unclear. Gonadotropin releasing hormone-1 (GnRH) neurons are essential for vertebrate reproduction. During development, these neurons emerge from the nasal placode and migrate through the cribriform plate into the brain. Both SDF-1 and GABA have been shown to regulate the rate of GnRH neuronal migration by accelerating and slowing migration, respectively. As such, this system was used to explore the mechanism by which these molecules act to produce coordinated cell movement during development. In the present study, GABA and SDF-1 are shown to exert opposite effects on the speed of cell movement by activating depolarizing or hyperpolarizing signaling pathways, GABA via changes in chloride and SDF-1 via changes in potassium. GABA and SDF-1 were also found to act synergistically to promote linear rather than random movement. The simultaneous activation of these signaling pathways, therefore, results in tight control of cellular speed and improved directionality along the migratory pathway of GnRH neurons.

Studying Cancer Cell Invasion in 3D Micro-Structural Matrices

Cellular mechanisms and external factors interact with each other during cell migration. To study cell-extracellular matrix interactions involved in cancer invasion, a collagen hydrogel-based three-dimensional (3D) model was adapted for cell culture and migration assays, in an attempt to recapitulate the mesenchymal stroma invasion. When different breast cancer cell lines were seeded inside the 3D gel, the fibrous collagen matrix only allowed the migration of highly metastatic cells.Controlling the polymerization of collagen monomer solution through concentration and ionic conditions yielded differences in the resulting fiber thickness and network structure, and, at the same time, variance of mechanical properties of the gel. Cells in the live state and the surrounding collagen fiber networks were monitored using confocal imaging techniques, followed by 3D cell tracking. Quantitative analysis revealed that cell movement speed and directionality had certain dependence on the matrix micro-structure and mechanical strength. Cells moved faster and more directionally as a group in a gel formed with lower collagen concentration or under lower PH value during polymerization. Stream-like cell aggregates were observed in matrices with thicker collagen fibers, while individual cell movement prevailed in other matrices. Pharmacological interventions targeting at various cell migration mechanisms, including cell-activated collagen matrix degradation and cytoskeletal dynamics, showed different drug effectiveness, depending on the matrix micro-structure. The findings indicate that the multimodality of cancer cell migration is related to the characteristics of the micro-environment.

Moving Live Dissociated Neurons With an Optical Tweezer

The use of an optical tweezer for moving dissociated neurons was studied. The main features of the tweezers are outlined as well as the general principles of its operation. Infrared beams at 980 and 1064 nm were used, focused so as to make a trap for holding neurons and moving them. Absorption by cells at those wavelengths is very small. Experiments were done to evaluate nonsticky substrate coatings, from which neurons could be easily lifted with the tweezers. The maximum speed of cell movement as a function of laser power was determined. Detailed studies of the damage to cells as a function of beam intensity and time of exposure were made. The 980 nm beam was much less destructive, for reasons that are not understood, and could be used to safely move cells through distances of millimeters in times of seconds. An illustrative application of the use of the tweezers to load neurons without damage into plastic cages on a glass substrate was presented. The conclusion is that optical tweezers are an accessible and practical tool for helping to establish neuron cultures of cells placed in specific locations.

PTEN regulates motility but not directionality during leukocyte chemotaxis

The localization at opposite cell poles of phosphatidylinositol-3 kinases and PTEN (phosphatase and tensin homolog on chromosome 10) governs Dictyostelium chemotaxis. To study this model in mammalian cells, we analyzed the dynamic redistribution of green fluorescent protein (GFP)-tagged PTEN chimeras during chemotaxis. N- or C-terminus GFP-tagged PTEN was distributed homogeneously in the cytoplasm of chemotaxing PTEN-negative Jurkat cells and PTEN-positive HL60 cells. Moreover, we did not detect uropod accumulation of endogenous PTEN in chemoattractant-stimulated HL60 cells. Cell fractionation indicated that both endogenous and ectopically expressed PTEN were confined largely to the cytosol, and that chemoattractant stimulation did not alter this location. PTEN re-expression in Jurkat cells or PTEN depletion by specific siRNA in HL60 cells did not affect cell gradient sensing; PTEN nonetheless modulated chemoattractant-induced actin polymerization and the speed of cell movement. The results suggest a role for PTEN in regulating actin polymerization, but not directionality during mammalian cell chemotaxis.

Immediate and long-term galvanotactic responses of Amoeba proteus to dc electric fields.

The long-term and immediate galvanotactic responses of Amoeba proteus to the direct current electric fields (dcEFs) were studied with the methods of computer-aided image analysis. It was found that in contrast to earlier reports, amoebae continued locomotion towards cathode (the negative pole) for hours and the increase in the field strength in the range 300-600 mV/mm caused the straightening of cell trajectories accompanied by the decreased frequency of the lateral pseudopods formation and lesser change in the speed of cell movement. In the cell regions pointing to the anode, the formation of new pseudopodia was prevented and the higher cEFs strength the more extended were the regions in which formation of new pseudopods was inhibited. Replacement of calcium with magnesium in the extracellular medium reduced the galvanotactic cell responses. Research on the localisation and kinetics of the primary cell responses to the dcEF or to change in its direction revealed that the primary cell responses occurred at the anode oriented cell regions. The cell response to the field reversal appeared to be localised and to take place in less than 1 sec. First the retraction and withdrawal of the anode-directed pseudopodium was observed whereas the uroid (cell tail) moved for 10-40 sec in the original direction before it begun to react to the field reversal. The exposure of amoebae to the dcEFs sensitised them to the reversion in the field direction and induced an acceleration of cell responses. The results presented are difficult to reconcile with the attempt to explain the cell galvanotaxis as a consequence of the membrane protein lateral electrophoresis or electroosmosis. It is suggested that the lateral electrophoresis of ions and the modification of ionic conditions at the vicinity of ion channels may be involved in the induction of fast responses of cells to external dcEFs.

Immediate and long-term galvanotactic responses of Amoeba proteus to dc electric fields

The long-term and immediate galvanotactic responses of Amoeba proteus to the direct current electric fields (dcEFs) were studied with the methods of computer-aided image analysis. It was found that in contrast to earlier reports, amoebae continued locomotion towards cathode (the negative pole) for hours and the increase in the field strength in the range 300–600 mV/mm caused the straightening of cell trajectories accompanied by the decreased frequency of the lateral pseudopods formation and lesser change in the speed of cell movement. In the cell regions pointing to the anode, the formation of new pseudopodia was prevented and the higher cEFs strength the more extended were the regions in which formation of new pseudopods was inhibited. Replacement of calcium with magnesium in the extracellular medium reduced the galvanotactic cell responses. Research on the localisation and kinetics of the primary cell responses to the dcEF or to change in its direction revealed that the primary cell responses occurred at the anode oriented cell regions. The cell response to the field reversal appeared to be localised and to take place in less than 1 sec. First the retraction and withdrawal of the anode-directed pseudopodium was observed whereas the uroid (cell tail) moved for 10–40 sec in the original direction before it begun to react to the field reversal. The exposure of amoebae to the dcEFs sensitised them to the reversion in the field direction and induced an acceleration of cell responses. The results presented are difficult to reconcile with the attempt to explain the cell galvanotaxis as a consequence of the membrane protein lateral electrophoresis or electroosmosis. It is suggested that the lateral electrophoresis of ions and the modification of ionic conditions at the vicinity of ion channels may be involved in the induction of fast responses of cells to external dcEFs. Cell Motil. Cytoskeleton 45:10–26, 2000. © 2000 Wiley-Liss, Inc.

Adhesion, alignment, and migration of cultured Schwann cells on ultrathin fibronectin fibres

Individual fibres of fibronectin (Fn-fibres), an extracellular matrix cell adhesion glycoprotein, were produced from a purified solution of fibronectin. These fibres range from 0.5–7 μm in width and have been engineered to produce mats (Fn-mats) by using a unidirectional shear force to orientate the fibres. Fn-fibres have been shown to promote alignment by contact guidance of human dermal fibroblasts, neurites, macrophages, and epitenon fibroblasts. Fn-mats have been used to orientate and enhance the regeneration of peripheral nerve components. We investigated cell spreading, orientation, formation of focal contacts, and the speed of cell movement on individual Fn-fibres, glass-covered with poly-L-lysine and poly-L-lysine/laminin/Fn. Fibronectin fibres significantly promoted cell spreading and the speed of cell migration with alignment of focal contacts and F-actin filaments to the axis of the fibres. The study reveals the potential of Fn-fibres to guide and direct cellular behaviour by contact guidance. The increase in migration and other behaviour exhibited by Schwann cells on Fn-fibres justifies the use of Fn-mats for peripheral nerve repair and is clinically important in that atrophy of the target organ, which is the most common failure of nerve repair, may be minimised. Cell Motil. Cytoskeleton 42:331–343, 1999. © 1999 Wiley-Liss, Inc.

Orchestration of neuronal migration by activity of ion channels, neurotransmitter receptors, and intracellular Ca2+ fluctuations

The real-time observation of cell movement in acute cerebellar slices reveals that granule cells alter their shape concomitantly with changes in the mode and rate of migration as they traverse different cortical layers. Although the origin of local environmental cues responsible for these position-specific changes in migratory behavior remains unclear, several signaling mechanisms involved in controlling granule cell movement have emerged. The onset of one such mechanism is marked by the expression of voltage-gated ion channels and neurotransmitter receptors in postmitotic cells prior to the initiation of their migration. Granule cells start their radial migration after the expression of N-type Ca2+ channels and the N-methyl-D-aspartate subtype of glutamate receptors on the plasmalemmal surface. Blockade of the channel or receptor activity significantly decreases the rate of cell movement, indicating that the activation of these membrane constituents provides an essential signal for the translocation of granule cells. Another signal that controls the rate of cell migration is embedded in the combined amplitude and frequency components of Ca2+ fluctuations in the somata of migrating granule cells. Interestingly, each phase of Ca2+ fluctuation controls a separate phase of saltatory movement in the granule cells: The cells move forward during the phase of transient Ca2+ elevation and remain stationary during the troughs. Consequently, the changes in the amplitude and frequency components of Ca2+ fluctuations directly affect granule cell movement: Reducing the amplitude or frequency of Ca2+ fluctuations slows down the speed of cell movement, while the enhancement of these components accelerates migration. These findings suggest that signaling molecules present in the local cellular milieu encountered on the migratory route control the shape and motility of granule cells by modifying Ca2+ fluctuations in the soma through the activation of specific ion channels and neurotransmitter receptors.

Epidermal growth factor alters fibroblast migration speed and directional persistence reciprocally and in a matrix-dependent manner.

Growth factors stimulate sustained cell migration as well as inducing select acute motility-related events such as membrane ruffling and disruption of focal adhesions. However, an in-depth understanding of the characteristics of sustained migration that are regulated by growth factor signals is lacking: how the biochemical signals are related to physical processes underlying locomotion, and how these events are coordinately influenced by interplay between growth factor and matrix substratum signals. To address these issues, we studied sustained migration of NR6 fibroblasts on a complex human matrix substratum, Amgel, comparing effects of epidermal growth factor (EGF) treatment across a range of Amgel levels. In the absence of EGF, cell migration speed and directional persistence are relatively independent of Amgel level, whereas in the presence of EGF speed is increased at intermediate Amgel levels but not at low and high Amgel levels while directional persistence is decreased at intermediate but not at low and high Amgel levels. The net effect of EGF is to increase the frequency of changes in the cell direction, and at the same time to slightly increase the path-length and thereby greatly enhance random dispersion of cells. Despite increasing migration speed during long-term sustained migration EGF treatment does not lead to significantly increased absolute rates of membrane extension in contrast to its well-known elicitation of membrane ruffling in the short term. However, EGF treatment does decrease cell spread area, yielding an apparent enhancement of specific membrane extension rate, i.e. normalized to cell spread area. Cell movement speed and directional persistence are thus, respectively, directly related and indirectly related to the increase in specific membrane extension rate (alternatively, the decrease in cell spread area) induced by EGF treatment during sustained migration. These results indicate that growth factor and matrix substrata coordinately regulate sustained cell migration through combined governance of underlying physical processes.

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.

Adhesion, orientation, and movement of cells cultured on ultrathin fibronectin fibers.

This study examined the behavior of rat tendon fibroblasts, baby hamster kidney fibroblasts, macrophage-like P388D1 cells, and neurons from rat dorsal root ganglia, cultured on fibronectin strands 0.2-5 micrograms in diameter. We investigated cell spreading, orientation, formation of focal contacts, the speed of cell movement, and the speed of neurite outgrowth in cells cultured on fibronectin strands, glass covered with fibronectin, and plain, nontreated glass. Fibronectin strands significantly promoted cell spreading and caused a marked alignment of all kinds of cells to the direction of the fiber. The fibers caused the alignment of actin filaments in fibroblasts and focal contacts in fibroblasts and macrophages and increased polymerization of F-actin in cells. Fibronectin fibers also increased the speed and persistence of cell movement and the rate of neurite outgrowth. Macrophages grown on fibronectin fibers produced numerous actin-rich microspikes and adopted a polarized, migratory phenotype. These findings indicate that fibronectin strands, resembling natural components of the extracellular matrix, are more effective in activating various types of cells then two-dimensional, fibronectin-covered substrata. The results also confirm the suitability of the three-dimensionally oriented fibronectin form for use in clinical practice.

Analysis of Optical Density Wave Propagation and Cell Movement during Mound Formation inDictyostelium discoideum

Aggregation fields ofDictyosteliumamoebae 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 μm/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.

Activation of macrophage-like cells by multiple grooved substrata. Topographical control of cell behaviour.

We studied the influence of substrata topography on the behaviour of murine P388D1 macrophage cell line. Cells were plated on plain fused silica substrata or substrata with microfabricated grooves of varying depth and width. Cell spread area, elongation, orientation and F-actin content were measured on plain substratum and 6 sets of gratings. The speed and persistence of cell movement were also studied. We found that patterned substrata substantially activated cell spreading and elongation and significantly increased the persistence and speed of cell movement, shallow grooves being more effective than deep ones. The contact of cells with micropatterned substrata significantly increased the F-actin content in cells. The sensitivity of LPS (lipopolisaccharide) stimulated and unstimulated macrophages to topographical cues was also compared.

Experimental specification of cell sorting, tissue spreading, and specific spatial patterning by quantitative differences in cadherin expression.

The sorting-out of embryonic cells from a cell mixture and the selective spreading of one cell population over the surface of another have been attributed to various causes. These include differentials in chemotaxis, in cellular adhesiveness, in cell surface contractility, in speed of cell movement, and in the timing of postulated changes in cellular adhesive and motile properties. One of us earlier predicted on mathematical grounds that two motile cell types differing only in the level of expression of a single cell adhesion system should not only segregate from one another but also arrange themselves with the less cohesive cells enveloping a core of the more cohesive ones. To test these predictions, we combined two populations of L cells transfected with P-cadherin cDNA and expressing this homophilic adhesion molecule in substantially differing amounts. When the two cell populations were intermixed, they segregated to approach a sphere-within-a-sphere configuration, the cell population expressing more P-cadherin forming islands which fused to become an internal "medulla." When the two cell populations were first formed into separate aggregates which were subsequently allowed to fuse, the cell population expressing more P-cadherin was enveloped by its partner, which formed an external "cortex." These observations confirm the early prediction and support the conclusion that both morphogenetic movements and the specific anatomical configurations to which they lead can be determined by particular sets of intercellular adhesive intensities, regardless of how these are generated and in the absence of differentials in other parameters.