The Aurora kinases are highly conserved serine/threonine kinases that play essential roles throughout mitosis. In metazoans, these functions are mediated by Aurora A and B at the spindle poles and the equatorial region respectively. I show here that Dictyostelium contains a single Aurora kinase, DdAurora that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box and localizes to the spindle poles during early mitosis. Like Aurora B, DdAurora localizes to centromeres in metaphase, the central spindle during anaphase and the cleavage furrow at the end of cytokinesis. In addition to these known features of Aurora A and B, I found that DdAurora remains associated with centromeres during anaphase and telophase which has not been shown in any other organisms. INCENP is known to be an important binding partner of Aurora B. In Dictyostelium the conserved C-terminal IN-box domain of DdINCENP is essential for its interaction with DdAurora and for the localization of DdAurora to the central spindle. In contrast, the centromeric and spindle pole localization of DdAurora does not require an interaction with DdINCENP. Surprisingly, a truncated DdINCENP protein lacking the IN-box domain can still localize on centromeres and the central spindle even though it does not bind to DdAurora. I also found that the localization of DdAurora to the central spindle requires Kif12, a protein similar to mitotic kinesin like protein 2 (MKLP2). However, this requirement is suppressed by the overexpression of GFP-DdINCENP. GFP-DdINCENP can localize to the central spindle in the absence of Kif12 and it probably recruits DdAurora to the same location through their strong interaction. Finally, I demonstrated that Myosin II heavy chain is important for the proper localization of the DdAurora/DdINCENP complex to the cleavage furrow during late cytokinesis. With the exception of DdINCENP, no other binding partner or substrate of DdAurora has been identified in Dictyostelium. By performing large-scale immunoprecipitation in wild-type cells, I identified several potential binding partners/substrates of DdAurora, including topoisomerase B and HspA. Future esearch on these proteins may help to elucidate DdAurora function in different stages of M phase.

No description available.

During development, Dictyostelium discoideum cells assume muticellularity via their collective aggregation. Cell-cell adhesion is required for morphogenesis, cell differentiation, cell sorting and gene expression during development. TgrC1 is a heterophilic cell adhesion molecule which is indispendable for complete development. TgrC1 can be considered as the most important cell adhesion molecule for D. discoideum development because deletion of the tgrC1 gene completely arrests development at the loose aggregate stage and inhibits fruiting body formation. In order to investigate the biological role of TgrC1 during development, I have chosen to identify and charactize the extracellular heterophilic partner and the cytoplasmic binding partner(s) of TgrC1. Using different biochemical approaches, we identified TgrB1 as the heterophilic binding partner of TgrC1 and demonstrated that their association is mediated through IPT/TIG domains in the extracellular region of both proteins. Both tgrB1 and tgrC1 share the same transcriptional promoter and their spatiotemporal expression pattern is identical during development. We also examined the assembly of TgrC1-TgrB1 complexes via the split green fluorescence protein complementation assay and the fluorescence resonance energy transfer approach. Whereas TgrC1 is capable of forming cis-homodimers spontaneously, cis-homodimerization of TgrB1 depends on its trans-interaction with TgrC1. A model of the assembly process has been proposed. To investigate signalling events initiated by the interaction between TgrB1 and TgrC1, pull-down assays were employed and led to the identification of myosin heavy chain kinase C as the cytoplamic partner of TgrC1. Mutational analysis showed that the basic residues in the short cytoplasmic domain of TgrC1 are critical to the binding with MHCK-C. Disruption of the interation between MHCK-C and TgrC1 results in an alteration of cell motility at the aggregation stage and aberrant cell sorting in slugs. These studies have highlighted the role of TgrB1-TgrC1 complexes in the regulation of morphogenesis during Dictyostelium development.

The slime mould Dictyostelium discoideum contains a novel cAMP-binding protein, CABPl, that is composed of two subunits. Anti-CABPl monoclonal antibodies were used to isolate a cDNA from a $ lambda$gtll expression library. In hybrid selection experiments, this cDNA was found to be complementary to mRNAs encoding both CABPl subunits and also to share some homology with two polypeptides, p34 and p31, that copurify with CABPl. Further analysis showed that the two CABPl polypeptides are identical except for an additional 37 amino acids present in the larger polypeptide. Both polypeptides are encoded by a single gene and the production of the two CABPl mRNAs is accomplished by an unusual splicing event. The p34 and p31 polypeptides are smaller but highly homologous to the two CABPl polypeptides and are probably produced in a similar fashion. The carboxyl halves of the four polypeptides show a 50% similarity to two polypeptides of a tellurium anion resistance determinant encoded on a bacterial plasmid.

Following starvation, Dictyostelium discoideum cells aggregate, a response that requires chemotaxis to cyclic AMP (cAMP) and the relay of the cAMP signal by the activation of adenylyl cyclase (ACA). Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG does have a role in early development. When the responses of rasG⁻ cells to cAMP were compared with the responses of rasC⁻ strain, these studies revealed that signal transduction through RasG is more important in chemotaxis and early gene expression, but that signal transduction through RasC is more important in ACA activation. Characterization of a rasC⁻/rasG⁻ mutant revealed that both cAMP chemotaxis and adenylyl cyclase (ACA) activation were negligible in this strain. The ectopic expression of carA from the actin 15 promoter restored early developmental gene expression to the rasC⁻/rasG⁻ strain, rendering it suitable for an analysis of cAMP signal transduction. Since there was negligible signaling through either the cAMP chemotactic pathway or the adenylyl cyclase activation pathway in this strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The mutational analysis of Switch I and Switch II regions also defined the key residues that generate functional differences between RasC and RasG. Rap1 is also activated in response to cAMP but its position in the signal transduction cascade was clarified by the finding that its activation was totally abolished in rasC⁻/rasG⁻/[act15]:carA and in rasG⁻ cells, but only slightly reduced in rasC⁻ cells. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC/rasG⁻4act15]:carA strain identifies RasG⁻/RasC⁻ as the presumptive monomeric GTPases required for this activation. The phenotypes of the vegetative ras null mutants were also examined. The results indicate that RasG plays an important role in cytokinesis. The partial absence of chemotaxis to folate in rase cells compared to the total absence of chemotaxis to folate in rasC⁻/rasG⁻, and rasC⁻/rasG⁻/[act15]:carA cells suggests a compensatory role of RasC for RasG during this process, a similar phenomenon to that observed for cAMP chemotaxis by aggregating cells.

Protein structure and functions are tightly regulated. Studying the integration of multiple modifications in single systems is a novel approach. Hisactophilin protein from Dictyostelium discoideum, is an actin binding protein that serves to induce formation of actin filaments and is regulated by protonation and myristoylation. Utilizing hisactophilin as a model, I determined the effect of pH and myristoyl-switching on actin binding and filament induction using fluorescence spectroscopy, light scattering, and time-course electron microscopy. Results revealed the accessible myristoyl group slows binding and the rate of actin polymerization compared to when the group is sequestered. Hisactophilin induces pH-dependent actin aggregates before reorganizing them into filaments and bundles. Hisactophilin mutants impact initial actin binding and the kinetics of the aggregated state. I determined the cooperativity of myristoylation and protonation as interdependent protein regulatory mechanisms, their impact on actin binding and proposed a novel mechanism for actin polymerization as a result of these integrated regulators. / NSERC

During development, Dictyostelium discoideum cells assume muticellularity via their collective aggregation. Cell-cell adhesion is required for morphogenesis, cell differentiation, cell sorting and gene expression during development. TgrC1 is a heterophilic cell adhesion molecule which is indispendable for complete development. TgrC1 can be considered as the most important cell adhesion molecule for D. discoideum development because deletion of the tgrC1 gene completely arrests development at the loose aggregate stage and inhibits fruiting body formation. In order to investigate the biological role of TgrC1 during development, I have chosen to identify and charactize the extracellular heterophilic partner and the cytoplasmic binding partner(s) of TgrC1. Using different biochemical approaches, we identified TgrB1 as the heterophilic binding partner of TgrC1 and demonstrated that their association is mediated through IPT/TIG domains in the extracellular region of both proteins. Both tgrB1 and tgrC1 share the same transcriptional promoter and their spatiotemporal expression pattern is identical during development. We also examined the assembly of TgrC1-TgrB1 complexes via the split green fluorescence protein complementation assay and the fluorescence resonance energy transfer approach. Whereas TgrC1 is capable of forming cis-homodimers spontaneously, cis-homodimerization of TgrB1 depends on its trans-interaction with TgrC1. A model of the assembly process has been proposed. To investigate signalling events initiated by the interaction between TgrB1 and TgrC1, pull-down assays were employed and led to the identification of myosin heavy chain kinase C as the cytoplamic partner of TgrC1. Mutational analysis showed that the basic residues in the short cytoplasmic domain of TgrC1 are critical to the binding with MHCK-C. Disruption of the interation between MHCK-C and TgrC1 results in an alteration of cell motility at the aggregation stage and aberrant cell sorting in slugs. These studies have highlighted the role of TgrB1-TgrC1 complexes in the regulation of morphogenesis during Dictyostelium development.

Following starvation, Dictyostelium discoideum cells aggregate, a response that requires chemotaxis to cyclic AMP (cAMP) and the relay of the cAMP signal by the activation of adenylyl cyclase (ACA). Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG does have a role in early development. When the responses of rasG⁻ cells to cAMP were compared with the responses of rasC⁻ strain, these studies revealed that signal transduction through RasG is more important in chemotaxis and early gene expression, but that signal transduction through RasC is more important in ACA activation. Characterization of a rasC⁻/rasG⁻ mutant revealed that both cAMP chemotaxis and adenylyl cyclase (ACA) activation were negligible in this strain. The ectopic expression of carA from the actin 15 promoter restored early developmental gene expression to the rasC⁻/rasG⁻ strain, rendering it suitable for an analysis of cAMP signal transduction. Since there was negligible signaling through either the cAMP chemotactic pathway or the adenylyl cyclase activation pathway in this strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The mutational analysis of Switch I and Switch II regions also defined the key residues that generate functional differences between RasC and RasG. Rap1 is also activated in response to cAMP but its position in the signal transduction cascade was clarified by the finding that its activation was totally abolished in rasC⁻/rasG⁻/[act15]:carA and in rasG⁻ cells, but only slightly reduced in rasC⁻ cells. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC/rasG⁻4act15]:carA strain identifies RasG⁻/RasC⁻ as the presumptive monomeric GTPases required for this activation. The phenotypes of the vegetative ras null mutants were also examined. The results indicate that RasG plays an important role in cytokinesis. The partial absence of chemotaxis to folate in rase cells compared to the total absence of chemotaxis to folate in rasC⁻/rasG⁻, and rasC⁻/rasG⁻/[act15]:carA cells suggests a compensatory role of RasC for RasG during this process, a similar phenomenon to that observed for cAMP chemotaxis by aggregating cells.

Univ., Diss., 2006--Kassel

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.