The cell surface is an active area where extracellular molecules meet their receptors and affect the cellular fate by inducing for example cell proliferation and adhesion. Syndecans and integrins are two transmembrane molecules that have been suggested to fine-tune these activities, possibly in cooperation. Syndecans are proteoglycans, i.e. proteins with specific types of carbohydrate chains attached. These chains are glycosaminoglycans and either heparan sulfate (HS) or chondroitin sulfate (CS). Syndecans are known to influence cell adhesion and signaling. Integrins in turn, are important adhesion molecules that connect the extracellular matrix with the cytoskeleton, and hence can regulate cell motility. In an attempt to study how the two types of glycosaminoglycans attached to syndecan-1 can interact with integrins, a cell based model system was used and functional motility assays were performed. The results showed that HS, but not CS, on the cell surface was capable of regulating integrin-mediated cell motility. Regulation of intracellular signaling is crucial to prevent abnormal cellular behavior. In the second part of this thesis, the aim was to see how the presentation of glycosaminoglycan chains to the FGF signaling complex could affect the cellular response. When attached to the plasma membrane via syndecan-1, CS chains could support the intracellular signaling, although not promoting as strong signals as HS. When glycosaminoglycans were attached to free ectodomains of syndecan-1, both types of chains sequestered FGF2 from the receptors to the same extent, pointing towards functional overlap between CS and HS. To further study the interplay between HS and CS, their roles in the formation of pharyngeal cartilage in zebrafish were established. HS was important during chondrocyte intercalation and CS in the formation of the surrounding extracellular matrix. Further, the balance between the biosynthetic enzymes determined the ratio of HS and CS, and HS biosynthesis was prioritized over CS biosynthesis. The results presented in this thesis provide further insight into the regulation of HS biosynthesis, as well as the roles of both HS and CS on the cell surface. It is evident, that in certain situations there is a strict requirement for a certain HS structure, albeit in other situations there is a functional overlap between HS and CS.

Phosphoinositide-specific phospholipase C (PLC) is involved in Ca2+ mediated signalling events that lead to altered cellular status. PLC activation causes hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and generates two second messengers, inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. Each has distinct role in depending on the cell type in mammalian cells, IP3 binds to intracellular receptors, stimulating the release of sequestered Ca2+. DAG remains in the membrane, where it can activate members of the protein kinase C (PKC) family. In plant absence of PKC keeps the question open as to what is the role of DAG in plants. The role of IP3 apart form triggering calcium release is not known, although the phosphorylated product of IP3 by groups of kinases has been implicated in certain nuclear signalling pathway. Using various sequence-analysis methods on plant PLC sequences, we identified two conserved motifs in known PLC sequences. The identified motifs are located in the C2 domain of plant PLCs and are not found in any other protein. These motifs are specifically found in the Ca2+ binding loops and form adjoining beta strands. Further, we identified certain conserved residues that are highly distinct from corresponding residues of animal PLCs. The motifs reported here could be used to annotate plant-specific phospholipase C sequences. Furthermore, we demonstrated that the C2 domain alone is capable of targeting PLC to the membrane in response to a Ca2+ signal. We also showed that the binding event results from a change in the hydrophobicity of the C2 domain upon Ca2+ binding. Bioinformatic analyses revealed that all PLCs from Arabidopsis and rice lack a transmembrane domain, myristoylation and GPI-anchor protein modifications. Our bioinformatic study indicates that plant PLCs are located in the cytoplasm, the nucleus and the mitochondria. Our results suggest that there are no distinct isoforms of plant PLCs, as have been proposed to exist in the soluble and membrane associated fractions. The same isoform could potentially be present in both subcellular fractions, depending on the calcium level of the cytosol. we have used Saccharomyces cerevisiae as a model system to investigate physiological function of PLC in regulation of lipid metabolism. S. cerevisiae synthesizes membrane phospholipids via a pathway which appears to be similar to that of higher eukaryotes. The synthesis of glycerolipid begins with the formation of phosphatidic acid which is quantitatively a minor lipid but is responsible for the repression of UNAINO-containing phospholipid biosynthetic gene by governing localization of Opi1. When the levels of phosphatidic acid are lowered which causes translocation of Opi1 from endoplasmic reticulum membrane to nucleus, where it binds to INO2 of the INO2-INO4 activator complex thereby attenuating transcriptional activation. The expression of phospholipid biosynthetic gene is affected by many conditions which include carbon source, nutrient availability, growth stage, pH and temperature. The well studied conditions which regulate phospholipid biosynthetic genes transcription are through exogenous supplementation of inositol, which is achieved by lowering of phosphatidic acid levels by its utilization for the synthesis of phosphatidylinositol. Since inositol was able to change regulates phospholipid biosynthetic gene we proposed to investigate inositol triphosphate role in such regulation. We overexpressed a plant phospholipase C in yeast to study its effect on lipid biosynthesis. The overexpressed yeast cells were subjected to microarray analysis and the result were confirmed by Q-PCR. The result obtained indicated that there was decrease in the expression of UNAINO-containing genes. To further validate our observation we carried out an in vivo assay to determined activity of enzyme involved in phospholipid biosynthesis. These results were in accordance with our expression analysis further supporting our hypothesis. Our study indicates that phospholipase c regulates phospholipid biosynthesis at transcription level in response to various stimuli. Overall, these data suggest that the C2 domain of plant PLC plays a vital role in calcium signalling. Further it can be inferred from this study that PI-PLC regulates lipid metabolism in S. cerevisiae.

Background: Subtilin is a peptide antibiotic (lantibiotic) natively produced by Bacillus subtilis ATCC6633. It is encoded in a gene cluster spaBTCSIFEGRK (spa-locus) consisting of four transcriptional units: spaS (subtilin pre-peptide), spaBTC (modification and export), spaIFEG (immunity) and spaRK (regulation). Despite the pioneer understanding on subtilin biosynthesis, a robust platform to facilitate subtilin research and improve subtilin production is still a poorly explored spot. Results: In this work, the intact spa-locus was successfully integrated into the chromosome of Bacillus subtilis W168, which is the by far best-characterized Gram-positive model organism with powerful genetics and many advantages in industrial use. Through systematic analysis of spa-promoter activities in B. subtilis W168 wild type and mutant strains, our work demonstrates that subtilin is basally expressed in B. subtilis W168, and the transition state regulator AbrB strongly represses subtilin biosynthesis in a growth phase-dependent manner. The deletion of AbrB remarkably enhanced subtilin gene expression, resulting in comparable yield of bioactive subtilin production as for B. subtilis ATCC6633. However, while in B. subtilis ATCC6633 AbrB regulates subtilin gene expression via SigH, which in turn activates spaRK, AbrB of B. subtilis W168 controls subtilin gene expression in SigH-independent manner, except for the regulation of spaBTC. Furthermore, the work shows that subtilin biosynthesis in B. subtilis W168 is regulated by the two-component regulatory system SpaRK and strictly relies on subtilin itself as inducer to fulfill the autoregulatory circuit. In addition, by incorporating the subtilin-producing system (spa-locus) and subtilin-reporting system (PpsdA-lux) together, we developed “online” reporter strains to efficiently monitor the dynamics of subtilin biosynthesis. Conclusions: Within this study, the model organism B. subtilis W168 was successfully established as a novel platform for subtilin biosynthesis and the underlying regulatory mechanism was comprehensively characterized. This work will not only facilitate genetic (engineering) studies on subtilin, but also pave the way for its industrial production. More broadly, this work will shed new light on the heterologous production of other lantibiotics.