Three-dimensional in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion

During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting in a myosin II regulatory light chain (RLC) null in which tips fail to form. In RLC-null mounds, ecmAO prestalk cells formed an initial cluster that began to move to the mound apex, but then arrested as a vertical column that extended from the mound's apex to its base. Mixing experiments with wild-type cells demonstrated that the RLC-null ecmAO prestalk-cell defect is cell autonomous. These observations define a specific mechanism for myosin's function in tip formation, namely a mechanical role in the upward movement of the ecmAO prestalk cluster. The wild-type data demonstrate that cell sorting can occur in two steps, suggesting that, in this Ax3 strain, spatially and temporally distinct cues may guide prestalk cells first to an initial cluster and then later to the tip.     

gp150蛋白在盘基网柄菌发育中的作用及粘附分子间关系的分析

饥饿的盘基网柄菌进入多细胞发育期 ,在发育早期 ,AK12 7细胞 (gp150突变细胞 )能表达DdCAD 1和 gp80两种粘附分子 ,但它们不足以促进细胞继续发育 ,发育停留在细胞疏松结合阶段。粘附分子 gp150调节的细胞与细胞间的粘着影响了细胞丘“突出”的形成 ,由此影响了盘基网柄菌多细胞发育的形态发生。TL93细胞 (DdCAD 1和gp80突变细胞 )能完成发育。主要原因是在细胞流发育阶段就表达了gp150分子 ,在细胞粘着的功能上有替代DdCAD 1和 gp80的作用。因此 gp150蛋白对盘基网柄菌多细胞发育有着不可或缺的作用     

Modelling Dictyostelium discoideum morphogenesis: the culmination

The culmination of the morphogenesis of the cellular slime mould Dictyostelium discoideum involves complex cell movements which transform a mound of cells into a globule of spores on a slender stalk. We show that cyclic AMP signalling and differential adhesion, combined with cell differentiation and slime production, are sufficient to produce the morphogenetic cell movements which lead to culmination. We have simulated the process of culmination using a hybrid cellular automata/partial differential equation model. With our model we have been able to reproduce the main features that occur during culmination, namely the straight downward elongation of the stalk, its anchoring to the substratum and the formation of the long thin stalk topped by the spore head. We conclude that the cyclic AMP signalling system is responsible for the elongation and anchoring of the stalk, but in a roundabout way: pressure waves that are induced by the chemotaxis towards cyclic AMP squeeze the stalk through the cell mass. This mechanism forces the stalk to elongate precisely in the direction opposite to that of the chemotactically moving cells. The process turns out to be ‘guided’ by inactive ‘pathfinder’ cells, which form the tip of the stalk. We show that the entire development is enacted by means of the aforementioned building blocks. This means that no global gradients or different modes of chemotaxis are needed to complete the culmination. MPEG movies of the simulations are available on-line: http://www-binf.bio. uu.nl/stan/bmb.     

A gene encoding a novel anti-adhesive protein is expressed in growing cells and restricted to anterior-like cells during development of Dictyostelium

The Dictyostelium gene ampA, initially identified by the D11 cDNA, encodes a novel anti-adhesive-like protein. The ampA gene product inhibits premature cell agglutination during growth and modulates cell-cell and cell-substrate adhesion during development. Analysis of the promoter indicates that cap site-proximal sequence directs ampA expression during both growth and early development. Expression following tip formation is controlled by more distal sequence, which contains TTGA repeats known to regulate prestalk cell gene expression in other promoters. Comparison of reporter gene expression and endogenous mRNA accumulation indicates that during growth the ampA gene is expressed in an increasing number of cells as a function of density. The number of cells expressing the ampA gene drops as development initiates, but the cells that continue to express the gene do so at high levels. These cells are initially scattered throughout the entire aggregate. By the tip formation stage, however, the majority of ampA-expressing cells are localized to the mound periphery, with only a few cells remaining scattered in the upper portion of the mound. In the final culminant, ampA is expressed only in the upper cup, lower cup, and basal disc. Although reporter expression is observed in cells that migrate anteriorly to a banded region just posterior to the tip, expression is rarely observed in the extreme tip. AmpA protein however, is localized to the tip as well as to ALCs during late development. The results presented here suggest that ampA gene expression is shut off in ALCs that continue along the prestalk differentiation pathway before they are added to the primordial stalk.     

Orientation of cells during slug formation inDictyostelium

Summary Scanning electron microscopic observations ofDictyostelium discoideum cell masses during slug formation revealed two populations around the anterior tip; one group of cells resembled elongated aggregation stream cells and their orientation suggested that they move to the tip, whereas the other group of cells were isodiametric and showed no obvious orientation. In seeking further evidence for a role of differential cAMP chemotaxis in the orientation and movement of slug cells the anterior prestalk cells were compared to the posterior prespore cells in two chemotaxis tests. When a cell mass is placed on cAMP agar the prestalk cells exhibited better movement to cAMP sources but when the gradient was generated in a diffusion chamber the prestalk cells did not. This evidence suggested that the cells which are better able to generate a cAMP gradient might form part of the anterior zone of the slug.     

Computational Modeling Reveals that a Combination of Chemotaxis and Differential Adhesion Leads to Robust Cell Sorting during Tissue Patterning

Robust tissue patterning is crucial to many processes during development. The "French Flag" model of patterning, whereby naïve cells in a gradient of diffusible morphogen signal adopt different fates due to exposure to different amounts of morphogen concentration, has been the most widely proposed model for tissue patterning. However, recently, using time-lapse experiments, cell sorting has been found to be an alternative model for tissue patterning in the zebrafish neural tube. But it remains unclear what the sorting mechanism is. In this article, we used computational modeling to show that two mechanisms, chemotaxis and differential adhesion, are needed for robust cell sorting. We assessed the performance of each of the two mechanisms by quantifying the fraction of correct sorting, the fraction of stable clusters formed after correct sorting, the time needed to achieve correct sorting, and the size variations of the cells having different fates. We found that chemotaxis and differential adhesion confer different advantages to the sorting process. Chemotaxis leads to high fraction of correct sorting as individual cells will either migrate towards or away from the source depending on its cell type. However after the cells have sorted correctly, there is no interaction among cells of the same type to stabilize the sorted boundaries, leading to cell clusters that are unstable. On the other hand, differential adhesion results in low fraction of correct clusters that are more stable. In the absence of morphogen gradient noise, a combination of both chemotaxis and differential adhesion yields cell sorting that is both accurate and robust. However, in the presence of gradient noise, the simple combination of chemotaxis and differential adhesion is insufficient for cell sorting; instead, chemotaxis coupled with delayed differential adhesion is required to yield optimal sorting.     

Unexpected localisation of cells expressing a prespore marker of Dictyostelium discoideum

Abstract. We show that the anterior, prestalk region of the Dictyostelium slug contains cells which express, or have expressed, a prespore-specific marker. We term these cells "prespore-like cells" (PLC). In newly formed slugs there is a sharp prespore/prestalk boundary, with very few PLC, but after several days of migration the clear demarcation between prespore and prestalk zones breaks down because the number of PLC increases dramatically. This is consistent with previous observations showing there to be rapid interchange of cells between the prestalk and prespore regions. This is not, however, their only source, as a scattering of PLC appear when separate prestalk and prespore regions first become apparent at the time of tip formation. Also, at culmination, there is respecification of "prespore" cells at the pre-stalk/prespore boundary to form part of the mature stalk. The existence of these cells, and of PLC, may explain why we find prespore-specific mRNAs in mature stalk cells.     

FrzCD, a methyl-accepting taxis protein from Myxococcus xanthus, shows modulated methylation during fruiting body formation.

The frizzy (frz) genes of Myxococcus xanthus are required to control directed motility during vegetative growth and fruiting body formation. FrzCD, a protein homologous to the methyl-accepting chemotaxis proteins from enteric bacteria, is modified by methylation in response to environmental conditions. Transfer of cells from rich medium to fruiting medium initially caused rapid demethylation of FrzCD. Subsequently, the amount of FrzCD increased, but most remained unmethylated. At about the time of mound formation (9 h), most of the FrzCD was converted to methylated forms. Dispersal of developing cells (10 h) in buffer led to the demethylation of FrzCD, whereas concentration of these cells caused methylation of FrzCD. Some mutants which were unable to form fruiting bodies still modified their FrzCD during incubation under conditions of starvation on a surface.     

Origins of the prestalk-prespore pattern in Dictyostelium development

Using cell-autonomous markers we have traced the origins of prespore cells and two types of prestalk cells (pstA and pstB cells) during slug formation. We show that cell sorting and positional information both contribute to Dictyostelium morphogenesis. The initial pattern established at the mound stage is topologically quite different from that of the slug. Confirming previous studies, we find that prespore cells occupy most of the aggregate but are absent from a thin layer at the base and from the emerging tip. PstB cells are almost entirely localized to the basal region during the early stages of tip formation. Thus prespore and pstB cell differentiation appear to occur in response to localized morphogenetic signals. In the case of pstB cells, these signals presumably emanate from the base and not, as might be expected, from the tip. When first detectable, pstA cells are scattered throughout the aggregate. They then appear to migrate to the apex, where the tip forms.     

Regulation of Dictyostelium morphogenesis by RapGAP3

Rap1 is a key regulator of cell adhesion and cell motility in Dictyostelium. Here, we identify a Rap1-specific GAP protein (RapGAP3) and provide evidence that Rap1 signaling regulates cell-cell adhesion and cell migration within the multicellular organism. RapGAP3 mediates the deactivation of Rap1 at the late mound stage of development and plays an important role in regulating cell sorting during apical tip formation, when the anterior-posterior axis of the organism is formed, by controlling cell-cell adhesion and cell migration. The loss of RapGAP3 results in a severely altered morphogenesis of the multicellular organism at the late mound stage. Direct measurement of cell motility within the mound shows that rapGAP3⁻ cells have a reduced speed of movement and, compared to wild-type cells, have a reduced motility towards the apex. rapGAP3⁻ cells exhibit some increased EDTA/EGTA sensitive cell-cell adhesion at the late mound stage. RapGAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation, which is dependent on F-actin polymerization. We suggest that the altered morphogenesis and the cell-sorting defect of rapGAP3⁻ cells may result in reduced directional movement of the mutant cells to the apex of the mound.     

Specific roles of Rac1 and Rac2 in motile functions of HT1080 fibrosarcoma cells

Rho family proteins are constitutively activated in the highly invasive human fibrosarcoma HT1080 cells. We now investigated the specific roles of Rac1 and Rac2 in regulating morphology, F-actin organization, adhesion, migration, and chemotaxis of HT1080 cells. Downregulation of Rac1 using specific siRNA probes resulted in cell rounding, markedly decreased spreading, adhesion, and chemotaxis of HT1080 cells. 2D migration on laminin-coated surfaces in contrast was not markedly affected. Selective Rac2 depletion did not affect cell morphology, cell adhesion, and 2D migration, but significantly reduced chemotaxis. Downregulation of both Rac1 and Rac2 resulted in an even more marked reduction, but not complete abolishment, of chemotaxis indicating distinct as well as overlapping roles of both proteins in chemotaxis. Rac1 thus is selectively required for HT1080 cell spreading and adhesion whereas Rac1 and Rac2 are both required for efficient chemotaxis.     

The impact of exclusion processes on angiogenesis models

Angiogenesis is the process by which new blood vessels form from existing vessels. During angiogenesis, tip cells migrate via diffusion and chemotaxis, new tip cells are introduced through branching, loops form via tip-to-tip and tip-to-sprout anastomosis, and a vessel network forms as endothelial cells, known as stalk cells, follow the paths of tip cells (a process known as the snail-trail). Using a mean-field approximation, we systematically derive one-dimensional non-linear continuum models from a lattice-based cellular automaton model of angiogenesis in the corneal assay, explicitly accounting for cell volume. We compare our continuum models and a well-known phenomenological snail-trail model that is linear in the diffusive, chemotactic and branching terms, with averaged cellular automaton simulation results to distinguish macroscale volume exclusion effects and determine whether linear models can capture them. We conclude that, in general, both linear and non-linear models can be used at low cell densities when single or multi-species exclusion effects are negligible at the macroscale. When cell densities increase, our non-linear model should be used to capture non-linear tip cell behavior that occurs when single-species exclusion effects are pronounced, and alternative models should be derived for non-negligible multi-species exclusion effects.

     

The Dictyostelium LIM domain-containing protein LIM2 is essential for proper chemotaxis and morphogenesis

We have identified limB, a gene encoding a novel LIM domain-containing protein, LIM2, in a screen for genes required for morphogenesis. limB null cells aggregate, although poorly, but they are unable to undergo morphogenesis, and the aggregates arrest at the mound stage. limB null cells exhibit an aberrant actin cytoskeleton and have numerous F-actin-enriched microspikes. The cells exhibit poor adhesion to a substratum and do not form tight cell-cell agglomerates in suspension. Furthermore, limB null cells are unable to properly polarize in chemoattractant gradients and move very poorly. Expression of limB from a prestalk-specific but not a prespore-specific promoter complements the morphogenetic defects of the limB null strain, suggesting that the limB null cell developmental defect results from an inability to properly sort prestalk cells. LIM2 protein is enriched in the cortex of wild-type cells, although it does not colocalize with the actin cytoskeleton. Our analysis indicates that LIM2 is a new regulatory protein that functions to control rearrangements of the actin cytoskeleton and is required for cell motility and chemotaxis. Our findings may be generally applicable to understanding pathways that control cell movement and morphogenesis in all multicellular organisms. Structure function studies on the LIM domains are presented.     

Biochemical and genetic analysis of pre-stalk specific acid phosphatase in Dictyostelium

The only known enzymatic marker of pre-stalk cells at the slug stage of development in Dictyostelium discoideum is an isozyme of acid phosphatase, AP2. There is another isozyme of acid phosphatase, AP1, which is present in vegetative cells and is not cell-type specific. We have purified these isozymes and find they differ in K_m and thermostability. Both isozymes are affected by mutations in a single locus, acpA. Two mutations in the acpA locus abolished all activity of both AP1 and AP2 while a third mutation reduced the activity and altered the thermostability of both isozymes. It is likely that acpA is the structural gene responsible for both AP1 and AP2. The cell-type specificity of AP2 appears to result from differences in the modification of the acpA gene product between pre-spore and pre-stalk cells. The resulting difference in AP2 provides a useful marker for pre-stalk cells.     

Glycogen degradation during migration of presumptive cell types in Dictyostelium discoideum.

During the time course of differentiation in Dictyostelium discoideum, glycogen was found to accumulate from the amoebae stage to the culmination stage of development. Upon sorocarp formation (23 h), glycogen was rapidly degraded. Ultramicrotechniques, utilizing amplification of glycogen by enzymatic cycling, were used to follow glycogen metabolism in pre-stalk and prespore cells during the differentiation cycle. Both cell types accumulated glycogen at nearly the same rate. By the pseudoplasmodium stage of development glycogen had accumulated to 50% of its maximum value, and no differences were found between pre-stalk and pre-spore cells. Glycogen was degraded as pre-stalk cells migrated into the position for stalk construction. At the culmination stage of development stalk cells near the base were devoid of glycogen while pre-stalk cells near the apex of the stalk showed no loss of glycogen. The complete loss of glycogen from stalk cells occurred over a distance occupied by approximately 100 cells, and over a time period of approx. 1 h. Pre-spore cells at the culmination stage showed no loss of glycogen even though separated from stalk cells by only a thin cellulose sheath. The degradation of prespore cell glycogen did not commence until stalk construction was completed and the pre-spore mass had reached the apex of the stalk. Pre-spore cells at the culmination stage contained high levels of glycogen while only 2 h later, total degradation had occurred.     

Molecular uncoupling of fractalkine-mediated cell adhesion and signal transduction. Rapid flow arrest of CX3CR1-expressing cells is independent of G-protein activation

Fractalkine is a novel multidomain protein expressed on the surface of activated endothelial cells. Cells expressing the chemokine receptor CX3CR1 adhere to fractalkine with high affinity, but it is not known if adherence requires G-protein activation and signal transduction. To investigate the cell adhesion properties of fractalkine, we created mutated forms of CX3CR1 that have little or no ability to transduce intracellular signals. Cells expressing signaling-incompetent forms of CX3CR1 bound rapidly and with high affinity to immobilized fractalkine in both static and flow assays. Video microscopy revealed that CX3CR1-expressing cells bound more rapidly to fractalkine than to VCAM-1 (60 versus 190 ms). Unlike VCAM-1, fractalkine did not mediate cell rolling, and after capture on fractalkine, cells did not dislodge. Finally, soluble fractalkine induced intracellular calcium fluxes and chemotaxis, but it did not activate integrins. Taken together these data provide strong evidence that CX3CR1, a seven-transmembrane domain receptor, mediates robust cell adhesion to fractalkine in the absence of G-protein activation and suggest a novel role for this receptor as an adhesion molecule.     

Characterization of the GbpD-activated Rap1 pathway regulating adhesion and cell polarity in Dictyostelium discoideum

The regulation of cell polarity plays an important role in chemotaxis. GbpD, a putative nucleotide exchange factor for small G-proteins of the Ras family, has been implicated in adhesion, cell polarity, and chemotaxis in Dictyostelium. Cells overexpressing GbpD are flat, exhibit strongly increased cell-substrate attachment, and extend many bifurcated and lateral pseudopodia. These cells overexpressing GbpD are severely impaired in chemotaxis, most likely due to the induction of many protrusions rather than an enhanced adhesion. The GbpD-overexpression phenotype is similar to that of cells overexpressing Rap1. Here we demonstrate that GbpD activates Rap1 both in vivo and in vitro but not any of the five other characterized Ras proteins. In a screen for Rap1 effectors, we overexpressed GbpD in several mutants defective in adhesion or cell polarity and identified Phg2 as Rap1 effector necessary for adhesion, but not cell polarity. Phg2, a serine/threonine-specific kinase, directly interacts with Rap1 via its Ras association domain.     

A Novel Ras-interacting Protein Required for Chemotaxis and Cyclic Adenosine Monophosphate Signal Relay in Dictyostelium

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced ~60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.