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Robert H Insall

Publications and source records attributed to Robert H Insall.

11 recordsLinked to original sources
PubMed2005

The Dictyostelium genome encodes numerous RasGEFs with multiple biological roles.

BACKGROUND: Dictyostelium discoideum is a eukaryote with a simple lifestyle and a relatively small genome whose sequence has been fully determined. It is widely used for studies on cell signaling, movement and multicellular development. Ras guanine-nucleotide exchange factors (RasGEFs) are the proteins that activate Ras and thus lie near the top of many signaling pathways. They are particularly important for signaling in development and chemotaxis in many organisms, including Dictyostelium. RESULTS: We have searched the genome for sequences encoding RasGEFs. Despite its relative simplicity, we find that the Dictyostelium genome encodes at least 25 RasGEFs, with a few other genes encoding only parts of the RasGEF consensus domains. All appear to be expressed at some point in development. The 25 genes include a wide variety of domain structures, most of which have not been seen in other organisms. The LisH domain, which is associated with microtubule binding, is seen particularly frequently; other domains that confer interactions with the cytoskeleton are also common. Disruption of a sample of the novel genes reveals that many have clear phenotypes, including altered morphology and defects in chemotaxis, slug phototaxis and thermotaxis. CONCLUSION: These results suggest that the unexpectedly large number of RasGEF genes reflects an evolutionary expansion of the range of Ras signaling rather than functional redundancy or the presence of multiple pseudogenes.

Actins↗
PubMed2005

Phospholipase D activity is essential for actin localization and actin-based motility in Dictyostelium.

PLD (phospholipase D) activity catalyses the generation of the lipid messenger phosphatidic acid, which has been implicated in a number of cellular processes, particularly the regulation of membrane traffic. In the present study, we report that disruption of PLD signalling causes unexpectedly profound effects on the actin-based motility of Dictyostelium. Cells in which PLD activity is inhibited by butan-1-ol show a complete loss of actin-based structures, accompanied by relocalization of F-actin into small clusters, and eventually the nucleus, without a visible fall in levels of F-actin. Addition of exogenous phosphatidic acid reverses the effects of butan-1-ol, confirming that these effects are caused by inhibition of PLD. Loss of motility correlates with complete inhibition of endocytosis and a reduction in phagocytosis. Inhibition of PLD caused a major decrease in the synthesis of PtdIns(4,5)P2, which could again be reversed by exogenously applied phosphatidic acid. Thus the essential role of PLD signalling in both motility and endocytosis appears to be mediated directly via regulation of PtdIns(4)P kinase activity. This implies that localized PLD-regulated synthesis of PtdIns(4,5)P2 is essential for Dictyostelium actin function.

Actins↗
PubMed2004

Regulation of WASP: PIP2 Pipped by Toca-1?

Cdc42 is a key regulator of cell polarity and actin dynamics. One of its effectors, WASP, initiates the assembly of new actin filaments. In this issue of Cell, show that a previously unknown regulator named Toca-1 is required for Cdc42 to activate WASP. This discovery changes our picture of how small GTPases and multiple other signals converge to stimulate actin polymerization and cell motility.

Actins↗
PubMed2004

Gdt2 regulates the transition of Dictyostelium cells from growth to differentiation.

BACKGROUND: Dictyostelium life cycle consists of two distinct phases - growth and development. The control of growth-differentiation transition in Dictyostelium is not completely understood, and only few genes involved in this process are known. RESULTS: We have isolated a REMI (restriction enzyme-mediated integration) mutant, which prematurely initiates multicellular development. When grown on a bacterial lawn, these cells aggregate before the bacteria are completely cleared. In bacterial suspension, mutant cells express the developmental marker discoidin Igamma even at low cell densities and high concentrations of bacteria. In the absence of nutrients, mutant cells aggregate more rapidly than wild type, but the rest of development is unaffected and normal fruiting bodies are formed. The disrupted gene shows substantial homology to the recently described gdt1 gene, and therefore was named gdt2. While GDT1 and GDT2 are similar in many ways, there are intriguing differences. GDT2 contains a well conserved protein kinase domain, unlike GDT1, whose kinase domain is probably non-functional. The gdt2 and gdt1 mRNAs are regulated differently, with gdt2 but not gdt1 expressed throughout development. The phenotypes of gdt2- and gdt1- mutants are related but not identical. While both initiate development early, gdt2- cells grow at a normal rate, unlike gdt1- mutants. Protein kinase A levels and activity are essentially normal in growing gdt2- mutants, implying that GDT2 regulates a pathway that acts separately from PKA. Gdt1 and gdt2 are the first identified members of a family containing at least eight closely related genes. CONCLUSIONS: We have isolated and characterised a new gene, gdt2, which acts to restrain development until conditions are appropriate. We also described a family of related genes in the Dictyostelium genome. We hypothesise that different family members might control similar cellular processes, but respond to different environmental cues.

Amino Acid Sequence↗
PubMed2004

The identification of Dictyostelium phosphoproteins altered in response to the activation of RasG.

Dictyostelium RasG has been implicated in the regulation of a variety of cellular processes, including the initiation of development, cell movement, and cytokinesis, but the molecular components of the signaling pathways involved are largely unknown. We used a tetracycline-regulated protein expression system to study the effect of activated RasG, RasG(G12T), expression on the phosphorylation state of Dictyostelium proteins. Over 70 vegetative phosphoprotein components were resolved by two-dimensional (2-D) immunoblot analysis and of these 16 phosphothreonine and three phosphotyrosine protein components were found to reproducibly change upon RasG(G12T) expression. Thirteen of these were recovered from 2-D gels and identified by mass spectrometry of in-gel tryptic digestions. The proteins identified include the signaling proteins RasGEF-R and protein kinase B, the adhesion protein DdCAD-1, the cytoskeletal protein actin, the mitochondrial division protein FtsZA, and proteins involved in translation and metabolism. In addition to the direct demonstration of the phosphorylation of putative downstream targets of RasG activation, these findings reveal previously undetected phosphorylation of several proteins.

Animals↗
PubMed2003

PIR121 regulates pseudopod dynamics and SCAR activity in Dictyostelium.

BACKGROUND: The WASP/SCAR family of adaptor proteins coordinates actin reorganization by coupling different signaling molecules, including Rho-family GTPases, to the activation of the Arp2/3 complex. WASP binds directly to Cdc42 through its GTPase binding domain (GBD), but SCAR does not contain a GBD, and no direct binding has been found. However, SCAR has recently been found to copurify with four other proteins in a complex. One of these, PIR121, binds directly to Rac. RESULTS: We have identified four of the members of this complex in Dictyostelium and disrupted the pirA gene, which encodes PIR121. The resulting mutant cells are unusually large, maintain an excessive proportion of their actin in a polymerized state and display severe defects in movement and chemotaxis. They also continually extend new pseudopods by widening and splitting existing leading edges rather than by initiating new pseudopods. Comparing these cells to scar null mutants shows behavior that is broadly consistent with overactivation of SCAR. Deletion of the pirA gene in a scar(-) mutant resulted in cells resembling their scar(-) parents with no obvious changes, confirming that PIR121 mainly acts through SCAR in vivo. Surprisingly given their hyperactive phenotype, we find that pirA(-) mutants contain very little intact SCAR protein despite normal levels of mRNA, suggesting a posttranscriptional downregulation of activated SCAR. CONCLUSIONS: Our results demonstrate a genetic connection between the pirA and scar genes. PIR121 appears to inhibit the activity of SCAR in the absence of activating signals. The location of the newly formed protrusions indicates that unregulated SCAR is acting at the edges of existing pseudopods, not elsewhere in the cell. We suggest that active SCAR protein released from the inhibitory complex is rapidly removed and that this is an important and novel mechanism for controlling actin dynamics.

Actins↗
PubMed2003

Parasexual genetics of Dictyostelium gene disruptions: identification of a ras pathway using diploids.

BACKGROUND: The relative ease of targeted gene disruption in the social amoeba Dictyostelium has stimulated its widespread use as an experimental organism for cell and developmental biology. However, the field has been hamstrung by the lack of techniques to recombine disrupted genes. RESULTS: We describe new techniques for parasexual fusion of strains in liquid medium, selection and maintenance of the resulting stable diploid strains, and segregation to make recombined haploids. We have used these techniques to isolate rasS/gefB double nulls. The phenotypes of these mutants are no more severe than either parent, with movement, phagocytosis and fluid-phase endocytosis affected to the same degree as in rasS or gefB single nulls. In addition, we have produced diploids from one AX2- and one AX3-derived parent, providing an axenic strain with fewer secondary phenotypes than has been previously available. CONCLUSIONS: The phenotype of the rasS/gefB double mutant suggests that the RasS and GefB proteins lie on the same linear pathway. In addition, axenic diploids and the techniques to generate, maintain and segregate them will be productive tools for future work on Dictyostelium. They will particularly facilitate generation of multiple mutants and manipulation of essential genes.

Animals↗
PubMed2003

Linked regulation of motility and integrin function in activated migrating neutrophils revealed by interference in remodelling of the cytoskeleton.

Neutrophils migrate rapidly by co-ordinating regulation of their beta2-integrin adhesion with turnover of filamentous F-actin. The seven-protein Arp2/3 complex regulates actin polymerisation upon activation by proteins of the WASP-family. To investigate links between actin polymerisation, adhesion, and migration, we used a novel osmotic-shock method to load neutrophils with peptides: (1). WASP-WA and Scar-WA (which incorporate the actin- and Arp2/3-binding regions of WASP and Scar1), to compete with endogenous WASP-family members; (2). proline rich motifs (PRM) from the ActA protein of L. monocytogenes or from vinculin, which bind vasodilator-stimulated phosphoprotein (VASP), a regulator of cytoskeleton assembly. In a flow system, rolling-adherent neutrophils were stimulated with formyl tri-peptide. This caused rapid immobilisation, followed by migration with increasing velocity, supported by activated beta2-integrin CD11b/CD18. Loading ActA PRM (but not vinculin PRM) caused concentration-dependent reduction in migration velocity. At the highest concentration, unstimulated neutrophils had elevated F-actin and were rigid, but could not change their F-actin content or shape upon stimulation. Scar-WA also caused marked reduction in migration rate, but WASP-WA had a lesser effect. Scar-WA did not modify activation-dependent formation of F-actin or change in shape. However, a reduction in rate of downregulation of integrin adhesion appeared to contribute to impaired migration. These studies show that interference in cytoskeletal reorganisation that follows activation in neutrophils, can impair regulation of integrin function as well as motility. They also suggest a role of the Arp2/3 complex and WASP-family in co-ordinating actin polymerisation and integrin function in migrating neutrophils.

Actin-Related Protein 2↗
PubMed2002

Pseudopodium dynamics and rapid cell movement in Dictyostelium Ras pathway mutants.

Loss of either of the Ras pathway members RasS or GefB causes growing Dictyostelium cells to move aberrantly rapidly. In this study, we describe the changes in motility that underlie these phenotypes using computer-assisted 3D dynamic image analysis. Unexpectedly, the two mutants use different mechanisms to achieve rapid migration. The rasS(-) cells' motility is characterised by highly dynamic cell morphology, with rapidly extending and retracting pseudopodia. The gefB(-) cells do not have an unusually dynamic morphology, and achieve their efficient translocation by the continual remodelling of an existing dominant anterior pseudopodium. In spite of these dramatic changes in pseudopodium behaviour, the underlying motility cycle of both mutants remains normal. The levels of F-actin in both mutant cell lines are significantly elevated with respect to the wild-type parental cells, suggesting a possible biochemical basis for these emphatic phenotypes.

Animals↗
PubMed2002

Methanol and acriflavine resistance in Dictyostelium are caused by loss of catalase.

Various chemicals with harmful effects are not themselves toxic, but are metabolized in vivo to produce toxic products. One example is methanol in Dictyostelium, which is lethal to cells containing the acrA gene, but relatively harmless to acrA mutants. This makes methanol resistance one of the tightest genetic selections in DICTYOSTELIUM: Loss of acrA also confers cross-resistance to unrelated compounds such as acriflavine and thiabendazole. We have used insertional mutagenesis to demonstrate that the acrA locus encodes the peroxisomal catalase A enzyme. Disruption of the catA gene results in parallel resistance to acriflavine. Molecular and biochemical studies of several previously characterized methanol-resistant strains reveal that each lacks catalase activity. One allele, acrA2, contains a 13 bp deletion which introduces a frameshift in the middle of the gene. The involvement of catalase in methanol resistance in Dictyostelium compares with its role in methanol metabolism in yeast and rodents. However, this is the first study to show that catalase is required for the toxicity of acriflavine. Our results imply that acriflavine and thiabendazole are precursors which must be oxidized to generate biologically active species. The catA/acrA gene is also a potentially invaluable negative selectable marker for Dictyostelium molecular genetics.

Acriflavine↗