Distribution of traction forces associated with shape changes during amoeboid cell migration.

Amoeboid motility results from the cyclic repetition of shape changes leading to periodic oscillations of the cell length (motility cycle). We analyze the dominant modes of shape change and their association to the traction forces exerted on the substrate using Principal Component Analysis (PCA) of time-lapse measurements of cell shape and traction forces in migrating Dictyostelium cells. Using wild-type cells (wt) as reference, we investigated Myosin II activity by studying Myosin II heavy chain null cells (mhcA-) and Myosin II essential light chain null cells (mlcE-). We found that wt, mlcE-and mhcA- cells utilize similar modes of shape changes during their motility cycle, although these shape changes are implemented at a slower pace in Myosin II null mutants. The number of dominant modes of shape changes is surprisingly few with only four modes accounting for 75% of the variance in all cases. The three principal shape modes are dilation/elongation, bending, and bulging of the front/back. The second mode, resulting from sideways protrusion/retraction, is associated to lateral asymmetries in the cell traction forces, and is significantly less important in mhcA- cells. These results indicate that the mechanical cycle of traction stresses and cell shape changes remains remarkably similar for all cell lines but is slowed down when myosin function is lost, probably due to a reduced control on the spatial organization of the traction stresses.

Signaling pathways controlling cell polarity and chemotaxis.

Many important biological processes, including chemotaxis (directional cell movement up a chemoattractant gradient), require a clearly established cell polarity and the ability of the cell to respond to a directional signal. Recent advances using Dictyostelium cells and mammalian leukocytes have provided insights into the biochemical and molecular pathways that control chemotaxis. Phosphoinositide 3-kinase plays a central and possibly pivotal role in establishing and maintaining cell polarity by regulating the subcellular localization and activation of downstream effectors that are essential for regulating cell polarity and proper chemotaxis. This review outlines our present understanding of these pathways.

Control of cell polarity and chemotaxis by Akt/PKB and PI3 kinase through the regulation of PAKa.

We demonstrate that PI3 kinase and protein kinase B (PKB or Akt) control cell polarity and chemotaxis, in part, through the regulation of PAKa, which is required for myosin II assembly. We demonstrate that PI3K and PKB mediate PAKa's subcellular localization, PAKa's activation in response to chemoattractant stimulation, and chemoattractant-mediated myosin II assembly. Mutation of the PKB phosphorylation site in PAKa to Ala blocks PAKa's activation and inhibits PAKa redistribution in response to chemoattractant stimulation, whereas an Asp substitution leads to an activated protein. Addition of the PI3K inhibitor LY294002 results in a rapid loss of cell polarity and the axial distribution of actin, myosin, and PAKa. These results provide a mechanism by which PI3K regulates chemotaxis.

Functional and regulatory analysis of the dictyostelium G-box binding factor.

The Dictyostelium discoidium G-box binding factor (GBF) is required for the induction of known postaggregative and cell-type-specific genes. gbf-null cells undergo developmental arrest at the loose-mound stage due to the absence of GBF-targeted gene transcription. GBF-mediated gene expression is activated by stimulation of cell-surface, seven-span cAMP receptors, but this activation is independent of heterotrimeric G-proteins. To further characterize GBF, we assayed a series of GBF mutants for their ability to bind a G-box in vitro and to complement the gbf-null phenotype. In vitro DNA-binding activity resides in the central portion of the protein, which contains two predicted zinc fingers. However, in vivo GBF function requires only one intact zinc finger. In addition, expression of some GBF mutants results in a partial complementation phenotype, suggesting that these mutants are hypomorphic alleles. We used a 2.4-kb GBF-promoter fragment to examine the regulation of GBF expression. GBF promoter-reporter studies confirmed the previous finding that GBF transcription is induced by continuous, micromolar extracellular cAMP. We also show that, like the activation of GBF-regulated transcription, the induction of GBF expression requires cell-surface cAMP receptors, but not heterotrimeric G-proteins. Finally, reporter studies demonstrated that induction of GBF-promoter-regulated expression does not require the presence of GBF protein, indicating that GBF expression is not regulated by a positive autoregulatory loop.

Regulated protein degradation controls PKA function and cell-type differentiation in Dictyostelium.

Cullins function as scaffolds that, along with F-box/WD40-repeat-containing proteins, mediate the ubiquitination of proteins to target them for degradation by the proteasome. We have identified a cullin CulA that is required at several stages during Dictyostelium development. culA null cells are defective in inducing cell-type-specific gene expression and exhibit defects during aggregation, including reduced chemotaxis. PKA is an important regulator of Dictyostelium development. The levels of intracellular cAMP and PKA activity are controlled by the rate of synthesis of cAMP and its degradation by the cAMP-specific phosphodiesterase RegA. We show that overexpression of the PKA catalytic subunit (PKAcat) rescues many of the culA null defects and those of cells lacking FbxA/ChtA, a previously described F-box/WD40-repeat-containing protein, suggesting CulA and FbxA proteins are involved in regulating PKA function. Whereas RegA protein levels drop as the multicellular organism forms in the wild-type strain, they remain high in culA null and fbxA null cells. Although PKA can suppress the culA and fbxA null developmental phenotypes, it does not suppress the altered RegA degradation, suggesting that PKA lies downstream of RegA, CulA, and FbxA. Finally, we show that CulA, FbxA, and RegA are found in a complex in vivo, and formation of this complex is dependent on the MAP kinase ERK2, which is also required for PKA function. We propose that CulA and FbxA regulate multicellular development by targeting RegA for degradation via a pathway that requires ERK2 function, leading to an increase in cAMP and PKA activity.

Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in dictyostelium.

We show that cells lacking two Dictyostelium class I phosphatidylinositol (PI) 3' kinases (PI3K and pi3k1/2-null cells) or wild-type cells treated with the PI3K inhibitor LY294002 are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated filamentous (F)-actin polymerization, and chemotax very slowly. PI3K is thought to produce membrane lipid-binding sites for localization of PH domain-containing proteins. We demonstrate that in response to chemoattractants three PH domain-containing proteins do not localize to the leading edge in pi3k1/2-null cells, and the translocation is blocked in wild-type cells by LY294002. Cells lacking one of these proteins, phdA-null cells, exhibit defects in the level and kinetics of actin polymerization at the leading edge and have chemotaxis phenotypes that are distinct from those described previously for protein kinase B (PKB) (pkbA)-null cells. Phenotypes of PhdA-dominant interfering mutations suggest that PhdA is an adaptor protein that regulates F-actin localization in response to chemoattractants and links PI3K to the control of F-actin polymerization at the leading edge during pseudopod formation. We suggest that PKB and PhdA lie downstream from PI3K and control different downstream effector pathways that are essential for proper chemotaxis.

The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.

cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on multicellular development when expressed in wild-type cells. These findings suggest that the phosphorylated C-terminus of cAR1 may be involved in regulating aspects of receptor-mediated processes, is not essential for GBF function, and may play a role in mediating subsequent development.

Protein tyrosine phosphatase PTP1 negatively regulates Dictyostelium STATa and is required for proper cell-type proportioning.

The protein tyrosine phosphatase PTP1, which mediates reversible phosphorylation on tyrosine, has been shown to play an important regulatory role during Dictyostelium development. Mutants lacking PTP1 develop more rapidly than normal, while strains that overexpress PTP1 display aberrant morphology. However, the signalling pathways involved have not been characterised. In reexamining these strains, we have found that there is an inverse correlation between levels of PTP1 activity, the extent of tyrosine phosphorylation on Dictyostelium STATa after treatment with cAMP, and the proportion of the slug population exhibiting STATa nuclear enrichment in vivo. This suggests that PTP1 acts to attenuate the tyrosine phosphorylation of STATa and downstream STATa-mediated pathways. Consistent with this, we show that when PTP1 is overexpressed, there is increased expression of a prestalk cell marker at the slug posterior, a phenocopy of STATa null slugs. In ptp1 null strains, STATa tyrosine phosphorylation and nuclear enrichment in the slug anterior is increased. There is also a change in the prestalk to prespore cell ratio. Synergy experiments suggest that this is due to a cell-autonomous defect in forming the subset of prespore cells that are located in the anterior prespore region.

An SH2-domain-containing kinase negatively regulates the phosphatidylinositol-3 kinase pathway.

SHK1 is a novel dual-specificity kinase that contains an SH2 domain in its C-terminal region. We demonstrate that SHK1 is required for proper chemotaxis and phagocytosis. Mutant shk1 null cells lack polarity, move very slowly, and exhibit an elevated and temporally extended chemoattractant-mediated activation of the kinase Akt/PKB. GFP fusions of the PH domain of Akt/PKB or the PH-domain-containing protein CRAC, which become transiently associated with the plasma membrane after a global stimulation with a chemoattractant, remain associated with the plasma membrane for an extended period of time in shk1 null cells. These results suggest that SHK1 is a negative regulator of the PI3K (phosphatidylinositol-3 kinase) pathway. Furthermore, when a chemoattractant gradient is applied to a wild-type cell, these PH-domain-containing proteins and the F-actin-binding protein coronin localize to its leading edge, but in an shk1 null cell they become randomly associated with the plasma membrane and cortex, irrespective of the direction of the chemoattractant gradient, suggesting that SHK1 is required for the proper spatiotemporal control of F-actin levels in chemotaxing cells. Consistent with such functions, SHK1 is localized at the plasma membrane/cortex, and we show that its SH2 domain is required for this localization and the proper function of SHK1.

HAPPY days for the Dictyostelium genome project.

Without the HAPPY map, the collaborators in the genome project would have found assembly to be extremely difficult, and the Dictyostelium genome sequence would perhaps have been left highly incomplete. With the HAPPY Map the YAC clones can be remapped and the original YAC skim strategy followed. In conclusion, this method has already made one community very happy and seems sure to make its mark in many other genome projects.

The membrane glycoprotein gp150 is encoded by the lagC gene and mediates cell-cell adhesion by heterophilic binding during Dictyostelium development.

gp150 is a membrane glycoprotein which has been implicated in cell-cell adhesion in the postaggregation stages of Dictyostelium development. An analysis of its tryptic peptides by mass spectrometry has identified gp150 as the product of the lagC gene, which was previously shown to play a role in morphogenesis and cell-type specification. Antibodies raised against the GST-LagC fusion protein specifically recognized gp150 in wild-type cells and showed that it is missing in lagC-null cells. Immunolocalization studies have confirmed its enrichment in cell-cell contact regions. In mutant cells that lack the aggregation stage-specific cell adhesion molecule gp80, gp150 is expressed precociously. Moreover, these cells acquire EDTA-resistant cell-cell binding during aggregation, suggesting a role for gp150 in this process. Cells in which the genes encoding gp80 and gp150 are both inactivated do not acquire EDTA-resistant cell adhesion during aggregation. Strains transformed with an actin 15::lagC construct express gp150 precociously, but do not show EDTA-resistant adhesion during early development. However, vegetative cells expressing gp150 can be recruited into aggregates of 16-h lagC-null cells. These results, together with those obtained with the cell-to-substratum binding assay, indicate that gp150 mediates cell-cell adhesion via heterophilic interactions with another component that accumulates during the aggregation stage.

An F-Box/WD40 repeat-containing protein important for Dictyostelium cell-type proportioning, slug behaviour, and culmination.

FbxA is a novel member of a family of proteins that contain an F-box and WD40 repeats and that target specific proteins for degradation via proteasomes. In fruiting bodies formed from cells where the fbxA gene is disrupted (fbxA(-) cells), the spore mass fails to fully ascend the stalk. In addition, fbxA(-) slugs continue to migrate under environmental conditions where the parental strain immediately forms fruiting bodies. Consistent with this latter behaviour, the development of fbxA(-) cells is hypersensitive to ammonia, the signaling molecule that regulates the transition from the slug stage to terminal differentiation. The slug comprises an anterior prestalk region and a posterior prespore region and the fbxA mRNA is highly enriched in the prestalk cells. The prestalk zone of the slug is further subdivided into an anterior pstA region and a posterior pstO region. In fbxA(-) slugs the pstO region is reduced in size and the prespore region is proportionately expanded. Our results indicate that FbxA is part of a regulatory pathway that controls cell fate decisions and spatial patterning via regulated protein degradation.

Dictyostelium: a model for regulated cell movement during morphogenesis.

Dictyostelium has played an important role in unraveling the pathways that control cell movement and chemotaxis. Recent studies have started to elucidate the pathways that control cell sorting, morphogenesis, and the establishment of spatial patterning in this system. In doing so, they provide new insights into how cell movements within a multicellular organism are regulated and the importance of pathways that are similar to those that regulate chemotaxis of cells on two-dimensional surfaces during aggregation.

The molecular genetics of chemotaxis: sensing and responding to chemoattractant gradients.

Chemotaxis plays a central role in various biological processes, such as the movement of neutrophils and macrophage during wound healing and in the aggregation of Dictyostelium cells. During the past few years, new understanding of the mechanisms controlling chemotaxis has been obtained through molecular genetic and biochemical studies of Dictyostelium and other experimental systems. This review outlines our present understanding of the signaling pathways that allow a cell to sense and respond to a chemoattractant gradient. In response to chemoattractants, cells either become polarized in the direction of the chemoattractant source, which results in the formation of a leading edge, or they reorient their polarity in the direction of the chemoattractant gradient and move with a stronger persistence up the gradient. Models are presented here to explain such directional responses. They include a localized activation of pathways at the leading edge and an "inhibition" of these pathways along the lateral edges of the cell. One of the primary pathways that may be responsible for such localized responses is the activation of phosphatidyl inositol-3 kinase (PI3K). Evidence suggests that a localized formation of binding sites for PH (pleckstrin homology) domain-containing proteins produced by PI3K leads to the formation of "activation domains" at the leading edge, producing a localized response.

A novel Akt/PKB-related kinase is essential for morphogenesis in Dictyostelium.

BACKGROUND: Dictyostelium Akt/PKB is homologous to mammalian Akt/PKB and is required for cell polarity and proper chemotaxis during early development. The kinase activity of Akt/PKB kinase is activated in response to chemoattractants in neutrophils and in Dictyostelium by the chemoattractant cAMP functioning via a pathway involving a heterotrimeric G protein and PI3-kinase. Dictyostelium contains several kinases structurally related to Akt/PKB, one of which, PKBR-1, is investigated here for its role in cell polarity, movement and cellular morphogenesis during development. RESULTS: PKBR-1 has a kinase and a carboxy-terminal domain related to those of Akt/PKB, but no PH domain. Instead, it has an amino-terminal myristoylation site, which is required for its constitutive membrane localization. Like Akt/PKB, PKBR-1 is activated by cAMP through a G-protein-dependent pathway, but does not require PI3-kinase, probably because of the constitutive membrane localization of PKBR-1. This is supported by experiments demonstrating the requirement for membrane association for activation and in vivo function of PKBR-1. PKBR-1 protein is found in all cells throughout early development but is then restricted to the apical cells in developing aggregates, which are thought to control morphogenesis. PKBR-1 null cells arrest development at the mound stage and are defective in morphogenesis and multicellular development. These phenotypes are complemented by Akt/PKB, suggesting functional overlap between PKBR-1 and Akt/PKB. Akt/PKB PKBR-1 double knockout cells exhibit growth defects and show stronger chemotaxis and cell-polarity defects than Akt/PKB null cells. CONCLUSIONS: Our results expand the previously known functions of Akt/PKB family members in cell movement and morphogenesis during Dictyostelium multicellular development. The results suggest that Akt/PKB and PKBR-1 have overlapping effectors and biological function: Akt/PKB functions predominantly during aggregation to control cell polarity and chemotaxis, whereas PKBR-1 is required for morphogenesis during multicellular development.

Role of Rac in controlling the actin cytoskeleton and chemotaxis in motile cells.

We have used the chemotactic ability of Dictyostelium cells to examine the roles of Rho family members, known regulators of the assembly of F-actin, in cell movement. Wild-type cells polarize with a leading edge enriched in F-actin toward a chemoattractant. Overexpression of constitutively active Dictyostelium Rac1B(61L) or disruption of DdRacGAP1, which encodes a Dictyostelium Rac1 GAP, induces membrane ruffles enriched with actin filaments around the perimeter of the cell and increased levels of F-actin in resting cells. Whereas wild-type cells move linearly toward the cAMP source, Rac1B(61L) and Ddracgap1 null cells make many wrong turns and chemotaxis is inefficient, which presumably results from the unregulated activation of F-actin assembly and pseudopod extension. Cells expressing dominant-negative DdRac1B(17N) do not have a well-defined F-actin-rich leading edge and do not protrude pseudopodia, resulting in very poor cell motility. From these studies and assays examining chemoattractant-mediated F-actin assembly, we suggest DdRac1 regulates the basal levels of F-actin assembly, its dynamic reorganization in response to chemoattractants, and cellular polarity during chemotaxis.

Just the right size: cell counting in Dictyostelium.

The regulation of tissue and organism size plays an essential, but poorly understood, role in multicellular development. Genes have been identified that affect body and organ size in a number of animals. Two recently identified genes, smlA and countin, are required for the proper function of a cell-counting mechanism that regulates organism size in the eukaryotic microorganism Dictyostelium discoideum. The discovery of this process now allows the study of size regulation in a simple multicellular system.

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.

Integration of signaling networks that regulate Dictyostelium differentiation.

In Dictyostelium amoebae, cell-type differentiation, spatial patterning, and morphogenesis are controlled by a combination of cell-autonomous mechanisms and intercellular signaling. A chemotactic aggregation of approximately 10(5) cells leads to the formation of a multicellular organism. Cell-type differentiation and cell sorting result in a small number of defined cell types organized along an anteroposterior axis. Finally, a mature fruiting body is created by the terminal differentiation of stalk and spore cells. Analysis of the regulatory program demonstrates a role for several molecules, including GSK-3, signal transducers and activators of transcription (STAT) factors, and cAMP-dependent protein kinase (PKA), that control spatial patterning in metazoans. Unexpectedly, two component systems containing histidine kinases and response regulators also play essential roles in controlling Dictyostelium development. This review focuses on the role of cAMP, which functions intracellularly to mediate the activity of PKA, an essential component in aggregation, cell-type specification, and terminal differentiation. Cytoplasmic cAMP levels are controlled through both the regulated activation of adenylyl cyclases and the degradation by a phosphodiesterase containing a two-component system response regulator. Extracellular cAMP regulates G-protein-dependent and -independent pathways to control aggregation as well as the activity of GSK-3 and the transcription factors GBF and STATa during multicellular development. The integration of these pathways with others regulated by the morphogen DIF-1 to control cell fate decisions are discussed.

PAKa, a putative PAK family member, is required for cytokinesis and the regulation of the cytoskeleton in Dictyostelium discoideum cells during chemotaxis.

We have identified a Dictyostelium discoideum gene encoding a serine/threonine kinase, PAKa, a putative member of the Ste20/PAK family of p21-activated kinases, with a kinase domain and a long NH(2)-terminal regulatory domain containing an acidic segment, a polyproline domain, and a CRIB domain. PAKa colocalizes with myosin II to the cleavage furrow of dividing cells and the posterior of polarized, chemotaxing cells via its NH(2)-terminal domain. paka null cells are defective in completing cytokinesis in suspension. PAKa is also required for maintaining the direction of cell movement, suppressing lateral pseudopod extension, and proper retraction of the posterior of chemotaxing cells. paka null cells are defective in myosin II assembly, as the myosin II cap in the posterior of chemotaxing cells and myosin II assembly into cytoskeleton upon cAMP stimulation are absent in these cells, while constitutively active PAKa leads to an upregulation of myosin II assembly. PAKa kinase activity against histone 2B is transiently stimulated and PAKa incorporates into the cytoskeleton with kinetics similar to those of myosin II assembly in response to chemoattractant signaling. However, PAKa does not phosphorylate myosin II. We suggest that PAKa is a major regulator of myosin II assembly, but does so by negatively regulating myosin II heavy chain kinase.