The Neurofibromatosis 2 tumor suppressor merlin in cytoskeleton organization and cell cycle regulation

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder, which predisposes to multiple tumours of the nervous system, typically schwannomas and meningiomas. Biallelic inactivation of the NF2 gene occurs both in sporadic and NF2-related schwannomas and in most meningiomas. The NF2 gene product merlin (or schwannomin) is structurally related to the ERM proteins, ezrin, radixin and moesin, which act as molecular linkers between the actin cytoskeleton and the plasma membrane. Merlin is a tumor suppressor that participates in cell cycle regulation. Merlin s phosphorylation status appears to be associated with its tumour suppressor activity, i.e. non-phosphorylated merlin functions as a tumour suppressor, whereas protein phosphorylation results in loss of functional activity. This thesis study was initiated to investigate merlin s role as a tumor suppressor and growth inhibitor. These studies show, that like many other tumor suppressors, also merlin is targeted to the nucleus at some stages of the cell cycle. Merlin s nuclear localization is regulated by cell cycle phase, contact inhibition and adhesion. In addition, a potential nuclear binding partner for merlin was identified, Human Enhancer of Invasion 10 (HEI10), a cyclin B interacting protein. Many tumor suppressors interact with microtubules and this thesis work shows that also merlin colocalizes with microtubules in mitotic structures. Merlin binds microtubules directly, and increases their polymerization in vitro and in vivo. In addition, primary mouse Schwann cells lacking merlin displays disturbed microtubule cytoskeleton. Fourth part of this thesis work began from the notion that PKA phosphorylates an unidentified site from the merlin N-terminus. Our studies show that serine 10 is a target for PKA and modulation of this residue regulates cytoskeletal organization, lamellipodia formation and cell migration. In summary, this thesis work shows that merlin s role is much more versatile than previously thought. It has a yet unidentified role in the nucleus and it participates in the regulation of both microtubules and the actin cytoskeleton. These studies have led to a better understanding of this enigmatic tumor suppressor, which eventually will aid in the design of specific drugs for the NF2 disease.

Causes and consequences of inbreeding in the ant Formica exsecta

Human actions cause destruction and fragmentation of natural habitats, predisposing populations to loss of genetic diversity and inbreeding, which may further decrease their fitness and survival. Understanding these processes is a main concern in conservation genetics. Yet data from natural populations is scarce, particularly on invertebrates, owing to difficulties in measuring both fitness and inbreeding in the wild. Ants are social insects, and a prime example of an ecologically important group for which the effects of inbreeding remain largely unstudied. Social insects serve key roles in all terrestrial ecosystems, and the division of labor between the females in the colonies queens reproduce, workers tend to the developing brood probably is central to their ecological success. Sociality also has important implications for the effects of inbreeding. Despite their relative abundance, the effective population sizes of social insects tend to be small, owing to the low numbers of reproductive individuals relative to the numbers of sterile workers. This may subject social insects to loss of genetic diversity and subsequent inbreeding depression. Moreover, both the workers and queens can be inbred, with different and possibly multiplicative consequences. The aim of this study was to investigate causes and consequences of inbreeding in a natural population of ants. I used a combination of long-term field and genetic data from colonies of the narrow-headed ant Formica exsecta to examine dispersal, mating behavior and the occurrence of inbreeding, and its consequences on individual and colony traits. Mating in this species takes place in nuptial flights that have been assumed to be population-wide and panmictic. My results, however, show that dispersal is local, with queens establishing new colonies as close as 60 meters from their natal colony. Even though actual sib-mating was rare, individuals from different but related colonies pair, which causes the population to be inbred. Furthermore, multiple mates of queens were related to each other, which also indicates localized mating flights. Hence, known mechanisms of inbreeding avoidance, dispersal and multiple mating, were not effective in this population, as neither reduced inbreeding level of the future colony. Inbreeding had negative consequences both at the individual and colony level. A queen that has mated with a related male produces inbred workers, which impairs the colony s reproductive success. The inbred colonies were less productive and, specifically, produced fewer new queens, possibly owing to effects of inbreeding on the caste determination of female larvae. A striking finding was that males raised in colonies with inbred workers were smaller, which reflects an effect of the social environment as males, being haploid, cannot be inbred themselves. The queens produced in the inbred colonies, in contrast, were not smaller, but their immune response was up-regulated. Inbreeding had no effect on queen dispersal, but inbred queens had a lower probability of successfully founding a new colony. Ultimately, queens that survived through the colony founding phase had a shorter lifespan. This supports the idea that inbreeding imposes a genetic stress, leading to inbreeding depression on both the queen and the colony level. My results show that inbreeding can have profound consequences on insects in the wild, and that in social species the effects of inbreeding may be multiplicative and mediated through the diversity of the social environment, as well as the genetic makeup of the individuals themselves. This emphasizes the need to take into account all levels of organization when assessing the effects of genetic diversity in social animals.

Population structure and evolution in the ant Plagiolepis pygmaea and its two social parasites Plagiolepis xene and Plagiolepis grassei

Social behaviour affects dispersal of animals and is an important modifier of genetic population structures. The female sex is often philopatric, which maintains coancestry within the breeding groups and promotes cooperative behaviours. This enables also inclusive fitness returns from altruism and explains why some individuals sacrifice personal reproduction for the good of others in social insects such as ants. However, reduced dispersal and population substructuring at the level of colonies may also entail inbreeding, loss of genetic diversity, and vulnerability. In addition, the most vulnerable ants are species that are evolved to parasitize colonies of other ants, and which compromise between abilities to disperse and the efficiency to parasitize the host. On the other hand, certain social organisations of ant colonies may facilitate a species to disperse outside its natural range and become a pest. Altogether, knowledge on genetic structuring of ant populations, as well as the evolution of their life histories can contribute to conservation biology and population management. The aim of this thesis was to investigate population structures and phylogenetic evolution of the ant Plagiolepis pygmaea and its two obligatory, workerless social parasites (inquilines) P. xene and P. grassei with genetic markers and DNA sequence data. The results support the general assumption that populations of inquiline parasites are highly fragmented and genetically vulnerable. Comparison of the two parasites suggests that differences in their relative abundance may follow from their interaction with the host, i.e. how well the species is adapted to reproduce in the host colonies. The results also indicate that the most recent free living ancestor to these two parasite species is their common host. This is considered to provide evidence for the controversial issue of sympatric speciation. Further, given that the level of adaptations to parasitic life history depends on the evolutionary time since the free-living ancestor, the results establish a link between species rarity and its evolutionary age. The populations of the host species P. pygmaea displayed significantly reduced dispersal both among the females (queens) and males, and high levels of inbreeding which may enhance worker altruism. In addition, the queens were found to mate with multiple males. Given the high relatedness between the queens and their mates, this occurs probably for non-genetic reasons, e.g. without benefits associated in genetically more diverse offspring. The results hence caution that the contribution of non-genetic factors to the prevailing mating patterns and genetic population structures should not be underestimated.

Väkivalta, antisemitismi ja konflikti Mel Gibsonin elokuvassa The Passion of The Christ

Tutkielman tarkoituksena on tutkia Mel Gibsonin elokuvaa The Passion of the Christ. Tarkastelussa mukana on myös Gibsonin muu tuotanto. Tutkielma edustaa poikkitieteellistä lähestymistapaa käyttäen eksegetiikan apuvälineitä kuten lähdekritiikkiä, mutta myös elokuvatutkimuksen välineitä. Tutkimuskysymyksiä nousee lähinnä kaksi, joista seuraa kolmas kysymys: 1. Onko elokuva juutalaisvastainen? Tarkastelen lähdekritiikin avulla mitä evankeliumeja Gibson on käyttänyt elokuvassaan. Mitä muita lähteitä hän on käyttänyt? Mikä on Gibsonin omaa ilmaisua? 2. Miksi elokuva on väkivaltainen? Mitä väkivalta palvelee elokuvassa? 3. Millainen on elokuvamaailman konflikti? Pohdin elokuvamaailman sisälle rakennettua konfliktia, mutta viittaan sillä myös konfliktiin, jonka elokuva itsessään synnytti. Kysymysten ratkaisu vaatii elokuvassa käytettyjen lähteiden tutkimista, mutta myös kysymysten tarkastelua osana laajempaa kokonaisuutta, jossa on mukana koko Gibsonin elokuvatuotanto. On myös selvää, ettei Gibson ole yhtä kuin hänen elokuvansa, mutta toisaalta hänen elokuviaan ei voi tarkastella irrotettuna ohjaajasta itsestään. Ensimmäisessä luvussa tarkastelen elokuvaa ilmiönä ja elokuvasta käytyä ennakkokeskustelua. Luvussa kaksi tarkastelen Gibsonin taustaa. Millaisista lähtökohdista Gibson lähti tekemään elokuvaa? Luvussa kolme esittelen käsikirjoituksesta alkavan elokuvan yleisen tuotantoprosessin. Tutkielman päälähteenä olen käyttänyt Brentanon kirjaa The Dolorous Passion of Our Lord Jesus Christ. Kirja pohjautuu 1800-luvulla eläneen katolisen nunnan, Anne Catherine Emmerichin näkyihin. Luvussa neljä tarkastelen Gibsonin muuta tuotantoa, ja tuon keskusteluun mukaan Scorsesen elokuvan Jeesuksesta. Gibsonin muu tuotanto on jäänyt tutkijoilta liian vähälle huomiolle. Elokuva The Passion of the Christ on nähtävä osana Gibsonin muuta tuotantoa. Näin ollen elokuvaa The Passion of the Christ voidaan ymmärtää paremmin. Luvussa viisi käyn elokuvan The Passion of the Christ läpi kappale kerrallaan tutkimalla, mitä lähteitä Gibson on käyttänyt elokuvassaan. Mitä hän on ottanut evankeliumeista, mitä Emmerichiltä ja mikä on hänen omaa ilmaisuaan? Luvussa kuusi käyn läpi elokuvan vastaanottoa niin raamatuntutkijoiden kuin suuren yleisön parissa. Tutkielmassa todetaan, ettei Gibson ole antisemitisti, vaan ksenofobinen rasisti. Hänen elokuvansa ovat ksenofobisesti rasistisia. Gibsonin kaikista elokuvista on löydettävissä itseään toistavia piirteitä, joissa esiintyy muukalaiskammoa ja väkivaltaa. Gibsonin nimittäminen antisemitistiksi ei tekisi Gibsonille oikeutta. Juutalaiset ovat vain osa laajempaa kokonaisuutta. Väkivalta palvelee kaikissa elokuvissa uuden, Jumalan valtakunnan syntymistä. Konflikti syntyy uuden ja vanhan valtakunnan kansalaisten välillä. Uhrien veren kautta syntyy Jumalan valtakunta. Johtopäätöksillä on merkitystä niin Gibsonin kuin hänen elokuviensa ymmärtämiselle. Elokuvan The Passion of the Christ tulevissa tutkimuksissa on otettava huomioon, ei vain Gibsonin tausta ja lähteet, vaan myös Gibsonin muu elokuvatuotanto.

Functional regulation of the Neurofibromatosis 2 tumor suppressor merlin

Neurofibromatosis 2 (NF2) is an autosomal dominant disorder manifested by the formation of multiple benign tumors of the nervous system. Affected individuals typically develop bilateral vestibular schwannomas which lead to deafness and balance disorders. The syndrome is caused by inactivation of the NF2 tumor suppressor gene, and mutation or loss of the NF2 product, merlin, is sufficient for tumorigenesis in both hereditary and sporadic NF2-associated tumors. Merlin belongs to the band 4.1 superfamily of cytoskeletal proteins, which also contain the related ezrin, radixin, and moesin (ERM) proteins. The ERM members provide a link between the cell cytoskeleton and membrane by connecting membrane-associated proteins to actin filaments. By stabilizing complexes in the cell cortex, the ERMs modulate morphology, growth, and migration of cells. Despite their structural homology, overlapping subcellular distribution, direct molecular association, and partial overlap of molecular interactions, merlin and ezrin exert opposite effects on cell proliferation. Merlin suppresses cell proliferation, whereas ezrin expression is linked to oncogenic activity. We hypothesized that the regions which differ between the proteins might explain merlin s specificity as a tumor suppressor. We therefore analyzed the regions, which are most diverse between merlin and ezrin; the N-terminal tail and the C-terminus. To determine the properties of the C-terminal region, we studied the two most predominant merlin isoforms together with truncation variants similar to those found in patients. We also focused on the evolutionally conserved C-terminal residues, E545-E547, that harbor disease causing mutations in its corresponding DNA sequence. In addition to inhibiting cell proliferation, merlin regulates cytoskeletal organization. The morphogenic properties of merlin may play a role in tumor suppression, since patient-derived tumor cells demonstrate cytoskeletal abnormalities. We analyzed the mechanisms of merlin-induced extension formation and determined that the C-terminal region of amino acids 538-568 is particularly important for the morphogenic activity. We also characterized the role of C-terminal merlin residues in the regulation of proliferation, phosphorylation, and intramolecular associations. In contrast to previous reports, we demonstrated that both merlin isoforms are able to suppress cell proliferation, whereas C-terminally mutated merlin constructs showed reduced growth inhibition. Phosphorylation serves as a mechanism to regulate the tumor suppressive activity of merlin. The C-terminal serine 518 is phosphorylated in response to both p21-activated kinase (PAK) and protein kinase A (PKA), which inactivates the growth inhibitory function of merlin. However, at least three differentially phosphorylated forms of the protein exist. In this study we demonstrated that also the N-terminus of merlin is phosphorylated by AGC kinases, and that both PKA and Akt phosphorylate merlin at serine 10 (S10). We evaluated the impact of this N-terminal tail phosphorylation, and showed that the phosphorylation state of S10 is an important regulator of merlin s ability to modulate cytoskeletal organization but also regulates the stability of the protein. In summary, this study describes the functional effect of merlin specific regions. We demonstrate that both S10 in the N-terminal tail and residues E545-E547 in the C-terminus are essential for merlin activity and function.