From actin monomers to bundles: The roles of twinfilin and α-actinin in Drosophila melanogaster development

The actin cytoskeleton is essential for a large variety of cell biological processes. Actin exists in either a monomeric or a filamentous form, and it is very important for many cellular functions that the local balance between these two actin populations is properly regulated. A large number of proteins participate in the regulation of actin dynamics in the cell, and twinfilin, one of the proteins examined in this thesis, belongs to this category. The second level of regulation involves proteins that crosslink or bundle actin filaments, thereby providing the cell with a certain shape. α-Actinin, the second protein studied, mainly acts as an actin crosslinking protein. Both proteins are conserved in organisms ranging from yeast to mammals. In this thesis, the roles of twinfilin and α-actinin in development were examined using Drosophila melanogaster as a model organism. Twinfilin is an actin monomer binding protein that is structurally related to cofilin. In vitro, twinfilin reduces actin polymerisation by sequestering actin monomers. The Drosophila twinfilin (twf) gene was identified and found to encode a protein functionally similar to yeast and mammalian twinfilins. A strong hypomorphic twf mutation was identified, and flies homozygous for this allele were viable and fertile. The adult twf mutant flies displayed reduced viability, a rough eye phenotype and severely malformed bristles. The shape of the adult bristle is determined by the actin bundles that are regularly spaced around the perimeter of the developing pupal bristles. Examination of the twf pupal bristles revealed an increased level of filamentous actin, which in turn resulted in splitting and displacement of the actin bundles. The bristle defect was rescued by twf overexpression in developing bristles. The Twinfilin protein was localised at sites of actin filament assembly, where it was required to limit actin polymerisation. A genetic interaction between twinfilin and twinstar (the gene encoding Cofilin) was detected, consistent with the model predicting that both proteins act to limit the amount of filamentous actin. α-Actinin has been implicated in several diverse cell biological processes. In Drosophila, the only function for α-actinin yet known is in the organisation of the muscle sarcomere. Muscle and non-muscle cells utilise different α-actinin isoforms, which in Drosophila are produced by alternative splicing of a single gene. In this work, novel α-actinin deletion alleles, including ActnΔ233, were generated, which specifically disrupted the transcript encoding the non-muscle α-actinin isoform. Nevertheless, ActnΔ233 homozygous mutant flies were viable and fertile with no obvious defects. By comparing α-actinin protein distribution in wild type and ActnΔ233 mutant animals, it could be concluded that non-muscle α-actinin is the only isoform expressed in young embryos, in the embryonic central nervous system and in various actin-rich structures of the ovarian germline cells. In the ActnΔ233 mutant, α-actinin was detected not only in muscle tissue, but also in embryonic epidermal cells and in certain follicle cell populations in the ovaries. The population of α-actinin protein present in non-muscle cells of the ActnΔ233 mutant is referred to as FC-α-actinin (Follicle Cell). The follicular epithelium in the Drosophila ovary is a well characterised model system for studies on patterning and morphogenesis. Therefore, α-actinin expression, regulation and function in this tissue were further analysed. Examination of the α-actinin localisation pattern revealed that the basal actin fibres of the main body follicle cells underwent an organised remodelling during the final stages of oogenesis. This involved the assembly of a transient adhesion site in the posterior of the cell, in which α-actinin and Enabled (Ena) accumulated. Follicle cells genetically manipulated to lack all α-actinin isoforms failed to remodel their cytoskeleton and translocate Ena to the posterior of the cell, while the actin fibres as such were not affected. Neither was epithelial morphogenesis disrupted. The reorganisation of the basal actin cytoskeleton was also disturbed following ectopic expression of Decapentaplegic (Dpp) or as a result of a heat shock. At late oogenesis, the main body follicle cells express both non-muscle α-actinin and FC-α-actinin, while the dorsal anterior follicle cells express only non-muscle α-actinin. The dorsal anterior cells are patterned by the Dpp and Epidermal growth factor receptor (EGFR) signalling pathways, and they will ultimately secrete the dorsal appendages of the egg. Experiments involving ectopic activation of EGFR and Dpp signalling showed that FC-α-actinin is negatively regulated by combined EGFR and Dpp signalling. Ubiquitous overexpression of the adult muscle-specific α-actinin isoform induced the formation of aberrant actin bundles in migrating follicle cells that did not normally express FC-α-actinin, provided that the EGFR signalling pathway was activated in the cells. Taken together, this work contributes new data to our knowledge of α-actinin function and regulation in Drosophila. The cytoskeletal remodelling shown to depend on α-actinin function provides the first evidence that α-actinin has a role in the organisation of the cytoskeleton in a non-muscle tissue. Furthermore, the cytoskeletal remodelling constitutes a previously undescribed morphogenetic event, which may provide us with a model system for in vivo studies on adhesion dynamics in Drosophila.

Cellernas cytoskelett är uppbyggt av olika typer av proteinfilament. De filament som är uppbyggda av proteinet aktin har en central funktion i många cellbiologiska processer, t.ex. muskelkontraktion och cellmigrering. Denna avhandling behandlar två proteiner som reglerar cellernas aktinbaserade cytoskelett. Proteinet twinfilin hör till de proteiner som reglerar balansen mellan koncentrationen av fritt aktin och aktinfilament. Twinfilin verkar genom att binda monomerer av aktin och förhindra dessa att bilda filament. Proteinet α-aktinin kopplar ihop aktinfilament med varandra, vilket ger cytoskelettet en viss tredimensionell struktur. Både twinfilin och α-aktinin förekommer hos både encelliga och flercelliga organismer. Encelliga organismer som har muterats till att sakna twinfilin respektive α-aktinin förökar sig normalt. Varken twinfilin eller α-aktinin är alltså nödvändiga för cellens överlevnad. I detta arbete har bananflugan Drosophila melanogaster använts som modell för att analysera twinfilinets respektive α-aktininets uppgift under utvecklingen av en multicellulär organism. I detta arbete isolerades bananflugans gen för twinfilin. Den biokemiska karakteriseringen av proteinet visade att bananflugans twinfilin fungerar på samma sätt som twinfilin isolerat från andra organismer (jäst och mus), d.v.s. binder monomerer av aktin och förhindrar att dessa bildar filament. Identifieringen av en flugmutant med en kraftigt reducerad nivå av twinfilin visade att twinfilin inte är nödvändigt för individens överlevnad. Däremot var borsten på flugans rygg tydligt missbildade. Formen på borstet bestäms av de knippen av aktinfilament som bildas i borstcellen under puppstadiet. En analys av borstcellerna hos twinfilinmutanten avslöjade en ökad produktion av aktinfilament jämfört med borstcellerna i normala puppor. Detta i sin tur leder till att knippena av aktinfilament inte har en normal struktur. Proteinet twinfilin kunde lokaliseras till de ställen i borstcellen där man vet att aktinfilament bildas. Liksom i jäst- och däggdjursceller har alltså även bananflugans twinfilin till uppgift att begränsa mängden aktinfilament som bildas. Manipulering av α-aktinin i odlade däggdjursceller har visat att α-aktinin har en funktion i många olika cellbiologiska processer. Däremot har analyser av bananflugor som muterats till att sakna α-aktinin i alla celler visat att endast muskelfibrer är absolut beroende av α-aktinin för att fungera normalt. I denna avhandling beskrivs en ny typ av mutation i genen för α-aktinin, vilken endast påverkar det α-aktinin som produceras i andra än muskelceller. Denna mutation har som resultat att de flesta celltyper, bl.a. könscellerna i flugans ovarier, saknar α-aktinin. Däremot fortsätter vissa andra typer av celler att producera α-aktinin, t.ex. ovariernas follikelceller. Produktionen av α-aktinin regleras alltså på olika sätt i olika typer av celler. α-aktininets funktion i ovariernas follikelceller undersöktes närmare. När den intracellulära lokaliseringen av α-aktinin i dessa celler analyserades, kunde man konstatera att en tidigare oupptäckt omorganisering av cellernas cytoskelett ägde rum under äggutvecklingens slutskede. Denna omorganisering av cytoskelettet visade sig vara beroende av funktionellt α-aktinin, eftersom cytoskelettet i follikelceller som helt saknade α-aktinin inte genomgick en normal omorganisering. Trots detta kunde follikelcellerna slutföra sin uppgift som normalt, vilken är att producera ett fungerade äggskal. Detta arbete är det första som visar att bananflugans α-aktinin har en funktion även i andra celler än muskelceller.