Characterization of small GTPase Cdc42 from the ectomycorrhizal fungus Suillus bovinus and Agrobacterium tumefaciens-mediated transformation of fungi

Ectomycorrhizal formation between the host tree, Pinus sylvestris and fungal symbiont, Suillus bovinus was investigated at the molecular level by isolating genes regulating the organization of the actin cytoskeleton in the fungal partner S. bovinus. An Agrobacterium tumefaciens mediated transformation (ATMT) system was developed for the ectomycorrhizal fungi in order to assign specific functions to the cloned molecules. The developed ATMT system was also used to transform a plant pathogenic fungus, Helminthosporium turcicum, to hygromycin B resistance. Small GTPases Cdc42 and Rac1, the regulators of actin cytoskeleton in eukaryotes were isolated from S. bovinus. Sbcdc42 and Sbrac1, are both expressed in vegetative and in the symbiotic hyphae of S. bovinus . Using IIF microscopy, Cdc42 and actin were co-localized at the tips of vegetative hyphae and were visualized in association with the plasma membrane in swollen cells typical to the symbiotic hyphae. These results suggest that the small GTPases Cdc42 may play a significant role in the polarized growth of S. bovinus hyphae and regulate fungal morphogenesis during ectomycorrhiza formation through reorganization of the actin cytoskeleton. The functional equality of Cdc42 was tested in yeast complementation experiments using a Saccharomyces cerevisiae temperature sensitive mutant, cdc42-1ts. The genomic clone of CDC42 was isolated from S. bovinus genomic DNA via specific primers for Cdc42. The analogous S. cerevisiae cdc42 mutations, dominant active G12V and dominant negative D118A, were generated in the Sbcdc42 gene by in-vitro mutagenesis. The ectomycorrhizal fungi, S. bovinus, P. involutus and H. cylindroporum were transformed using ATMT and phleomycin as a selectable marker. PCR screeing suggested that the T-DNA was inserted in all the three fungal genomes but the fate of integration could not be proved by Southern blot analysis. An alternative Agrobacterium strain, AGL-1 and selection marker, hygromycin was used to transform our model fungus S. bovinus. PCR and Southern analysis suggested an improved efficiency of transformation. All the transformed fungal colonies selected for hygromycin gave positives in PCR and the Southerns showed multiple or single copy T-DNA integrations into the S. bovinus genome. Using the same Agrobacterium strain and the selectable marker, a maize pathogen, H. turcicum was also subjected to ATMT. The H. turcicum transformation data suggested the single copy T-DNA integrations into the genome of the screened transformants that further confirms wider applicability of the ATMT. The plasmids carrying the wild-type (pHGCDC42) and the mutated Sbcdc42 alleles (pHGGV; pHGDA) under Agaricus bisporus gpd promoter were constructed in an A. tumefaciens vector. ATMT was used to transform S. bovinus with the plasmids carrying the wild-type and mutated Sbcdc42 alleles. The isolation of Sbcdc42 and Sbrac1 genes and some other functionally related genes from ectomycorrhizal fungus, S. bovinus will form the basis of future work to resolve the signalling pathway leading to ectomycorrhizal symbiosis. The development of ATMT system will be a valuable tool in analysing the exact function of signalling pathway components in ectomycorrhizal symbiosis or in plant pathogenic interactions. The transformation frequency and broad applicability along with the simplicity of T-DNA integration make Agrobacterium a valuable, new and a powerfull tool for targeted and insertional mutagenesis in these plant associated fungi. The developed ATMT systems should therefore make it possible to generate large number of transformants with tagged genes which could then be screened for their specific roles in symbiosis and pathogenecity, respectively.

Cellular Cholesterol Accumulation and Egress from Endosomal Compartments

One of the most important factors determining the development of atherosclerosis is the amount of LDL particles in the circulation. In general, LDL particles are clinically regarded as “bad cholesterol” since these particles get entrapped within the vascular wall, leading to atherosclerosis. Circulating HDL particles are conversely regarded as “good cholesterol” because of their ability to transport cholesterol from peripheral tissues to the liver for secretion as bile salts. Once inside the artery wall LDL particles are engulfed by macrophages, resulting in macrophage foam cells. If the macrophage foam cells are not able to efflux the cholesterol back into the bloodstream, the excessive cholesterol ultimately leads to cell death, and the deposition of cellular debris within the atherosclerotic lesion. The cells ability to secrete cholesterol is mainly dependent on the ABCA1 transporter (ATP-binding cassette transporter A1) which transfers cellular cholesterol to extracellular apoA-I (apolipoprotein A-I) particles, leading to the generation of nascent HDL particles. The process of atherosclerotic plaque development is therefore to a large extent a cellular one, in which the capacity of the macrophages in handling the excessive cholesterol load determines the progression of lesion development. In this work we have studied the cellular mechanisms that regulate the trafficking of LDL-derived cholesterol from endosomal compartments to other parts of the cell. As a basis for the study we have utilized cells from patients with Niemann-Pick type C disease, a genetic disorder resulting from mutations in the NPC1 and NPC2 genes. In these cells, cholesterol is entrapped within the endosomal compartment, and is not available for efflux. By identifying proteins that bypass the cholesterol trafficking defect, we were able to identify the small GTPase Rab8 as an important protein involved in ABCA1 dependent cholesterol efflux. In the study, we show that Rab8 regulates cholesterol efflux in human macrophages by facilitating intracellular cholesterol transport, as well as by regulating the plasma membrane availability of ABCA1. Collectively, these results give new insight in to atherosclerotic lesion development and intracellular cholesterol processing.

Rab8 and Rab8-interacting proteins as players in cell polarization

Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.

ORP1L, a new Rab7 effector and regulator of late endosome functions

Oxysterol binding protein (OSBP) homologues have been found in eukaryotic organisms ranging from yeast to humans. These evolutionary conserved proteins have in common the presence of an OSBP-related domain (ORD) which contains the fully conserved EQVSHHPP sequence motif. The ORD forms a barrel structure that binds sterols in its interior. Other domains and sequence elements found in OSBP-homologues include pleckstrin homology domains, ankyrin repeats and two phenylalanines in an acidic tract (FFAT) motifs, which target the proteins to distinct subcellular compartments. OSBP homologues have been implicated in a wide range of intracellular processes, including vesicle trafficking, lipid metabolism and cell signaling, but little is known about the functional mechanisms of these proteins. The human family of OSBP homologues consists of twelve OSBP-related proteins (ORP). This thesis work is focused on one of the family members, ORP1, of which two variants were found to be expressed tissue-specifically in humans. The shorter variant, ORP1S contains an ORD only. The N-terminally extended variant, ORP1L, comprises a pleckstrin homology domain and three ankyrin repeats in addition to the ORD. The two ORP1 variants differ in intracellular localization. ORP1S is cytosolic, while the ankyrin repeat region of ORP1L targets the protein to late endosomes/lysosomes. This part of ORP1L also has profound effects on late endosomal morphology, inducing perinuclear clustering of late endosomes. A central aim of this study was to identify molecular interactions of ORP1L on late endosomes. The morphological changes of late endosomes induced by overexpressed ORP1L implies involvement of small Rab GTPases, regulators of organelle motility, tethering, docking and/or fusion, in generation of the phenotype. A direct interaction was demonstrated between ORP1L and active Rab7. ORP1L prolongs the active state of Rab7 by stabilizing its GTP-bound form. The clustering of late endosomes/lysosomes was also shown to be linked to the minus end-directed microtubule-based dynein-dynactin motor complex through the ankyrin repeat region of ORP1L. ORP1L, Rab7 and the Rab7-interacting lysosomal protein (RILP) were found to be part of the same effector complex recruiting the dynein-dynactin complex to late endosomes, thereby promoting minus end-directed movement. The proteins were found to be physically close to each other on late endosomes and RILP was found to stabilize the ORP1L-Rab7 interaction. It is possible that ORP1L and RILP bind to each other through their C-terminal and N-terminal regions, respectively, when they are bridged by Rab7. With the results of this study we have been able to place a member of the uncharacterized OSBP-family, ORP1L, in the endocytic pathway, where it regulates motility and possibly fusion of late endosomes through interaction with the small GTPase Rab7.

Functional role of the Mso1p-Sec1p complex in membrane fusion regulation

Sec1/Munc18 (SM) protein family members are evolutionary conserved proteins. They perform an essential, albeit poorly understood function in SNARE complex formation in membrane fusion. In addition to the SNARE complex components, only a few SM protein binding proteins are known. Typically, their binding modes to SM proteins and their contribution to the membrane fusion regulation is poorly characterised. We identified Mso1p as a novel Sec1p interacting partner. It was shown that Mso1p and Sec1p interact at sites of polarised secretion and that this localisation is dependent on the Rab GTPase Sec4p and its GEF Sec2p. Using targeted mutagenesis and N- and C-terminal deletants, it was discovered that the interaction between an N-terminal peptide of Mso1p and the putative Syntaxin N-peptide binding area in Sec1p domain 1 is important for membrane fusion regulation. The yeast Syntaxin homologues Sso1p and Sso2p lack the N-terminal peptide. Our results show that in addition to binding to the putative N-peptide binding area in Sec1p, Mso1p can interact with Sso1p and Sso2p. This result suggests that Mso1p can mimic the N-peptide binding to facilitate membrane fusion. In addition to Mso1p, a novel role in membrane fusion regulation was revealed for the Sec1p C-terminal tail, which is missing in its mammalian homologues. Deletion of the Sec1p-tail results in temperature sensitive growth and reduced sporulation. Using in vivo and in vitro experiments, it was shown that the Sec1p-tail mediates SNARE complex binding and assembly. These results propose a regulatory role for the Sec1p-tail in SNARE complex formation. Furthermore, two novel interaction partners for Mso1p, the Rab GTPase Sec4p and plasma membrane phospholipids, were identified. The Sec4p link was identified using Bimolecular Fluorescence Complementation assays with Mso1p and the non-SNARE binding Sec1p(1-657). The assay revealed that Mso1p can target Sec1p(1-657) to sites of secretion. This effect is mediated via the Mso1p C-terminus, which previously has been genetically linked to Sec4p. These results and in vitro binding experiments suggest that Mso1p acts in cooperation with the GTP-bound form of Sec4p on vesicle-like structures prior to membrane fusion. Mso1p shares homology with the PIP2 binding domain of the mammalian Munc18 binding Mint proteins. It was shown both in vivo and in vitro that Mso1p is a phospholipid inserting protein and that this insertion is mediated by the conserved Mso1p amino terminus. In vivo, the Mso1p phospholipid binding is needed for sporulation and Mso1p-Sec1p localisation at the sites of secretion at the plasma membrane. The results reveal a novel layer of membrane fusion regulation in exocytosis and propose a coordinating role for Mso1p in connection with membrane lipids, Sec1p, Sec4p and SNARE complexes in this process.

Integrin trafficking regulated by Rab21 is necessary for cytokinesis

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.